NZ723509B2 - Factor VIII Compositions and Methods of Making and Using Same - Google Patents

Abstract

recombinant factor VIII fusion protein comprising a factor VIII polypeptide and an extended recombinant polypeptide (XTEN), wherein said factor VIII polypeptide comprises an Al domain, an A2 domain, an A3 domain, a C1 domain, a C2 domain and optionally all or a portion of a B domain, and wherein the XTEN is inserted within the FVIII polypeptide between two adjacent domains. he XTEN is inserted within the FVIII polypeptide between two adjacent domains.

Description

FACTOR VIII COMPOSITIONS AND METHODS OF MAKING AND USING SAME CROSS-REFERENCE TO RELATED APPLICATION This is a divisional application of New Zealand ation No. 628800, which claims priority to to U.S. Provisional Application Serial No. 61/599,400 filed February 15, 2012. The above documents are herein incorporated by reference in their entirety.

SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web, and is hereby incorporated by reference in its entirety. Said ASCII copy, created on July 11, 2012, is named 32887346.txt and is 13,344,768 Bytes in size.

BACKGROUND OF THE ION Factor VIII is an important component of the intrinsic pathway of the blood coagulation cascade. In the circulation, factor VIII is mainly xed to von Willebrand factor. Upon activation by thrombin, (Factor IIa), it dissociates from the complex to interact with factor IXa in the intrinsic coagulation cascade, which, in turn, activates factor X. Once removed from the von Willebrand factor complex, activated factor VIII is proteolytically inactivated by activated Protein C (APC), factor Xa, and factor IXa, and is quickly cleared from the blood . When complexed with normal von Willebrand factor protein, the half- life of factor VIII is approximately 12 hours, whereas in the absence of von Willebrand factor, the half-life of factor VIII is reduced to 2 hours (Tuddenham EG, et al., Br J Haematol. (1982) 52(2):259-267).

In hemophilia, the clotting of blood is disturbed by a lack of certain plasma blood clotting factors. ilia A is a deficiency of factor VIII, and is a ive sex-linked, X chromosome disorder that represents 80% of hemophilia cases. The standard of care for the management of hemophilia A is replacement y with recombinant factor VIII concentrates. Subjects with severe hemophilia A have circulating procoagulant factor VIII levels below 1-2% of normal , and are generally on lactic therapy with the aim of keeping factor VIII above 1% between doses, which can usually be achieved by giving factor VIII two to three times a week. Persons with moderately severe hemophilia (factor VIII levels of 2-5% of ) constitute 25-30% hemophilia incidents and manifest bleeding after minor trauma. Persons with mild ilia A (factor VIII levels of 5-40% of normal) comprise -20% of all hemophilia incidents, and develop bleeding only after significant trauma or surgery.

The in vivo activity of exogenously ed factor VIII is limited both by a short protein halflife and inhibitors that bind to the factor VIII and diminish or y hemostatic function.

Up to 30% of hemophilia A patients receiving ously-supplied factor VIII mount an IgG immune response s factor VIII (Towfighi, F., et al. Comparative measurement of anti-factor VIII antibody by da assay and ELISA reveals restricted isotype profile and epitope specificity. Acta ol (2005) 114:84-90), which can result in the complete inhibition of its procoagulant ty and/or promote more rapid nce of the factor VIII (Briet E et al. High titer inhibitors in severe haemophilia A. A meta-analysis based on eight long-term -up studies concerning inhibitors associated with crude or intermediate purity factor VIII products. Throm. Haemost. (1994) 72: 162-164).

The IgG antibodies, called FVIII inhibitors, are primarily directed towards the A2, A3 and C2 domains (Scandella D et al. zation of epitopes for human factor VIII tor antibodies by immunoblotting and antibody neutralization. Blood (1989) 74:1618-1626), but can arise against the A1, B and C1 domains, as well. As such, treatment options for patients with FVIII inhibitors are limited.

Large proteins such as factor VIII are normally given intravenously so that the ment is directly available in the blood stream. It has been preViously demonstrated that an unmodified factor VIII injected intramuscularly yielded a maximum circulating level of only 1.4% of the normal plasma level (Pool et al, Ineffectiveness of Intramuscularly Injected Factor VIII Concentrate in Two Hemophilic Patients. New England I. ne (1966) 275(10):547-548). Formulations that could be administered other than by the intravenous route would greatly simplify their use, increase safety, and result in substantial cost savings.

Chemical modifications to a therapeutic protein can modify its in Vivo clearance rate and subsequent serum half-life. One example of a common modification is the addition of a polyethylene glycol (PEG) moiety, typically coupled to the n Via an aldehyde or N-hydroxysuccinimide (NHS) group on the PEG reacting with an amine group (e.g. lysine side chain or the N-terminus). However, the conjugation step can result in the formation of heterogeneous product mixtures that require extraction, purification and/or other further processes, all of which ineVitably affect t yield and quality control.

Also, the pharmacologic function of coagulation factors may be hampered if amino acid side chains in the Vicinity of its binding site become modified by the PEGylation process. Other approaches include the genetic fusion of an EC domain to the therapeutic protein, which increases the size of the eutic protein, hence reducing the rate of clearance through the . In some cases, the Fc domain confers the ability to bind to, and be recycled from lysosomes by the FcRn receptor, resulting in increased pharmacokinetic half-life. Unfortunately, the Fc domain does not fold ntly during recombinant expression, and tends to form insoluble precipitates known as inclusion bodies. These ion bodies must be lized and onal protein must be renatured from the misfolded aggregate, which is a time-consuming, inefficient, and expensive process.

SUMMARY OF THE INVENTION The present ion relates to novel coagulation factor VIII fusion protein compositions and the uses thereof. Specifically, the compositions provided herein are particularly used for the treatment or improvement of a condition associated with hemophilia A, deficiencies of factor VIII, bleeding disorders and coagulopathies. In one aspect, the present invention provides compositions of isolated fusion ns comprising a factor VIII (FVIII) and one or more ed recombinant polypeptides (XTEN) wherein the fusion protein exhibits procoagulant activity. A subject XTEN useful for constructing such fusion proteins is typically a polypeptide with a non-repetitive sequence and unstructured conformation.

In one embodiment, one or more XTEN is linked to a coagulation factor FVIII (“CF”) ed from native human factor VIII, factor VIII B-domain deleted sequences I BDD”), and sequence variants thereof (all the ing collectively “FVIII” or “CF”), resulting in a recombinant factor VIII-XTEN fusion protein (“CFXTEN”). The factor VIII polypeptide component of the CFXTEN comprises an Al domain, an A2 domain, a Cl domain, a C2 domain, and optionally a B domain or a portion thereof In some embodiments, the FVIII is further characterized by delineation of the aforementioned domains to comprise an acidic a1, a2 and a3 spacer. In another embodiment, the present disclosure is directed to pharmaceutical compositions sing the fusion proteins and the uses thereof in s and regimens for treating factor VIII-related conditions. The CFXTEN compositions have ed pharmacokinetic and cologic properties compared to FVIII not linked to XTEN, which may permit more convenient dosing and improved efflcacy.

In a first aspect, the invention relates to recombinant factor VIII fusion proteins comprising a factor VIII polypeptide and one or more extended recombinant polypeptide (XTEN) linked to the factor VIII. In some embodiments, the invention provides recombinant factor VIII fusion proteins comprising a factor VIII polypeptide and at least one extended recombinant polypeptide (XTEN), wherein said factor VIII polypeptide comprises an Al domain including an al acidic spacer region, an A2 domain including an a2 acidic spacer region, an A3 domain including an a3 acidic spacer region, Cl , C2 domain and optionally all or a n of B domain, and wherein said at least one XTEN is linked to said factor VIII polypeptide at (i) the C-terminus of said factor VIII polypeptide; (ii) within B domain of said factor VIII polypeptide if all or a portion of B domain is present; (iii) within the Al domain of said factor VIII polypeptide; (iV) within the A2 domain of said factor VIII polypeptide; (V) within the A3 domain of said factor VIII polypeptide; (Vi) within the Cl domain of said factor VIII polypeptide; (Vii) within the C2 domain of said factor VIII polypeptide; (Viii) at the N—terminus of said factor VIII polypeptide, or (ix) between two domains of said factor VIII polypeptide, wherein the fusion protein retains at least about %, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% of the procoagulant actiVity, when measured by an in Vitro coagulation assay, compared to a corresponding factor VIII not linked to XTEN. In one embodiment, in the foregoing recombinant factor VIII fusion protein the at least one XTEN is linked to said factor VIII polypeptide at a site at or within 1 to 6 amino acids of a site selected from Table 5, Table 6, Table 7, Table 8, and Table 9. In other embodiments, the invention provides recombinant factor VIII fusion proteins comprising a factor VIII polypeptide and at least a first extended inant polypeptide (XTEN), wherein said factor VIII polypeptide ses an Al domain including an al acidic spacer region, an A2 domain including an a2 acidic spacer region, an A3 domain including an a3 acidic spacer , a Cl domain, a C2 domain and optionally all or a portion of a B domain, and wherein said first XTEN is linked to said factor VIII polypeptide at (i) the C- terminus of said factor VIII polypeptide; (ii) within the B domain of said factor VIII polypeptide if all or a portion of the B domain is present; (iii) within the Al domain of said factor VIII polypeptide; (iV) within the A2 domain of said factor VIII polypeptide; (V) within the A3 domain of said factor VIII polypeptide; (Vi) within the C1 domain of said factor VIII polypeptide; or (Vii) within the C2 domain of said factor VIII polypeptide; and when compared to a corresponding factor VIII protein not linked to XTEN, the fusion protein (a) retains at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 100%, 200%, 300%, 400%, or 500% of the procoagulant actiVity in an in Vitro coagulation assay bed herein or other such assays known in the art, and/or (b) exhibits d binding to an anti- factor VIII antibody in an in vitro binding assay described herein or other such assays known in the art. I n one embodiment, in the foregoing recombinant factor VIII fusion protein the at least one XTEN is linked to said factor VIII polypeptide at a site at or within 1 to 6 amino acids of a site selected from Table , Table 6, Table 7, Table 8, and Table 9. In other embodiments, the invention es recombinant factor VIII fusion proteins comprising a factor VIII polypeptide and at least a first extended recombinant polypeptide (XTEN), wherein said factor VIII polypeptide comprises an A1 domain including an al acidic spacer region, an A2 domain including an a2 acidic spacer region, an A3 domain including an a3 acidic spacer region, a C1 domain, a C2 domain and optionally all or a portion of a B domain, and wherein said first XTEN is linked to said factor VIII polypeptide at an insertion site selected from Table 6 and Table 7 and wherein the fusion n retains at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, 200%, 300%, 400%, or 500% of the procoagulant activity, when measured by an in vitro coagulation assay described herein or other such assays known in the art, compared to a corresponding factor VIII protein not linked to XTEN. Non-limiting examples of the factor VIII protein not linked to XTEN includes native FVIII, BDD FVIII, pBC100 and sequences from Table 1. In another embodiment of the recombinant factor VIII fusion protein, the factor VIII ptide has at least about 80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% sequence ty to a sequence selected from the group consisting of the sequences of Table 1, the sequence depicted in and the sequence depicted in when optimally aligned. In yet another embodiment, the fusion protein comprises at least another XTEN linked to said factor VIII ptide at the C-terminus of said factor VIII polypeptideor within or optionally replacing the B domain of said factor VIII polypeptide. In a c embodiment, the fusion protein comprises at least one XTEN sequence located within or optionally replacing the B domain of said factor VIII polypeptide. In another specific embodiment, the fusion protein comprises at least one XTEN sequence linked to said factor VIII polypeptide at the C- us of said factor VIII polypeptide. In one ment, the recombinant factor VIII fusion protein comprises a B-domain deleted variant of human factor VIII, wherein the B-domain deletion starts from a first position at about amino acid residue number 741 to about 750 and ending at a second position at amino acid e number 1635 to about 1648 with reference to full-length human factor VIII sequence as set forth in In another embodiment, the recombinant factor VIII fusion n comprises a first XTEN sequence linked to said factor VIII polypeptide at the C-terminus of said factor VIII polypeptide, and at least a second XTEN within or ing the B domain of said factor VIII polypeptide, wherein the second XTEN is linked to the C-terminal end of about amino acid residue number 741 to about 750 and to the N—terminal end of amino acid residue numbers 1635 to about 1648 with reference to full-length human factor VIII ce as set forth in wherein the cumulative length of the XTEN is at least about 100 amino acid residues. In one embodiment, in the foregoing fusion protein, the second XTEN links the factor VIII amino acids between N745 to P1640 or between S743 to Q1638 or between P747 to V1642 or between N745 and Q1656 or between N745 and S1657 or between N745 and T1667 or between N745 and Q1686 or between R747 and V1642 or between T751 and T1667.

In one embodiment, the recombinant factor VIII fusion protein comprises a sequence haVing at least about 80% sequence identity, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, to about 100% sequence identity compared to a sequence of comparable length selected from Table 21, when optimally aligned. In another embodiment, the recombinant factor VIII fusion protein ses at least a second XTEN, optionally a third XTEN, optionally a fourth XTEN, optionally a fifth XTEN and optionally a sixth XTEN, wherein each of the second, third, fourth, fifth, or sixth XTEN is linked to said factor VIII polypeptide at a second, third, fourth, fifth, or sixth site selected from the group consisting of an insertion site from Table 5, Table 6, Table 7 Table 8, and Table 9; a location within 6 amino acids of amino acid residue 32, 220, 224, 336, 339, 390, 399, 416, 603, 1656, 1711, 1725, 1905 and 1910 re factor VIII; a location between any two adjacent domains of said factor VIII polypeptide, wherein said two adjacent domains are selected from the group ting ofA1 and A2 s, A2 and B domains, B and A3 domains, A3 and C1 domains, and C1 and C2 domains; a location within the B domain of said factor VIII polypeptide, n the second XTEN is linked to the C-terminal end of about amino acid e number 741 to about 750 and to the N—terminal end of amino acid residue numbers 1635 to about 1648 of a native factor VIII sequence; and the C-terminus of said factor VIII polypeptide. In one embodiment, the first XTEN is separated from the second XTEN by at least 10 amino acids, at least 50 amino acids, at least 100 amino acids, at least 200 amino acids, at least 300 amino acids, or at least 400 amino acids. In one embodiment of the recombinant factor VIII fusion protein that comprises at least a second XTEN, optionally a third XTEN, optionally a fourth XTEN, optionally a fifth XTEN and optionally a sixth XTEN, each XTEN has at least about 80% sequence identity, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% sequence identity compared to an XTEN of comparable length ed from the group consisting of the ces in Table 4, Table 13, Table 14, Table 15, Table 16, and Table 17, when optimally aligned. In yet another embodiment of the recombinant factor VIII fusion protein that comprises at least a second XTEN, optionally a third XTEN, optionally a fourth XTEN, optionally a fifth XTEN and optionally a sixth XTEN, In red embodiments, the recombinant factor VIII fusion protein exhibits a terminal half-life at least about 3 hours, or 4 hours, or 6 hours, or 12 hours, or 13 hours, or 14 hours, or 16 hours, or 24 hours, or 48 hours, or 72 hours, or 96 hours, or 120 hours, or 144 hours, or 7 days, or 14 days, or 21 days when administered to a subject, n said t is selected from human and factor VIII/yon Willebrand factor double knock-out mouse. Further, in the ments of this paragraph, the fusion protein exhibits reduced binding to anti-factor VIII antibody or greater retained procoagulant activity, or both as compared to a ponding factor VIII not linked to XTEN. In one embodiment, the procoagulant activity of the recombinant factor VIII fusion protein is at least 30%, or 40%, 50%, 80%, 100%, 200%, 300%, 400%, or 500% greater procoagulant ty in the presence of the anti-FVIII antibody compared to a corresponding factor VIII not linked to XTEN when each are assayed by an in Vitro coagulation assay. In one embodiment, the reduced binding of the fusion protein to anti-factor VIII antibody is determined using a Bethesda assay using anti-factor VIII antibody selected from the group consisting of the dies of Table 10 and polyclonal antibody from a hemophilia A patient with factor VIII inhibitors, wherein the reduced binding and retained procoagulant actiVity of the fusion protein is eVidenced by a lower Bethesda titer of at least about 2, 4, 6, 8, 10, 12, 15, 20, 30, 40, 50, 60, 70, 80, 100, or 200 Bethesda units for the fusion protein compared to that for the factor VIII not linked to XTEN.

In one embodiment, the recombinant factor VIII fusion protein can, for example, comprise one or more XTEN wherein the XTEN has at least about 80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% sequence ty compared to one or more XTEN of comparable length selected from Table 4, Table 13, Table 14, Table 15, Table 16, and Table 17, when optimally aligned.

In another , the invention relates to recombinant factor VIII fusion proteins comprising FVIII and one or more XTEN in specific N— to C-terminus urations. In one embodiment of the CFXTEN composition, the invention provides a recombinant factor VIII fusion protein of formula I: (XTEN)X-CF-(XTEN)y I wherein independently for each occurrence, CF is a factor VIII as defined , including ces having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity with ced from Table 1; X is either 0 or 1 and y is either 0 or 1 wherein x+y 31; and XTEN is an extended recombinant polypeptide as described herein, including, but not limited to ces having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 4. Accordingly, the CFXTEN fusion composition can have XTEN-CF, XTEN-CF-XTEN, or CF-XTEN configurations.

In another ment of the CFXTEN composition, the invention provides a recombinant factor VIII fusion protein of formula II: (XTEN)X-(S)X-(CF)-(XTEN) y II wherein independently for each occurrence, CF is a factor VIII as defined herein, ing sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 1; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a ge sequence or amino acids compatible with restrictions sites; x is either 0 or 1 and y is either 0 or 1 wherein x+y 21; and XTEN is an extended inant polypeptide as described herein including, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 4.

In another embodiment of the CFXTEN composition, the invention provides a inant factor VIII fusion n, wherein the fusion protein is of formula III: (XTEN)X-(S)X-(CF)-(S)y-(XTEN)y 111 wherein independently for each occurrence, CF is a factor VIII as defined herein, including sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequence set for in Table 1; S is a spacer ce having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence or amino acids compatible with restrictions sites; x is either 0 or 1 and y is either 0 or 1 wherein x+y 21; and XTEN is an extended recombinant polypeptide as described herein including, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 4.

In another embodiment of the CFXTEN composition, the ion provides a recombinant factor VIII fusion protein of formula IV: (A1)-(XTEN)u-(A2)-(XTEN)V-(B)-(XTEN)W-(A3)-(XTEN)x-(C1)-(XTEN)y-(C2)-(XTEN)Z wherein independently for each occurrence, A1 is an A1 domain of FVIII; A2 is an A2 domain of FVIII; A3 is an A3 domain of FVIII; B is a B domain of FVIII which can be a fragment or a splice variant of the B domain; C1 is a C1 domain of FVIII; C2 is a C2 domain of FVIII; v is either 0 or 1; w is either 0 or 1; X is either 0 or 1; y is either 0 or 1; y is either 0 or 1 with the proviso that u + v + X + y+z 31; and XTEN is an extended recombinant ptide as described herein including, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to ces set forth in Table 4.

In another embodiment of the CFXTEN ition, the invention provides a recombinant factor VIII fusion protein of formula V: (XTEN)t-(S)a -(A1)-(S)b-(XTEN)u-(S)b-(A2)-(S)c-(XTEN)V-(S)c-(B)-(S)d-(XTEN)w-(S)d-(A3)—(S)e- x-(S)e-(C1)-(s)r(XTEN)y-(S)r(cz)-(S)g-(XTEN)Z V n independently for each occurrence, A1 is an A1 domain of FVIII; A2 is an A2 domain of FVIII; A3 is an A3 domain of FVIII; B is a B domain of FVIII which can be a fragment or a splice variant of the B domain; C1 is a C1 domain of FVIII; C2 is a C2 domain of FVIII; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence or amino acids compatible with restrictions sites; a is either 0 or 1; b is either 0 or 1; c is either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is either 0 or 1; g is either 0 or 1; t is either 0 or 1; u is either 0 or 1; v is either 0 or 1; W is 0 or 1, X is either 0 or 1; y is either 0 or 1; z is either 0 or 1 with the proviso that t + u + V + w+ X + y + z 31; and XTEN is an extended recombinant polypeptide as described herein including, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to ces set forth in Table 4. In another embodiment of formula V, the spacer ce is glycine or a sequence selected from Tables 11 and 12.

In another ment of the CFXTEN composition, the invention provides a recombinant factor VIII fusion protein of formula VI: (XTEN)u-(S)a-(A1)-(S)b-(XTEN)v-(S)b-(A2)-(S)c-(XTEN)w-(S)c-(A3)-(S)d-(XTEN)x-(S)d-(C1)- (s)e-(XTEN)y-(8)6-(C2)—(S)r(XTEN)Z VI wherein independently for each occurrence, A1 is an A1 domain of FVIII; A2 is an A2 domain of FVIII; A3 is an A3 domain of FVIII; C1 is a C1 domain of FVIII; C2 is a C2 domain of FVIII; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence or amino acids compatible with restrictions sites; a is either 0 or 1; b is either 0 or 1; c is either 0 or 1; dis either 0 or 1; e is either 0 or 1; fis either 0 or 1; uis either 0 or 1; Vis either 0 or 1; Wis 0 or 1, X is either 0 or 1; y is either 0 or 1; z is either 0 or 1 with the proviso that u + V + w+ X + y + z 31; and XTEN is an extended recombinant polypeptide as described herein including, but not d to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 4. In another embodiment of formula V, the spacer sequence is e or a sequence selected from Tables 11 and 12.

In another embodiment of the CFXTEN composition, the invention provides a recombinant factor VIII fusion protein of formula VII: (SP)-(XTEN)x-(CS)X-(S)x-(FVIII_1-745)-(S)y-(XTEN)y-(S)y-(FVIII_1640-2332)-(S)Z-(CS)Z- (XTEN)Z VII wherein independently for each occurrence, SP is a signal peptide, ably with sequence MQIELSTCFFLCLLRFCFS (SEQ ID NO: 1611), CS is a cleavage sequence listed in Table 12, S is a spacer ce having n 1 to about 50 amino acid residues that can ally include amino acids ible with restrictions sites, “FVIII_1-745” is residues 1-745 of Factor FVIII and “FVIII_1640-2332” is residues 1640-2332 of FVIII, X is either 0 or 1, y is either 0 or 1, and z is either 0 or 1, wherein x+y+z >2; and XTEN is an eXtended recombinant polypeptide as bed herein including, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity sequences set forth in Table 4. In one embodiment of formula VII, the spacer sequence is GPEGPS (SEQ ID NO: 1612). In another embodiment of formula V, the spacer sequence is glycine or a sequence selected from Tables 11 and 12.

In another embodiment of the CFXTEN composition, the invention provides a recombinant factor VIII fusion protein of formula VIII: (A1)—(S)a-(XTEN)V-(S)a-(A2)-(B1)—(S)b-(XTEN)w-(S)b-(B2)-(A3)—(S)c-(XTEN)x-(S)c-(C1)—(S)d- (XTEN)y-(S)d-(C2)-(S)e-(XTEN)Z VIII wherein independently for each occurrence, A1 is an A1 domain of FVIII; A2 is an A2 domain of FVIII; B1 is a fragment of the B domain that can have from residue 741 to 743-750 of FVIII or atively from about residue 741 to about residues 745 of FVIII; B2 is a fragment of the B domain that can have from residues 1635-1686 to 1689 of FVIII or alternatively from about residue 1640 to about residues 1689 of FVIII; A3 is an A3 domain of FVIII; C1 is a C1 domain of FVIII; C2 is a C2 domain of FVIII; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence or amino acids compatible with ctions sites; a is either 0 or 1; b is either 0 or 1; c is either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is either 0 or 1; u is either 0 or 1; V is either 0 or 1; W is 0 or 1, X is either 0 or 1; y is either 0 or 1; z is either 0 or 1 With the proviso that u + V + w+ X + y + z 21; and XTEN is an extended recombinant polypeptide as described herein including, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 4. In one embodiment of formula VIII, the spacer sequence is GPEGPS (SEQ ID NO: 1612). In another embodiment of a V, the spacer sequence is glycine or a sequence selected from Tables 11 and 12.

In r embodiment of the CFXTEN composition, the invention provides a recombinant factor VIII fusion protein of formula IX: (A1N)'(S)a'(XTEN)I'(S)b'(A1C)'(A2 N)-(S)c-(XTEN)u-(S)d-(A2c)-(BN)-(S)e-(XTEN)v-(S)r(Bc)-(A3N)- (S)g'(XTEN)W'(S)h'(A3C)'(C1N)'(S)i'(XTEN)X'(S)j'(C1C)'(C2N)'(S)k'(XTEN)y'(S)1'(C2C)'(S)m'(XTEN)z Wherein independently for each occurrence, AlN is a nt of the A1 domain from at least e number 1 red relative to native, mature FVIII) to no more than residue number 371, A1C is a fragment of the A1 domain from at least residue number 2 to no more than residue number 372, With the priViso that no sequence of the A1N fragment is duplicated in the A1C is a fragment; A2N is a nt of the A2 domain from at least residue number 373 to no more than residue number 739, Me is a fragment of the A2 domain from at least residue number 374 to no more than residue number 740, With the priViso that no ce of the A2N fragment is duplicated in the Me is a fragment; BN is a fragment of the B domain from at least residue number 741 to no more than residue number 1647, BC is a fragment of the B domain from at least e number 742 to no more than residue number 1648, With the priViso that no sequence of the BN fragment is duplicated in the BC is a fragment; A3N is a fragment of the A3 domain from at least residue number 1649 to no more than residue number 2019, A3C is a fragment of the A3 domain from at least residue number 1650 to no more than residue number 2019, With the priViso that no sequence of the A3N fragment is duplicated in the A3C is a fragment; ClN is a fragment of the C1 domain from at least residue number 2020 to no more than e number 2171, C1C is a fragment of the C1 domain from at least residue number 2021 to no more than residue number 2172, With the priViso that no sequence of the ClN fragment is ated in the C1C is a fragment; C2N is a fragment of the C2 domain from at least e number 2173 to no more than residue number 2331, C2C is a fragment of the C2 domain from at least residue number 2174 to no more than e number 2332, with the priviso that no sequence of the C2N fragment is duplicated in the C2C is a fragment; S is a spacer sequence having between 1 to about 50 amino acid es that can optionally include a cleavage sequence or amino acids compatible with restrictions sites; a is either 0 or 1; b is either 0 or 1; c is either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is either 0 or 1; g is either 0 or 1; h is either 0 or 1; i is either 0 or 1; j is either 0 or 1; k is either 0 or 1; l is either 0 or 1; m is either 0 or 1; t is either 0 or 1; u is either 0 or 1; V is either 0 or 1; W is 0 or 1, X is either 0 or 1; y is either 0 or 1; z is either 0 or 1 with the proviso that t + u + V + w+ X + y + z 31; and XTEN is an extended recombinant polypeptide as described herein including, but not limited to sequences having at least about 80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% sequence identity compared to one or more XTEN of able length ed from Table 4. In one embodiment of formula IX, the spacer sequence is GPEGPS (SEQ ID NO: 1612).

In another embodiment of formula V, the spacer sequence is e or a sequence selected from Tables 11 and 12. In another embodiment of formula IX, Z is 1. In another embodiment of the fusion protein of formula IX V is 1 and the XTEN is linked to the C-terminal end of about amino acid residue number 741 to about 750 and to the N—terminal end of amino acid residue numbers 1635 to about 1648 with reference to full-length human factor VIII sequence as set forth in In another embodiment of the fusion n of formula IX, the sum of t, u, v, W, X, y, and 2 equals 2, 3, 4, 5, or 6. In another embodiment of formula IX, the sum of t, u, v, W, X, y, and 2 equals 2, and v is 1 and z is 1. In another embodiment of the fusion protein of formula IX, the sum of t, u, v, W, X, y, and 2 equals 3, v and 2 each equal 1, and either t, u, W, X or y is 1. In another embodiment of formula IX, the sum of t, u, v, W, X, y, and 2 equals 4, v and W and 2 each equal 1, and two of t, u, X or y is 1. In another embodiment of the fusion protein of formula IX, the cumulative length of the XTENs is n about 84 to about 3000 amino acid residues. In another embodiment of formula IX, at least one XTEN is inserted immediately downstream of an amino acid which corresponds to an amino acid in mature native human factor VIII selected from the group consisting of amino acid residue number 32, 220, 224, 336, 339, 399, 416, 603, 1656, 1711, 1725, 1905 and 1910. In another embodiment of the fusion protein a IX, each XTEN is linked to said fusion protein at sites selected from Table 5, Table 6, Table 7, Table 8, and Table 9. In another embodiment of the fusion protein formula IX, each XTEN has at least about 80%, or about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% sequence identity compared to an XTEN of comparable length selected from the group consisting of the ces in Table 4, Table 13, Table 14, Table 15, Table 16, and Table 17, when optimally d.

In another embodiment of the CFXTEN composition, the invention provides a first recombinant factor VIII polypeptide of formula X: (A1)—a1—(A2)—a2—[B] X and a second polypeptide comprising Formula XI: a3 — (A3) — (Cl) - (C2) XI wherein the first polypeptide and the second polypeptide are fused or exist as a heterodimer; wherein, Al is an Al domain of factor VIII; A2 is an A2 domain of factor VIII; [B] is a B domain of factor VIII, a fragment thereof, or is deleted; A3 is an A3 domain of factor VIII; Cl is a Cl domain of factor VIII; C2 is a C2 domain of factor VIII; al, a2, and a3 are acidic spacer regions; wherein the Al domain ses an XTEN permissive loop-l (Al -1) region and an XTEN permissive loop-2 (Al -2) region; wherein the A2 domain comprises an XTEN permissive loop-l (A2-l) region and an XTEN permissive loop-2 (A2- 2) region; n the A3 domain comprises an XTEN permissive loop-l (A3-l) region and an XTEN permissive loop-2 (A3 -2) region; wherein an XTEN sequence is inserted into at least one of the regions Al -1, Al -2, A2-l, A2-2, A3-l, or A3 -2; and wherein the recombinant factor VIII protein exhibits procoagulant activity. In one embodiment of the dimer, the first ptide and the second polypeptide form a single polypeptide chain sing the formula (Al) al (A2) a2 [B] [a3] (A3) — (C1) — (C2). In one embodiment of the foregoing, “fused” means a peptidic bond.

In another embodiment of the CFXTEN ition, the invention provides a first recombinant factor VIII polypeptide of formula X: (Al)—al—(A2)—a2—[B] X and a second polypeptide comprising Formula XI: a3 — (A3) — (Cl) - (C2) XI wherein the first polypeptide and the second polypeptide are fused or exist as a heterodimer; wherein, Al is an Al domain of factor VIII; A2 is an A2 domain of factor VIII; [B] is a B domain of factor VIII, a fragment thereof, or is d; A3 is an A3 domain of factor VIII; Cl is a Cl domain of factor VIII; C2 is a C2 domain of factor VIII; al, a2, and a3 are acidic spacer regions; wherein an XTEN ce is inserted into a3; and wherein the recombinant factor VIII n exhibits procoagulant ty. In one embodiment of the heterodimer, the first polypeptide and the second polypeptide form a single polypeptide chain comprising the formula (Al) al (A2) a2 [B] [a3] (A3) (C1) (C2). In one embodiment of the foregoing, “fused” means a peptidic bond. in embodiments of the foregoing formulae X and XI polypeptides, the XTEN permissive loops are contained within e-exposed, flexible loop structures, and wherein Al-l is located between beta strand 1 and beta strand 2, Al -2 is located between beta strand 11 and beta strand l2, A2-l is located between beta strand 22 and beta strand 23, A2-2 is located between beta strand 32 and beta strand 33, A3-l is located n beta strand 38 and beta strand 39 and A3-2 is located between beta strand 45 and beta strand 46, according to the secondary structure of mature factor VIII stored as Accession Number 2R7E of the DSSP database. In other embodiments of the foregoing formulae X and \I polypeptides, the surface-exposed, flexible loop ure comprising Al-l corresponds to a region in native mature human factor VIII from about amino acid 15 to about amino acid 45. In other embodiments ofthe foregoing formulae X and Xi polypeptides the Al -1 corresponds to a region in native mature human factor VIII from about amino acid 18 to about amino acid 41. In other embodiments of the foregoing formulae X and XI polypeptides, the surface-exposed, flexible loop structure comprising A1-2 corresponds to a region in native mature human factor VIII from about amino acid 201 to about amino acid 232. In other embodiments of the foregoing formulae X and XI polypeptides the A1-2 corresponds to a region in native mature human factor VIII from about amino acid 218 to about amino acid 229. In other embodiments of the foregoing formulae X and XI polypeptides, the surface-exposed, flexible loop structure comprising A2-1 corresponds to a region in native mature human factor VIII from about amino acid 395 to about amino acid 421. In other embodiments of the foregoing ae X and XI polypeptides, the A2-1 corresponds to a region in native mature human factor VIII from about amino acid 397 to about amino acid 418. In other embodiments of the foregoing ae X and XI polypeptides, the surface-exposed, flexible loop structure comprising A2-2 corresponds to a region in native mature human factor VIII from about amino acid 577 to about amino acid 635. In other ments of the foregoing formulae X and XI polypeptides, the A2-2 corresponds to a region in native mature human factor VIII from about amino acid 595 to about amino acid 607. In other embodiments of the foregoing formulae X and XI polypeptides, the surface-exposed, flexible loop structure comprising A3-1 corresponds to a region in native mature human factor VIII from about amino acid 1705 to about amino acid 1732. In other embodiments of the foregoing formulae X and XI polypeptides, the A3-1 corresponds to a region in native mature human factor VIII from about amino acid 1711 to about amino acid 1725. In other embodiments of the ing ae X and XI polypeptides, the the surface-exposed, flexible loop structure comprising A3-2 corresponds to a region in native mature human factor VIII from about amino acid 1884 to about amino acid 1917. In other embodiments of the foregoing formulae X and XI ptides, the A3-2 corresponds to a region in native mature human factor VIII from about amino acid 1899 to about amino acid 1911. in other embodiments of the foregoing formulae X and XI polypeptides, an XTEN sequence is inserted into at least two of the s A1-1, A1-2, A2-1, A2-2, A3-1, or A3-2. In other embodiments of the in0'0. formulae X and XI polypeptides, an XTEN sequence is inserted ately downstream of an amino acid which corresponds to an amino acid in mature native human factor VIII selected from the group consisting of amino acid residue number 32, 220, 224, 336, 339, 399, 416, 603, 1656, 1711, 1725, 1905 and 1910. In other embodiments of the foregoing formulae X and Xl polypeptides, an additional XTEN sequence is inserted into the a3 acidic spacer region. In other embodiments of the foregoing formulae X and XI polypeptides, an additional XTEN sequence is inserted into the a3 acide spacer ately ream of an amino acid which corresponds to amino acid 1656. In other embodiments of the foregoing formulae X and XI polypeptides, the A1 domain comprises an XTEN permissive loop-1 (A1- 1) region and an XTEN permissive loop-2 (A1-2) region wherein the A2 domain comprises an XTEN permissive loop-1 (A2-1) region and an XTEN permissive loop-2 (A2-2) region, and wherein the A3 domain comprises an XTEN permissive loop-1 (A3-1) region and an XTEN permissive loop-2 (A3 -2) region, and wherein an additional XTEN sequence is inserted into at least one of the regions A1-1, A1-2, A2-1, A2-2, A3-1, or A3 -2. In other embodiments of the foregoing formulae X and XI polypeptides, an additional XTEN sequence is inserted immediately downstream of an amino acid which corresponds to an amino acid in mature native human factor VIII selected from the group consisting of amino acid residue number 32, 220, 224, 336, 339, 390, 399, 416, 603, 1656, 1711, 1725, 1905 and 1910. In the ing embodiments of formulae X and Xi polypeptides, the fusion protein exhibits at least about %, 40%, 50%, 60%, 70%, or 80%, or 90% of the procoagulant activity of the corresponding factor VIII not linked to XTEN, wherein the procoagulant activity is assayed by an in vitro coagulation assay.

In all embodiments, the ptide can, for example, exhibit an in vitro procoagulant activity exceeding 0.5 IU/ml, or 1.0, or 1.5, or 2.0 IU/ml When expressed in cell-culture medium and d by an in vitro coagulation assay. The procoagulant activity can be measured by a chromogenic assay, a one stage clotting assay (e. g., a aPTT) or both.

In some ments, Wherein the recombinant factor VIII fusion n comprises a factor VIII and at least a first and a second XTEN, the at least first XTEN is separated from the at least second XTEN by at least 10 amino acids, at least 50 amino acids, at least 100 amino acids, at least 200 amino acids, at least 300 amino acids, or at least 400 amino acids. $026} In preferred embodiments, the recombinant factor VIII fusion protein comprising a factor VIII and at least a first XTEN and, optionally, at least a second, or optionally at least a third, or ally at least a fourth XTEN, the fusion protein exhibits d binding to an anti-factor VIII antibody as ed to the corresponding factor VIII not linked to XTEN. The reduced binding can be assessed either in vivo or by an in vitro assay. In one embodiment, the in vitro assay is an ELISA assay, Wherein the binding of an anti-FVIII antibody to the fusion protein is reduced at least about 5%, 10%, 15%, 20%, %, 30%, 35% or at least about 40% or more compared to a FVIII not linked to XTEN. In another embodiment, the in vitro assay is a Bethesda assay Wherein the d binding of the fusion protein is evidenced by a lower da titer of at least about 2, 4, 6, 8, 10, 12, 15, 20, 30, 40, 50, 60, 70, 80, 100, or 200 Bethesda units for the fusion protein ed to that for a factor VIII not linked to XTEN. In the in vitro assays, the anti-factor VIII antibody is selected from an antibody of Table 10 and polyclonal antibody from a hemophilia A patient With factor VIII inhibitors. In particular embodiments of a recombinant factor VIII fusion protein comprising a factor VIII and at least a first and a second XTEN exhibiting reduced binding to a factor VIII inhibitor antibody, the first XTEN is linked to said factor VIII polypeptide Within a C2 domain of said factor VIII polypeptide, and the second XTEN is linked to said factor VIII polypeptide Within an A1 or A2 domain of said factor VIII ptide, Wherein said fusion protein exhibits reduced binding to a factor VIII inhibitor antibody as compared to the corresponding factor VIII not linked to XTEN, Wherein the factor VIII inhibitor antibody is capable of binding to an epitope located Within the A1, A2 or C2 domain, and further Wherein the fusion protein exhibits procoagulant activity. In one embodiment of the foregoing fusion protein, the second XTEN is linked to said factor VIII polypeptide with in the A2. domain of the factor VIII ptide and the factor VIII inhibitor antibody binds to the A2 domain of the factor VIII polypeptide. In another embodiment of the foregoing fusion protein, the second XTEN is linked to said factor VIII polypeptide Within the (.2 domain of the factor VIII polypeptide and the factor VIII inhibitor antibody binds to the C2 domain of the factor VIII polypeptide. The binding of an anti—factor VIII antibody to the fusion protein is d by at least about 5%, I094), I596, 20%, 25%, 30%, 35% or 40% compared to the eorreoponding factor VIII not linlt’ed to XTEN when assayed by an ELISA assay, wherein the anti~factor \HII antibody is selected from the group consisting of the dies in Table l0 and a polyclonal antibody from a hemophilia A subject with factor Vlll inhibitors. The foregoing fusion proteins can r comprise at least three XTEN s, wherein the at least third XTEN is linked to the factor VIII at a site selected from within or replacing the B domain, at the {TL-terminus, and at or Within 1, 2, 3, 4, 5, or 6 amino acids of an insertion site selected from Table 7 or Table 9. in the ments with reduced binding to anti-factor Vlll antibodies, the fusion protein has greater procoagulant activity in the presence of the anti—FVlll antibody of at least l0%, 20%, 30%, 40%., 50%, 80%, l00%, 200%, 300%, 400%, or 500% or more compared to a corresponding factor VIII not linked to XTEN when assayed by an in Vll't’O coagulation assay (eg, a chromogenic or one—stage clotting assay). {0027} in all embodiments, the XTEN of the fusion n can, for example, be characterized in that the XTEN comprise at least 36, or at least 42, or at least 72, or at least 96, or at least 144, or at least 288, or at least 400, or at least 500, or at least 576, or at least 600, or at least 700, or at least 800, or at least 864, or at least 900, or at least 1000, or at least 2000, to about 3000 amino acid residues or even more residues; the sum of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues constitutes at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% of the total amino acid residues of the XTEN; the XTEN is substantially non-repetitive such that (i) the XTEN contains no three contiguous amino acids that are identical unless the amino acids are serine; (ii) at least about 80% of the XTEN sequence consists of non-overlapping sequence motifs, each of the sequence motifs comprising about 9 to about 14, or about 12 amino acid residues consisting of four to six amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), wherein any two contiguous amino acid residues do not occur more than twice in each of the non-overlapping sequence motifs; or (iii) the XTEN sequence has a subsequence score of less than 10; the XTEN has r than 90%, or greater than 95%, or greater than 99% random coil formation as determined by GOR algorithm; the XTEN has less than 2% alpha helices and 2% beta-sheets as determined by Chou-Fasman thm; the XTEN lacks a predicted T-cell epitope When ed by TEPITOPE algorithm, wherein the PE old score for said prediction by said algorithm has a threshold of —9, and wherein said fusion n exhibits a terminal half-life that is longer than at least about 12 h, or at least about 24 h, or at least about 48 h, or at least about 72 h, or at least about 96 h, or at least about 120 h, or at least about 144 h, or at least about 21 days or greater. In one embodiment, the recombinant factor VIII fusion protein ses at least a second, or at least a third, or at least a fourth XTEN, Which can be identical or different to the other XTEN. According to a different approach, the at least one, at least a second, or at least a third, or at least a fourth XTEN of the CFXTEN fusion protein each have at least about 80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% sequence ty compared to one or more XTEN of comparable length selected from Table 4, Table 13, Table 14, Table , Table 16, and Table 17, When optimally aligned. In yet another ent approach, the at least one, at least a second, or at least a third, or at least a fourth XTEN of the CFXTEN fusion n each have at least 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% ce identity compared to a sequence selected from AE42_1, AE42_2, AE42_3, AG42_1, AG42_2, AG42_3, , AE144_1A, AE144_2A, 2B, AE144_3A, AE144_3B, AE144_4A, AE144_4B, AE144_5A, AE144_6B, AG144_1, AG144_2, A, AG144_B, AG144_C, AG144_F, AG144_3, 4, AE288_1, AE288_2, AG288_1, and AG288_2. {0028} In one embodiment, the factor ‘v’lll component of the CFXTEN recombinant factor Vlll fusion protein eon’iprisies one, two or three amino acid substitutions selected from residues R1648, Yl680, and R1689, numbered relative to mature human factor VIII, wherein the substitutions are selected from alanine, glycine, and phenylalanine. Non-limiting examples ofsaid substitutions include Rl648A, YléSQE, and R1689A. {002,9} ln another embodiment, the CFXTEN fusion protein exhibits an apparent molecular weight lactor of at least about L3, or at least about two, or at least about three, or at least about four, or at least about five, or at least about six, or at least about seven, or at least about eight, or at least about nine, or at least about 10, when ed by size ion chromatography or comparable method.

In some embodiments of the CFXTEN fusion proteins, one or more of the XTEN is to the FVIII Via one or two cleavage sequences that each is cleavable by a mammalian protease selected from the group consisting of factor XIa, factor XIIa, kallikrein, factor VIIa, factor IXa, factor Xa, factor Ila (thrombin), Elastase-2, MMP-12, MMP13, MMP-17 and MMP-20, n cleavage at the cleavage sequence by the mammalian protease releases the factor VIII sequence from the XTEN sequence, and wherein the released factor VIII sequence exhibits an increase in gulant activity compared to the ved fusion protein. In one embodiment, the cleavage ce(s) are cleavable by factor XIa.

According to a different approach, the CFXTEN fusion proteins comprise at least three XTENs located at different locations of the factor VIII polypeptide, wherein said different locations are selected from: an insertion location at or Within 1 to 6 amino acids from a site selected from Table 5, Table 6, Table 7 Table 8, and Table 9; a location at or Within 1 to 6 amino acids of amino acid residue 32, 220, 224, 336, 339, 390, 399, 416, 603, 1656, 1711, 1725, 1905 and 1910 ofmature factor VIII; a location between any two adjacent domains in the factor VIII ce, wherein said two adjacent domains are selected from the group consisting of A1 and A2, A2 and B, B and A3, A3 and C1, and C1 and C2; a on Within an internal B domain deletion starting from a first position at about amino acid residue number 741 to about 750 and ending at a second position at amino acid residue number 1635 to about 1648 With nce to full-length human factor VIII sequence as set forth in and the C-terminus of the factor VIII sequence, wherein the cumulative length of the multiple XTENs is at least about 100 to about 3000 amino acid residues and wherein the fusion protein retains at least about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90% of the procoagulant activity compared to the corresponding factor VIII not linked to XTEN, wherein the procoagulant activity is assayed by an in Vitro coagulation assay. In one embodiment of the foregoing, the fusion protein exhibits a prolonged terminal half-life when administered to a subject as compared to a corresponding factor VIII polypeptide lacking said XTEN, wherein said fusion n exhibits a terminal half-life at least about 3 hours, or 4 hours, or 6 hours, or 12 hours, or 13 hours, or 14 hours, or 16 hours, or 24 hours, or 48 hours, or 72 hours, or 96 hours, or 120 hours, or 144 hours, or 7 days, or 14 days, or 21 days when administered to a subject. In one embodiment, the subject is selected from the group consisting of human and a factor VIII/von rand factor double out mouse. In one embodiment of the foregoing, the fusion protein does not comprise a sequence ed from GTPGSGTASSSP (SEQ ID NO: 31), GSSTPSGATGSP (SEQ ID NO: 32), GSSPSASTGTGP (SEQ ID NO: 33), GASPGTSSTGSP (SEQ ID NO: 34), and GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSG SETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTST EPSEGSAP (SEQ ID NO: 59). In another embodiment of the foregoing, the fusion protein does not n an XTEN sequence consisting of GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSG SETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTST EPSEGSAP (SEQ ID NO: 59), PGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTS STGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTP SS (SEQ ID NO: 71), or PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSG ATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGA SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTG SPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSG TASSSPGSSTPSGATGS (SEQ ID NO: 80).

In a timber aspect, the invention concerns CFXTEN fusion proteins with enhanced cokinetic properties, including enhanced parameters ed to FVIII not linked to XTEN, wherein the enhanced properties include but are not limited to longer terminal half-life, larger area under the curve, increased time in which the blood concentration remains within the therapeutic , increased time between consecutive doses results in blood concentrations within the therapeutic window, and decreased dose in IU over time that can be administered compared to a FVIII not linked to XTEN, yet still result in a blood concentration above a old concentration needed for a gulant effect.

In some embodiments, a CFXTEN fusion proteins exhibit a prolonged terminal half-life when administered to a subject as compared to a corresponding factor VIII polypeptide lacking said XTEN.

The subject can be a human or a mouse, such as a factor VIII/von Willebrand factor double knock-out mouse. In one embodiment of the ing, the CFXTEN exhibits a terminal half-life that is at least about two-fold, or about three fold, or about four-fold, or about five-fold, or about 10-fold, or about 20- fold longer when administered to a subject compared to the corresponding factor VIII not linked to XTEN. In one embodiment, the CFXTEN fusion protein exhibits a terminal half-life at least about 3 hours, or 4 hours, or 6 hours, or 12 hours, or 13 hours, or 14 hours, or 16 hours, or 24 hours, or 48 hours, or 72 hours, or 96 hours, or 120 hours, or 144 hours, or 7 days, or 14 days, or 21 days when administered to the subject. In other embodiments, the enhanced pharmacokinetic property of the fiJsion proteins of the embodiments is the property of maintaining a circulating blood concentration of gulant fusion protein in a subject in need thereof above a threshold concentration of 0.01 IU/ml, or 0.05 IU/ml, or 0.1 IU/ml, or 0.2 IU/ml, or 0.3 IU/ml, or 0.4 IU/ml or 0.5 IU/ml for a period that is at least about two fold, or at least about three-fold, or at least about four-fold, or at least about five-fold, or at least about six-fold, or at least about eight-fold, or at least about ten-fold, or at least about 20-fold, or at least about 40-fold, or at least about 60-fold longer ed to the corresponding FVIII not linked to XTEN and stered to a subject at a comparable dose. The increase in half-life and time spent above the threshold concentration permits less frequent dosing and decreased amounts of the fusion protein (in moles equivalent) that are administered to a subject, compared to the corresponding FVIII not linked to XTEN.

In one embodiment, administration of a subject fusion protein to a subject using a therapeutically- ive dose regimen s in a gain in time of at least two-fold, or at least three-fold, or at least four- fold, or at least five-fold, or at least six-fold, or at least eight-fold, or at least 10-fold, or at least about 20- fold, or at least about 40-fold, or at least about d or higher between at least two consecutive CmaX peaks and/or Cmin troughs for blood levels of the fusion protein compared to the corresponding FVIII not linked to the XTEN and stered using a comparable dose regimen to a subject.

In preferred embodiments, the CFXTEN fusion proteins retain at least about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90% of the procoagulant activity compared to the corresponding factor VIII not linked to XTEN, wherein the procoagulant ty is assayed by an in vitro coagulation assay such as, but not limited to a chromogenic assay or a one- or two-stage clotting assay.

According to a different ch, the invention provides recombinant factor VIII fusion proteins comprising a factor VIII polypeptide and at least one extended recombinant polypeptide (XTEN), wherein said factor VIII polypeptide comprises A1 domain, A2 domain, A3 domain, C1 domain, C2 domain and optionally all or a portion of B domain, and wherein said at least one XTEN is linked to said factor VIII ptide at an insertion site ed form residue s 18-32, or 40, or 4, or 336-403, or 599, or 745-1640, or 1656-1728, or 1796-1804, or 1900-1912, or 2171-2332; and wherein the fusion protein retains at least about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90% of the procoagulant activity compared to the corresponding factor VIII not linked to XTEN. In one embodiment of the foregoing, the fusion protein comprises at least a second XTEN, or at least a third, or at least a fourth XTEN wherein the XTEN are linked to the factor VIII at a site at or within 1 to 6 amino acids of a site ed from Table 5, Table 6, Table 7, Table 8, and Table 9. In another embodiment, the invention provides an recombinant factor VIII fusion protein further comprising at least a second XTEN, or at least a third, or at least a fourth XTEN linked to said FVIII polypeptide at an insertion site selected from Table 5, Table 6, Table 7, Table 8, Table 9, at or within 6 amino acids to the N— or C-terminus side of an ion location at one or more insertion locations from Figure 8 and within one or more insertion ranges from Figure 9 wherein at least two XTEN are separated by an amino acid sequence of at least 100 to about 400 amino acids.

The invention provides CFXTEN wherein the XTEN have a Ratio XTEN Radii of at least 2.3 or at least 2.5, and are separated by an amino acid ce of at least about 20 amino acid residues, or at least about 50, or at least about 100, or at least about 200, or at least about 300, or at least about 400 amino acid residues. In other ments, the CFXTEN comprise at least four XTEN n the XTEN have a Ratio XTEN Radii of at least 2.3, or at least 2.5, or at least 2.8, and wherein at least three of the four of the XTEN linked to the fusion protein are separated by an amino acid sequence of at least about 20 amino acid residues, or at least about 50, or at least about 100, or at least about 200, or at least about 300, or at least about 400 amino acid residues, and the fourth XTEN is linked within the B domain (or a fragment thereof) or within the C domain (or the terminus f).

In some embodiments, the subject compositions are configured to have reduced binding affinity for a clearance receptor in a subject as compared to the corresponding FVIII not linked to the XTEN. In one embodiment, the CFXTEN fusion protein exhibits binding affinity for a clearance receptor of the FVIII in the range of about 0.01%-30%, or about 0.1% to about 20%, or about 1% to about 15%, or about 2% to about 10% of the binding affinity of the corresponding FVIII not linked to the XTEN. In another embodiment, a fusion n with reduced affinity for a clearance receptor has reduced active clearance and a corresponding increase in half-life of at least about 2-fold, or 3-fold, or at least 4-fold, or at least about 5-fold, or at least about 6-fold, or at least about 7-fold, or at least about 8-fold,or at least about 9- fold, or at least about 10-fold, or at least about 12-fold, or at least about 15 -fold, or at least about 17-fold, or at least about 20-fold longer ed to the corresponding FVIII that is not linked to the XTEN.

In an embodiment, the invention provides a recombinant factor VIII fusion protein sing FVIII and one or more XTEN wherein the fusion protein exhibits increased solubility of at least three- fold, or at least about four-fold, or at least about five-fold, or at least about six-fold, or at least about seven-fold, or at least about eight-fold, or at least about nine-fold, or at least about ten-fold, or at least about 15-fold, or at least a 20-fold, or at least 40-fold, or at least 60-fold at physiologic conditions compared to the FVIII not linked to XTEN.

In a further aspect, the invention provides a pharmaceutical composition comprising the fusion protein of any of the embodiments described herein and a pharmaceutically acceptable carrier.

In another embodiment, the invention es a method of treating a coagulopathy in a subject, comprising administering to said subject a composition comprising a clotting effective amount of the pharmaceutical composition. In one embodiment of the , after said administration, a blood concentration of procoagulant factor VIII is maintained at about 0.05, or 1, or 1.5 IU/ml or more for at least 48 hours after said administration. In another embodiment, the invention provides a method of clotting blood in a t, comprising contacting a ng effective amount of the pharmaceutical composition with the blood.

In another ment, the invention es a method of treating a coagulopathy in a t with circulating inhibitors of factor VIII, comprising administering to said subject a composition comprising a therapeutically effective amount of the pharmaceutical composition of CFXTEN, wherein the composition exhibits greater procoagulant activity in said subject compared to a composition comprising the corresponding factor VIII not linked to XTEN and administered using a able amount. In one embodiment of the method, the coagulopathy is hemophilia A. In another embodiment, the coagulopathy is the result of trauma or surgery or infection.

The invention provides a method of treating a bleeding episode in a subject, comprising administering to said subject a composition comprising a clotting ive amount of the CFXTEN pharmaceutical composition, wherein the clotting effective amount of the fusion protein arrests a bleeding episode for a period that is at least three-fold, or at least four-fold, or at least five-fold longer compared to a corresponding factor VIII not linked to XTEN and administered using a comparable amount to said subject. Non-limiting examples of a corresponsing factor VIII not linked to XTEN e native FVIII, the sequences of Table l, BDD-FVIII, and the pCB0114 FVIII.

In another ment, the invention provides a CFXTEN inant factor VIII fusion n for use in a pharmaceutical regimen for treating a ilia A patient, said regimen comprising a pharmaceutical ition comprising a CFXTEN fusion protein. In one embodiment of the pharmaceutical regimen, the regimen r ses the step of determining the amount of ceutical composition comprising the CFXTEN needed to achieve hemostasis in the hemophilia A t. In another embodiment, the pharmaceutical regimen for treating a hemophilia A t comprises administering the pharmaceutical composition in two or more successive doses to the subject at an effective amount, wherein the administration results in at least a 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 90% greater improvement of at least one, two, or three parameters associated with the hemophilia A disease compared to the factor VIII not linked to XTEN and administered using a comparable dose. Non-limited examples of parameters improved include blood concentration of procoagulant FVIII, a reduced activated partial prothrombin (aPTT) assay time, a reduced age or two-stage clotting assay time, delayed onset of a bleeding episode, a reduced chromogenic assay time, a reduced bleeding assay time, resolution of a bleeding event, or a reduced Bethesda titer to native FVIII.

In another aspect, the invention provides isolated nucleic acid sequences encoding the fusion proteins of any one of the embodiments of the CFXTEN fusion protein. In one embodiment, the isolated nucleic acid is the complement of a sequence encoding a CFXTEN fusion protein of the embodiments.

In one embodiment, the ed nucleic acid further comprises a sequence encoding a signal peptide, n said sequence is ATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGT (SEQ ID NO: 1613), or the complement thereof In another embodiment, the invention provides an expression vector comprising the nucleic acid encoding the fusion protein, or the complement f. In another embodiment, the ion provides an isolated host cell comprising the foregoing expression vector. In another embodiment, the invention provides a method of producing the fusion protein of any of the embodiments, comprising providing a host cell comprising the expression vector; ing the host cell to effect production of the fusion protein; and recovering the fusion protein.

In one ment, the invention provides an isolated fusion protein comprising a polypeptide having at least about 80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% ce identity ed to a sequence of comparable length selected from Table 21, when optimally aligned.

In another embodiment, the invention provides an ed nucleic acid comprising a polynucleotide sequence selected from (a) a sequence having at least about 80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% sequence identity compared to a sequence of comparable length selected from Table 21, when optimally aligned, or (b) the complement of the polynucleotide of (a). In another ment, the isolated nucleic acid comprises the sequence ATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGT (SEQ ID NO: 1613) linked to the 5’ end of the nucleic acid of (a) or the complement of the sequence linked to the 3’ end of (b).

It is specifically contemplated that the recombinant factor VIII fusion proteins can exhibit one or more or any combination of the properties disclosed herein.

INCORPORATION BY REFERENCE All publications, patents, and patent applications ned in this cation are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS The features and advantages of the invention may be further explained by reference to the following detailed description and accompanying drawings that sets forth illustrative embodiments. shows a schematic entation of the FVIII architecture and spatial arrangement of the domains during processing and clotting, and is intended to ent both native FVIII and B domain deleted variants. The A1 domain ranges from residue 1 to 372 (numbering relative to the mature form of FVIII sequence NCBI Protein RefS eq NP_000123 and encompassing a1 residues), A2 domain ranges from residue 373 to 740, B domain ranges from residue 741 to 1648, A3 domain ranges from residue 1649 to 2019 (encompassing a3 acidic region), C1 domain ranges from 2020 to 2172, and the C2 domain ranges from residue 2173 to 2332. BDD variants include deletions between the range 741 to 1648, leaVing some or no t es, with a non-limiting BDD remnant sequence being SFSQNPPVLKRHQR (SEQ ID NO: 1614). shows the domain architecture of a single chain FVIII prior to processing. Arrows te the sites at es R372, R740, R1648, and R1689 that are cleaved in the processing and conversion of FVIII to FVIIIa. shows the FVIII molecule that has been processed into the heterodimer by the cleavage at the R1648 residue, with the a3 acidic region of the A3 domain indicated on the N—terminus of the A3. shows the FVIII molecule processed into the FVIIIa heterotrimer by the cleavage at the R372, R740, and R1689 residues. is a schematic of the ation cascade, showing the intrinsic and extrinsic arms leading to the common pathway. depicts the amino acid ce of mature human factor VIII (SEQ ID NO: 1592). depicts a factor VIII sequence with a deletion of a portion of the B domain (SEQ ID NO: 1593). illustrates several examples of CFXTEN configurations of FVIII linked to XTEN (the latter shown as thick, wavy . In all cases, the FVIII can be either native or a BDD form of FVIII, or a single chain form in which the entire B domain, including the native cleavage sites are removed. shows, left to right, three variations of single chain factor VIII with XTEN linked to the N—terminus, the C-terminus, and two XTEN linked to the N— and C-terminus. shows six variations of mature heterodimer FVIII with, left to right, an XTEN linked to the N—terminus of the A1 domain; an XTEN linked to the C-terminus of the C2 domain; an XTEN linked to the N—terminus of the A1 domain and the inus of the C2 domain; an XTEN linked to the N-terminus of the A1 domain and to the N- terminus of the A3 ; an XTEN linked to the C-terminus of the C2 domain and to the N-terminus of the A3 domain via residual B domain amino acids; and an XTEN linked to the N-terminus of the A1 domain, the C-terminus of the A2 domain via residual B domain amino acids, and to the C-terminus of the C2 domain. shows, left to right, three variations of single chain factor VIII: an XTEN linked to the N—terminus of the A1 domain, an XTEN linked within a surface loop of the A1 domain and an XTEN linked within a e loop of the A3 domain; an XTEN linked within a surface loop of the A2 domain, an XTEN linked within a surface loop of the C2 domain and an XTEN linked to the C terminus of the C2 domain; an XTEN linked to the N—terminus of the A1 domain and within a surface loop of the C1 domain and to the C-terminus of the C domain. shows six variations of mature heterodimer FVIII with, left to right, an XTEN linked to the N-terminus of the A1 domain, an XTEN linked within a surface loop of the A1 domain, and an XTEN linked within a surface loop of the A3 domain; an XTEN linked within a surface loop of the A2 domain, and an XTEN linked within a surface loop of the C1 domain, and an XTEN linked to the C-terminus of the C2 domain; an XTEN linked to the N—terminus of the A1 , an XTEN linked within a e loop of the A1 domain, an XTEN linked within a e loop of the A3 domain, and an XTEN linked to the C-terminus of the C2 domain; an XTEN linked to the N—terminus of the A1 domain, an XTEN linked to the N—terminus of the A3 domain via residual amino acids of the B domain, and an XTEN linked within a surface loop of the C2 domain; an XTEN linked within a surface loop of the A2 domain, an XTEN linked to the N—terminus of the A3 domain via residual amino acids of the B , an XTEN linked within a surface loop of the C1 , and an XTEN linked to the C-terminus of the C2 ; and an XTEN linked within the B domain or between the residual B domain residues of the BDD variant (and the invention also contemplates a variation in which the XTEN replaces the entirety of the B domain, including all native cleavage sites, linking the A2 and A3 domains, resulting in a single chain form of factor VIII). This figure also embodies all variations in which one or more XTEN sequences are inserted within the B domain and the resulting fusions are cleaved at one or more sites (e.g., at R1648 site) during intracellular processing. is a graphic portrayal of a CFXTEN construct with an XTEN inserted within the B domain and linked to the inus of the C2 domain illustrating the unstructured characteristic of the XTEN g to random coil formation that can cover portions of the factor VIII proximal to the XTEN.

In the lower panel, the drawing depicts that when XTEN is in random coil, it can adopt a conformation resulting in steric hindrance that blocks binding of factor VIII inhibitor antibodies that would otherwise have affinity for epitopes al to the XTEN site of insertion. is a graphic portrayal of the various analyses performed on a FVIII B-domain d sequence to fy insertion sites for XTEN within the FVIII sequence. Each of lines A—H are on an arbitrary scale of Y axis values across the FVIII BDD ce such that low values represent areas with a high predicted tolerance for XTEN insertion, with the e numbers on the X axis. Line A shows the domain boundaries; all discontinuities in this line ent boundaries that are likely to accept XTEN.

Line B shows exon boundaries; i.e., each step in the line represents a new exon. Line C shown regions that were not visible in the X-ray structure due to a lack of order in the l. Lines labeled D represents multiple predictions of order that were calculated using the respective programs FoldIndex found on the Wide web site bip.weizmann.ac.il/fidbin/findex (last accessed February 23, 2011) (see Jaime Prilusky, Clifford E. Felder, Tzviya ZeeV-Ben-Mordehai, Edwin g, Oma Man, Jacques S.

Beckmann, Israel , and Joel L. Sussman, 2005, Bioinformatics based on the Kyte & Doolitlle algorithm, as well as RONN found on the World-Wide web site strubi.ox.ac.uk/RONN (last accessed February 23, 2011) (see Yang,Z.R., Thomson, R., McMeil, P. and Esnouf, R.M. (2005) RONN: the bio- basis function neural network technique applied to the detection of natively disordered regions in proteins Bioinformatics 21: 3369-3376. Lines E and F were calculated based on multiple sequence alignments of FVIII genes from 11 mammals available in GenBank. Line E represents the conservation of individual residues. Line F represent the conservation of 3 amino acid segments of FVIII. Lines G and H represent gaps and insertions observed in the multiple sequence alignment of 11 mammalian FVIII genes. Line J lists the XTEN insertion points by amino acid number that were obtained based by combining the multiple measurements above. depicts the sites in a FVIII B-domain deleted sequence (SEQ ID NO: 1594) identified as active insertion points for XTEN using the information ed in and as ed in the assays of Example 34. depicts the range of sites in a FVIII B-domain deleted sequence (SEQ ID NO: 1595) identified for insertion ofXTEN using the information depicted in and or Example 34 plus a span of amino acids around each ion point that are considered suitable for insertion of XTEN. is a schematic of the ly of a CFXTEN library created by identifying insertion points as described for FIGS. 7 ed by insertion of single XTEN (black bars) at the various insertion points using molecular biology techniques. The constructs are expressed and recovered, then evaluated for FVIII ty and pharmacokinetic ties to identify those CFXTEN configurations that result in enhanced properties. is a schematic of the assembly of a CFXTEN ent y in which segments of FVIII BDD domains, either singly or linked to various lengths ofXTEN (black bars) are assembled in a combinatorial fashion into libraries of genes encoding the CFXTEN, which can then be evaluated for FVIII actiVity and pharmacokinetic properties to identify those CFXTEN configurations that result in enhanced properties. illustrates several examples of CFXTEN configurations with XTEN (shown as thick, wavy lines), with certain XTEN releasable by inserting cleavage sequences (indicated by black triangles) that are cleavable by gulant proteases. A illustrates a scFVIII with two terminal releasable XTENS. B illustrates the same uration as A but with an additional non-releasable XTEN linking the A3 and C1 domains. C illustrates a mature heterodimer FVIII with two terminal releasable XTEN. D illustrates the same configuration as 10C but with an additional leasable XTEN linking the A3 and C1 domains. is a schematic flowchart of representative steps in the ly, production and the evaluation of an XTEN. is a schematic flowchart of representative steps in the assembly of a CFXTEN polynucleotide construct encoding a fusion protein. Individual oligonucleotides 501 are annealed into sequence motifs 502 such as a 12 amino acid motif (“12-mer”), which is ligated to additional sequence motifs from a library to create a pool that encompasses the desired length of the XTEN 504, as well as d to a smaller concentration of an oligo ning BbsI, and KpnI restriction sites 503. The resulting pool of ligation products is rified and the band with the desired length ofXTEN is cut, resulting in an isolated XTEN gene with a stopper sequence 505. The XTEN gene is cloned into a stuffer vector. In this case, the vector encodes an optional CBD sequence 506 and a GFP gene 508. Digestion is then performed with BbsI/HindIII to remove 507 and 508 and place the stop codon. The resulting product is then cloned into a BsaI/HindIII digested vector containing a gene encoding the FVIII, resulting in the gene 500 encoding an FVIII-XTEN fusion n. is a schematic flowchart of representative steps in the assembly of a gene encoding fusion protein comprising a CF and XTEN, its expression and recovery as a fusion protein, and its evaluation as a candidate CFXTEN product. illustrates the use of donor XTEN sequences to produce truncated XTENs. A provides the sequence of AG864 (SEQ ID NO: 1596), with the underlined sequence used to generate a ce length of 576 (SEQ ID NO: 1597). B provides the sequence of AG864 (SEQ ID NO: 1598), with the underlined sequence used to te a sequence length of 288 (SEQ ID NO: 1599).

C provides the ce of AG864 (SEQ ID NO: 1600), with the underlined sequence used to generate a sequence length of 144 (SEQ ID NO: 1601). D provides the sequence of AE864 (SEQ ID NO: 1602), with the underlined sequence used to generate a sequence length of 576 (SEQ ID NO: 1603). E provides the sequence of AE864 (SEQ ID NO: 1604), with the underlined sequence used to generate a sequence length of 288 (SEQ ID NO: 1605). F provides the ce of AE864 (SEQ ID NO: 1606) used to generate four ces of 144 length (SEQ ID NOS 1607-1610, respectively, in order of appearance) (the double underline indicates the first amino acid in the 144 ce with the single underline representing the balance of that sequence). is a schematic representation of the design of Factor VIII-XTEN expression vectors with different strategies introducing XTEN elements into the FVIII coding sequence. A shows an expression vector encoding XTEN fused to the 3’ end of the ce encoding FVIII. B s an expression vector ng an XTEN element inserted into the middle of the coding sequence encoding a single FVIII. C depicts an expression vector encoding two XTEN elements: one inserted internal to the FVIII coding sequence, and the other fused to the 3’ end of the FVIII coding sequence. illustrates the process of combinatorial gene assembly of genes ng XTEN. In this case, the genes are assembled from 6 base nts and each fragment is available in 4 different codon versions (A, B, C and D). This allows for a tical diversity of 4096 in the assembly of a 12 amino acid motif. shows the pharmacokinetic profile (plasma concentrations) in cynomolgus monkeys after single doses of different compositions of GFP linked to unstructured polypeptides of varying length, stered either subcutaneously or intravenously, as described in Example 41. The compositions were GFP-L288, GFP-L576, GFP-XTEN_AF576, 76 and XTEN_AD836-GFP. Blood samples were analyzed at various times after injection and the concentration of GFP in plasma was measured by ELISA using a polyclonal antibody against GFP for capture and a biotinylated preparation of the same polyclonal antibody for detection. Results are presented as the plasma concentration versus time (h) after dosing and show, in particular, a considerable increase in half-life for the XTEN_AD836-GFP, the composition with the longest sequence length of XTEN. The construct with the shortest ce length, the GFP-L288 had the shortest half-life. shows an GE gel of samples from a stability study of the fusion protein of XTEN_AE864 fused to the N—terminus of GFP (see Example 42). The GFP-XTEN was incubated in cynomolgus plasma and rat kidney lysate for up to 7 days at 37°C. In addition, GFP-XTEN administered to cynomolgus monkeys was also ed. Samples were withdrawn at O, 1 and 7 days and analyzed by SDS PAGE followed by ion using Western analysis with dies against GFP. shows results of a size exclusion chromatography analysis of glucagon-XTEN construct samples measured t protein standards of known molecular weight, with the graph output as absorbance versus retention volume, as described in Example 40. The glucagon-XTEN constructs are 1) glucagon-Y288; 2) glucagonY-144; 3) glucagon-Y72; and 4) glucagon-Y36. The results indicate an increase in apparent molecular weight with sing length ofXTEN moiety (see Example 40 for data). shows results of a Western blot of proteins expressed by cell culture of cells transformed with constructs as designated (Example 25). The samples in lanes 1-12 were: MW Standards, FVIII (42.5 ng), pBCOIOOB, pBC0114A, pBC0100, pBC0114, pBC0135, pBC0136, pBC0137, pBC0145, pBC0149, and pBC0146, respectively. Lanes 8, 9 and 12 show bands consistent with a FVIII with a C-terminal XTEN288, with an estimated MW of 95 kDa. Lanes 7 and 11 show bands consistent with a FVIII with a C-terminal , with an estimated MW of 175 kDa. Lanes 2-6 show bands consistent with FVIII and heavy chain. Lanes 10 and 23 show bands consistent with heavy chain. Lane 7 shows a band consistent with heavy chain and an attached XTEN42. shows the results of FVIII assay on samples ed from FVIII and von Willebrand factor double knock-out mice with hydrodynamic plasmid DNA injection, as detailed in Example 36. is a graphic and tabular portrayal of the pharmacokinetic properties of rBDD-FVIII and the d CFXTEN fusion proteins pBC0145 and pBC0146 (with C-terminal XTEN) administered to either HemA or FVIII/VWF double knock-out mice as described in Example 30, showing the enhanced half-life of the CFXTEN in both strains of mice. is a c and r portrayal of the pharmacokinetic properties of rBDD-FVIII and the CFXTEN fusion proteins pSD0050 and pSDOO62 (with internal inserted XTEN) administered to either HemA (A) or FVIII/VWF double knock-out mice (B) using a cell culture PK assay in HemA mice. Dose, 5-minute ry, and half-life (Tl/2) are shown, as bed in Example 32, underscoring the enhanced recovery and half-life of the CFXTEN compared to the ve control FVIII in both strains of mice. is a graphic depiction of a titration of GMA8021 FVIII inhibitor using the pBC0114 BDD-FVIII AND CFXTEN construct LSDOO49.002 with three 144 amino acid XTEN insertions at residues 18, 745 and 2332. The data indicate a shift of imately 0.7 order of magnitude in the amount of antibody in [Lg/ml required to inhibit the CFXTEN to the 50% level, compared to FVIII positive control. is a schematic of the logic flow chart of the algorithm SegScore. In the figure the following legend applies: i, j - counters used in the control loops that run through the entire sequence; HitCount- this variable is a counter that keeps track of how many times a subsequence encounters an identical subsequence in a block; SubSeqX - this variable holds the subsequence that is being checked for redundancy; SubSeqY - this variable holds the subsequence that the SubSeqX is d against; BlockLen - this variable holds the user determined length of the block; SegLen - this le holds the length of a segment. The program is hardcoded to generate scores for subsequences of s 3, 4, 5, 6, 7, 8, 9, and 10; Block - this variable holds a string of length BlockLen. The string is composed of letters from an input XTEN sequence and is determined by the position of the i counter; SubSeqList - this is a list that holds all of the generated subsequence scores. depicts the application of the algorithm SegScore to a hypothetical XTEN of 11 amino acids (SEQ ID NO: 1591) in order to ine the repetitiveness. An XTEN sequence consisting ofN amino acids is divided into N—S+1 subsequences of length S (S=3 in this case). A pair-wise ison of all subsequences is performed and the average number of identical subsequences is calculated to result in the subsequence score of 1.89. is a graph of the individual construct values of the ratio of FVIII activity in the assayed CFXTEN to that of the pBC114 FVIII positive control after exposure to the GMA8021 antibody to FVIII, d according to the number ofXTEN in the construct fusion protein (see Example 28). The results show an essentially linear relationship in the ability of the CFXTEN to retain FVIII actiVity with increasing number of incorporated XTEN. depicts the primary ce and domain structure of mature B-domain deleted (BDD) human FVIII construct (Example 46). The location of the introduced NheI and Clal restriction sites is shown. Note that the amino acid numbering corresponds to the amino acid positions in the y sequence of mature FVIII (). Individual s are bounded by gray lines/boxes with domain identification in gray text. Acidic regions (a1, a2, a3) are indicated with dashed boxes. Solid wedges/triangles indicate sites of thrombin cleavage in the activation of FVIII to FVIIIa. Unfilled /triangle indicates the site of intracellular proteolytic processing to the two-chained form of FVIII. ns indicate sites of ed glycosylation. Circles indicate sites of Tyr sulfation. Unique non- native restriction sites (Nhel, GCTAG; Clal, ATCGAT) introduced into cDNA to facilitate XTEN insertion/recombination are highlighted in gray with double underline. provides graphical representation of the FVIII construct bed in , indicating the domain organization and the location of native and non-native restriction sites. shows the graphical w outputs for structural datasets 2R7E, 3CDZ, and PMOO76106. Accessible Solvent Areas (ASA) for the amino acids in domains A1, A2, A3, C1 and C2 are shown. Analyses were performed on X-ray crystallographic coordinates 3CDZ (Ngo et al., Structure 16: 597-606 (2008)) and 2R7E (Shen et al., Blood 111:1240-1247 (2008)) deposited in the Protein Data Bank maintained by the Research Collaboratory for Structural Bioinformatics (RCSB; http://www.rcsb.org/pdb), as well as on atomic coordinates PMOO76106 for the predicted refined FVIII structure derived from a molecular dynamics simulation study (Venkateswarlu, BMC Struct. Biol. 10:7 (2010)) deposited in the n Model Database //mi.caspur.it/PMDB/main.php) maintained by Consorzio Interuniversitario per le Applicazioni di Supercalcolo per Universita e Riserca (CASPUR) and the Department of Biochemical Sciences of the University of Rome. shows a structural representation of the location ofXTEN insertion sites. The central drawing corresponding to the l structure of FVIII (PDB: 2R7E) is surrounded by detailed View of domains A1, A2, A3, C1 and C2. Beta strands and alpha helices are shown as ribbon entation.

Loops are shown as alpha carbon pipes. The amino acids at XTEN insertion sites are shown as CPK sphere representation. The number in each graph indicate the location of the XTEN insertion sites according to the numbering in . shows a ural representation of the location ofXTEN insertion sites shown in wherein the resulting inant FVIII protein ys FVIII actiVity. shows a structural representation of the location ofXTEN ion sites shown in wherein the resulting recombinant FVIII protein displays FVIII actiVity. shows a structural representation of the location ofXTEN insertion sites shown in wherein the resulting recombinant FVIII protein displays FVIII activity. shows a ClustalW multiple sequence alignment of domains A], A2, A3, C1 and C2 of FVIII showing the location ofXTEN insertions resulting in recombinant FVIII proteins ying FVIII actiVity (black box, white text) or displaying no FVIII actiVity (grey box, bold text). shows a DSSP graphical representation of the secondary structure of the two polypeptide chains in a native active human FVIII crystal structure deposited under the identifier 2R7E at the Protein Data Bank (see e 47). Amino acid sequence numbering is the same as in the protein sequence in . The beta sheet regions are shown as filled arrows and are designated B1 to B66.

The location of the XTEN permissive loops is d by crosshatched boxes. Domain Al XTEN permissive loops are ated Loop Al-l and Loop Al -2. Domain A2 XTEN permissive loops are designated Loop A2-l and Loop A2-2. Domain A3 XTEN sive loops are designated Loop A3-l and Loop A3-2. shows a DSSP graphical representation of the secondary structure of the two polypeptide chains in a native active human FVIII crystal structure deposited under the identifier 2R7E at the Protein Data Bank (see Example 47). Amino acid sequence numbering is the same as in the n sequence in . The beta sheet regions are shown as filled arrows and are designated B1 to B66.

The location of the XTEN permissive loops is denoted by atched boxes. Domain Al XTEN permissive loops are designated Loop Al-l and Loop Al -2. Domain A2 XTEN permissive loops are designated Loop A2-l and Loop A2-2. Domain A3 XTEN permissive loops are designated Loop A3-l and Loop A3-2. shows a ClustalW multiple sequence alignment of domains A], A2, A3, C1 and C2 of FVIII showing the location ofXTEN ions resulting in recombinant FVIII proteins displaying FVIII actiVity (black box, white text) or displaying no FVIII actiVity (grey box, bold text). The locations of the XTEN permissive loops are indicated by dashed rectangles (see Example 47).

. A presents a front View structural representation of human FVIII (PDB:2R7E) g the location of domains Al, A2, A3, C1 and C2 (circled in dashed lined) and the locations of XTEN permissive loops Al -1, Al -2, A2-l, A2-2, A3-l and A3-2 highlighted as CPK sphere representations. B presents a side View structural representation of human FVIII R7E) showing the location of domains Al, A2, A3, C1 and C2 (circled in dashed lined) and the locations of XTEN sive loops Al -1, Al -2, A2-l, A2-2, A3-l and A3-2 highlighted as CPK sphere representations. shows the top View structural representations of isolated human FVIII (PDB:2R7E) A domains g the location ofXTEN permissive loops highlighted as CPK sphere entations.

B, 42D and 42F show side View structural entations of isolated human FVIII (PDB:2R7E) A domains showing the location ofXTEN permissive loops highlighted as CPK sphere representations. shows sequences of various factor VIII B-domain deletions and individual mutations.

Lines 4-10 show s B-domain deletions with indicated XTEN linking the flanking B-domain residual or A3 domain residues. The R1648A mutation is indicated by arrow in line 5 and 8, while the Y] 68OF on is indicated by arrow in lines 8-10. is a bar graph of chromogenic and aPTT assay activity of various CFXTEN with single XTEN insertions (Example 49). is a bar graph of chromogenic and aPTT assay actiVity of various CFXTEN with 2 XTEN insertions (Example 49).

FIG 46 is a bar graph of chromogenic and aPTT assay actiVity of various CFXTEN with 3 XTEN insertions (Example 49). is a graph of plasma levels in DKO mice of various administered CFXTEN with single XTEN insertions compared to a BDD-FVIII control, demonstrating the 10- to d longer half-life achieved by the XTEN insertions at various locations (Example 50). is a graph of plasma levels in DKO mice of various administered CFXTEN with one, two, and three XTEN insertions compared to a BDD-FVIII control, demonstrating the increases in half- life achieved by the inclusion of additional XTEN insertions ed to single or two insertions (Example 51). are graphs of the plotted inhbition curves for remaining factor VIII procoagulant actiVity in samples assayed in the Bethesda assay with three hemophilia patient sera (FIGS. 49A—C) or sheep anti-FVII (D) described in Example 52, demonstrating a clear left-shift of the tion curve for the two CFXTEN molecules compared to the FVIII not linked to XTEN.

DETAILED DESCRIPTION OF THE INVENTION Before the ments of the invention are described, it is to be understood that such ments are provided by way of example only, and that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. Numerous ions, changes, and substitutions will now occur to those d in the art without departing from the invention.

Unless otherwise defined, all technical and scientific terms used herein have the same g as commonly understood by one of ry skill in the art to which this invention belongs. gh s and materials similar or equivalent to those described herein can be used in the practice or testing of the present ion, suitable methods and materials are described below. In case of conflict, the patent specification, ing definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention.

DEFINITIONS In the context of the present application, the following terms have the meanings ascribed to them unless specified otherwise: As used in the specification and claims, the singular forms cc :9 66 a an” and “the” include plural references unless the context clearly dictates ise. For e, the term “a cell” includes a plurality of cells, including mixtures thereof.

The terms “polypeptide”, de”, and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been d, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation With a labeling component.

As used herein, the term “amino acid” refers to either natural and/or unnatural or synthetic amino acids, including but not limited to both the D or L optical isomers, and amino acid analogs and peptidomimetics. Standard single or three letter codes are used to ate amino acids.

The term n," When used in reference to a factor VIII polypeptide refers to either a full length domain or a functional fragment thereof, for example, full length or functional fragments of the A1 domain, A2 domain, A3 domain, B domain, C1 domain, and/or C2 domain of factor VIII.

The term “natural L-amino acid” means the L optical isomer forms of glycine (G), proline (P), alanine (A), valine (V), leucine (L), isoleucine (I), nine (M), cysteine (C), phenylalanine (F), tyrosine (Y), tryptophan (W), ine (H), lysine (K), arginine (R), glutamine (Q), asparagine (N), glutamic acid (E), aspartic acid (D), serine (S), and threonine (T).

The term aturally occurring,” as applied to sequences and as used herein, means polypeptide or polynucleotide sequences that do not have a counterpart to, are not complementary to, or do not have a high degree of homology With a Wild-type or lly-occurring sequence found in a mammal. For example, a non-naturally occurring polypeptide or fragment may share no more than 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50% or even less amino acid sequence identity as compared to a natural sequence When suitably aligned.

] The terms “hydrophilic” and “hydrophobic” refer to the degree of y that a substance has With water. A hydrophilic substance has a strong affinity for water, tending to dissolve in, mix With, or be wetted by water, While a hydrophobic substance substantially lacks affinity for water, tending to repel and not absorb water and g not to dissolve in or mix With or be wetted by water. Amino acids can be characterized based on their hydrophobicity. A number of scales have been developed. An example is a scale developed by Levitt, M, et al., J Mol Biol (1976) 104:59, which is listed in Hopp, TP, et al., Proc Natl Acad Sci U S A (1981) 78:3 824. Examples of “hydrophilic amino acids” are arginine, lysine, threonine, alanine, asparagine, and glutamine. Of particular interest are the hydrophilic amino acids aspartate, glutamate, and serine, and glycine. Examples of “hydrophobic amino acids” are tryptophan, tyrosine, phenylalanine, methionine, leucine, isoleucine, and valine.

A “fragment” When applied to a protein, is a truncated form of a native biologically active n that s at least a n of the therapeutic and/or biological activity. A “variant”. When applied to a protein is a protein With sequence homology to the native biologically active protein that retains at least a portion of the therapeutic and/or biological activity of the biologically active protein. For example, a variant protein may share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity compared with the reference biologically active protein. As used herein, the term “biologically active protein moiety” includes proteins modified deliberately, as for example, by site directed nesis, synthesis of the encoding gene, insertions, or accidentally through mutations.

The term nce variant” means polypeptides that have been modified compared to their native or original sequence by one or more amino acid insertions, deletions, or substitutions. Insertions may be d at either or botlt termini of the protein, and/or may be positioned within internal regions of the amino acid sequence. A non—limiting example is insertion of an XTEN ce within the sequence ot‘the ically—active payload protein. In deletion variants, one or more amino acid residues in a polypeptide as described herein are d. on variants, therefore, include all fragments of a payload polypeptide sequence. in substitution variants, one or more amino acid residues of a polypeptide are removed and replaced with alternative residues. in one aspect, the substitutions are conservative in nature and conservative substitutions of this type are well known in the art.

As used herein, “internal XTEN” refers to XTEN ces that have been inserted into the sequence of the ation factor. Internal XTENs can be ucted by insertion of an XTEN sequence into the sequence of a coagulation factor such as FVIII, either by insertion between two adjacent amino acids within a domain (“intradomain”) or between two domains (“interdomain”) of the coagulation factor or wherein XTEN replaces a partial, internal sequence of the ation factor.

As used , “terminal XTE ” refers to XTEN sequences that have been fused to or in the N— or C-terminus of the coagulation factor or to a proteolytic cleavage sequence or linker at the N— or C- terminus of the coagulation factor. Terminal XTENs can be fused to the native termini of the coagulation factor. Alternatively, terminal XTENs can replace a portion of a terminal ce of the coagulation factor.

The term “XTEN release site” refers to a ge sequence in CFXTEN fusion proteins that can be recognized and cleaved by a mammalian protease, effecting release of an XTEN or a portion of an XTEN from the CFXTEN fusion protein. As used , “mammalian protease” means a protease that normally exists in the body fluids, cells or tissues of a mammal. XTEN release sites can be engineered to be d by various mammalian proteases . “XTEN release proteases”) such as FXIa, FXIIa, kallikrein, FVIIIa, , FXa, FIIa (thrombin), Elastase-2, MMP-12, MMP13, MMP-l7, MMP-20, or any protease that is present during a clotting event. Other equivalent proteases (endogenous or exogenous) that are capable of izing a defined cleavage site can be utilized. The ge sites can be adjusted and tailored to the protease utilized.

The term “within”, when referring to a first polypeptide being linked to a second polypeptide, encompasses linking that connects the N—terminus of the first or second polypeptide to the C-terminus of the second or first polypeptide, respectively, as well as insertion of the first polypeptide into the sequence of the second polypeptide. For example, when an XTEN is linked “within” a domain of a factor VIII ptide, the XTEN may be linked to the N—terminus, the C-terminus, or may be inserted in said domain.

As used herein, the term “site,” when used to refer to an ion site of an XTEN within or to a biological polypeptide such as a factor VIII, ents the amino acid position at which the XTEN is linked. When numbered sites are described, such as a first, second, third, fourth, fifth, or sixth site for the insertion of an XTEN within or to the factor VIII, each site will be understood to ent a distinct site in the factor VIII; e.g., the second site is a different factor VIII location from the first site, the third site is different from the second and the first, etc.

“Activity” or “procoagulant activity” as applied to form(s) of a CFXTEN polypeptide provided herein, refers to the ability to bind to a target coagulation n substrate or cofactor and promote a clotting event, whether measured by an in vitro, ex vivo or in vivo assay. Such assays include, but are not limited to, age clotting assays, two-stage clotting assays, genic assays, and ELISA assays.

“Biological actiVity” refers to an in vitro or in vivo biological function or effect, including but not limited to either receptor or ligand binding, or an effect on coagulation generally known in the art for the FVIII ation factor, or a cellular, physiologic, or clinical response, including arrest of a bleeding episode.

As used herein, the term "ELISA" refers to an enzyme-linked immunosorbent assay as described herein or as ise known in the art.

A “host cell” includes an individual cell or cell culture which can be or has been a recipient for the subject vectors. Host cells include progeny of a single host cell. The progeny may not necessarily be completely identical (in morphology or in genomic of total DNA complement) to the al parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in Vivo with a vector of this invention. ted” when used to describe the various polypeptides disclosed herein, means ptide that has been identified and separated and/or recovered from a component of its natural environment.

Contaminant components of its natural environment are materials that would typically interfere with stic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, does not require tion” to distinguish it from its naturally occurring counterpart. In on, a “concentrated”, “separated” or “diluted” polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is generally greater than that of its naturally occurring counterpart. In general, a polypeptide made by recombinant means and expressed in a host cell is ered to be “isolated.” ] An “isolated” polynucleotide or polypeptide-encoding nucleic acid or other polypeptide- encoding nucleic acid is a nucleic acid le that is identified and separated from at least one inant nucleic acid molecule with which it is ordinarily associated in the natural source of the polypeptide-encoding nucleic acid. An isolated polypeptide-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated polypeptide-encoding nucleic acid molecules therefore are distinguished from the specific polypeptide-encoding nucleic acid molecule as it exists in natural cells. However, an isolated polypeptide-encoding nucleic acid le includes polypeptide-encoding nucleic acid molecules contained in cells that ordinarily express the polypeptide where, for example, the nucleic acid le is in a chromosomal or extra-chromosomal location different from that of natural cells.

A “chimeric” protein contains at least one fusion polypeptide comprising at least one region in a different position in the sequence than that which occurs in nature. The regions may normally exist in te proteins and are brought together in the fusion polypeptide; or they may normally exist in the same n but are placed in a new arrangement in the fusion ptide. A chimeric protein may be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship. gated”, “linked,” “fused,” and “fusion” are used interchangeably herein. These terms refer to the joining together of two or more chemical elements, sequences or components, by whatever means ing chemical conjugation or recombinant means. For example, a promoter or enhancer is operably linked to a coding ce if it affects the transcription of the sequence. Generally, “operably linked” means that the DNA sequences being linked are contiguous, and in reading phase or in-frame.

An “in-frame fusion” refers to the joining of two or more open g frames (ORFs) to form a uous longer ORF, in a manner that maintains the correct reading frame of the original ORFs.

Thus, the resulting recombinant fusion n is a single protein containing two or more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature).

] In the context of polypeptides, a “linear sequence” or a “sequence” is an order of amino acids in a polypeptide in an amino to carboxyl terminus direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide. A “partial sequence” is a linear sequence of part of a polypeptide that is known to se additional residues in one or both directions.

“Heterologous” means derived from a genotypically distinct entity from the rest of the entity to which it is being compared. For example, a e rich sequence removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous e rich sequence. The term “heterologous” as applied to a polynucleotide, a polypeptide, means that the polynucleotide or polypeptide is derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared.

The terms “polynucleotides”, “nucleic acids”, “nucleotides” and nucleotides” are used interchangeably. They refer to a polymeric form of tides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof Polynucleotides may have any three- dimensional structure, and may perform any function, known or unknown. The following are non- limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, mal RNA, mes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and tide analogs. If present, modifications to the nucleotide structure may be ed before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

The term “complement of a polynucleotide” denotes a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference ce, such that it could hybridize with a reference sequence with complete fidelity.

“Recombinant” as applied to a polynucleotide means that the polynucleotide is the product of various combinations of in vitro cloning, restriction and/or ligation steps, and other procedures that result in a construct that can potentially be expressed as a recombinant protein in a host cell.

The terms “gene” and “gene fragment” are used hangeably herein. They refer to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated. A gene or gene fragment may be genomic or cDNA, as long as the polynucleotide ns at least one open reading frame, which may cover the entire coding region or a segment thereof A n gene” is a gene composed of at least two heterologous polynucleotides that are linked together.

] “Homology” or “homologous” or “sequence identity” refers to sequence similarity or interchangeability between two or more cleotide sequences or between two or more polypeptide sequences. When using a program such as BestFit to determine sequence identity, similarity or homology between two different amino acid sequences, the default settings may be used, or an appropriate scoring matrix, such as blosum45 or 80, may be selected to optimize identity, similarity or gy scores. ably, polynucleotides that are gous are those which hybridize under stringent conditions as defined herein and have at least 70%, preferably at least 80%, more preferably at least 90%, more ably 95%, more preferably 97%, more preferably 98%, and even more preferably 99% sequence identity compared to those sequences. Polypeptides that are homologous preferably have sequence identities that are at least 70%, preferably at least 80%, even more preferably at least 90%, even more preferably at least 95-99%, and most preferably 100% identical.

”Ligation” refers to the process of forming phosphodiester bonds between two nucleic acid fragments or genes, linking them together. To ligate the DNA fragments or genes together, the ends of the DNA must be ible with each other. In some cases, the ends will be directly compatible after endonuclease digestion. However, it may be necessary to first convert the staggered ends commonly produced after endonuclease digestion to blunt ends to make them compatible for ligation.

The terms “stringent conditions” or “stringent ization conditions” includes reference to ions under which a polynucleotide will hybridize to its target sequence, to a ably greater degree than other sequences (e.g., at least 2-fold over background). lly, stringency of hybridization is expressed, in part, with reference to the temperature and salt concentration under which the wash step is carried out. lly, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short polynucleotides (e. g., 10 to 50 nucleotides) and at least about 60°C for long polynucleotides (e. g., r than 50 nucleotides)—for example, “stringent conditions” can include ization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and three washes for 15 min each in 0.1><SSC/1% SDS at 60°C to 65°C. Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C may be used. SSC tration may be varied from about 0.1 to 2><SSC, with SDS being present at about 0.1%. Such wash temperatures are lly selected to be about 5°C to °C lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.

The Tm is the temperature (under defined ionic th and pH) at which 50% of the target ce hybridizes to a perfectly matched probe. An equation for calculating Tm and conditions for nucleic acid ization are well known and can be found in Sambrook, J. et a]. “Molecular Cloning: A Laboratory Manual,” 3ml edition, Cold Spring Harbor Laboratory Press, 2001. Typically, blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 ug/ml. Organic solvent, such as formamide at a tration of about 35-50% v/v, may also be used under particular circumstances, such as for RNA:DNA hybridizations. Useful ions on these wash conditions will be readily apparent to those of ordinary skill in the art.

The terms nt identity,” percentage of sequence identity,” and “% identity,” as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent ty may be measured over the length of an entire defined polynucleotide sequence, or may be measured over a shorter length, for e, over the length of a fragment taken from a larger, defined polynucleotide ce, for instance, a fragment of at least 45, at least 60, at least 90, at least 120, at least 150, at least 210 or at least 450 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured. The percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of matched positions (at which identical residues occur in both polypeptide sequences), dividing the number of matched ons by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. When sequences of different length are to be compared, the shortest sequence defines the length of the window of comparison. Conservative substitutions are not ered when calculating sequence identity.

“Percent (%) sequence identity,” with respect to the polypeptide sequences identified , is defined as the percentage of amino acid residues in a query sequence that are identical with the amino acid residues of a second, reference polypeptide ce or a portion thereof, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not ering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using ly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can ine appropriate parameters for ing alignment, including any algorithms needed to achieve maximal ent over the full length of the sequences being compared. Percent identity may be measured over the length of an entire defined polypeptide sequence, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.

Such lengths are exemplary only, and it is understood that any nt length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

The term epetitiveness” as used herein in the context of a polypeptide refers to a lack or limited degree of internal homology in a peptide or polypeptide sequence. The term “substantially non- repetitive” can mean, for example, that there are few or no instances of four contiguous amino acids in the sequence that are identical amino acid types or that the polypeptide has a subsequence score (defined infra) of 10 or less or that there is no a pattern in the order, from N- to C-terminus, of the ce motifs that constitute the polypeptide sequence. The term “repetitiveness” as used herein in the context of a polypeptide refers to the degree of internal homology in a peptide or polypeptide sequence. In contrast, a “repetitive” sequence may contain multiple identical copies of short amino acid sequences. For instance, a polypeptide ce of interest may be divided into n-mer sequences and the number of identical sequences can be counted. Highly repetitive sequences contain a large fraction of identical sequences while non-repetitive sequences contain few identical sequences. In the context of a polypeptide, a ce can contain multiple copies of shorter sequences of defined or variable length, or motifs, in which the motifs themselves have non-repetitive ces, rendering the full-length ptide substantially petitive. The length of polypeptide within which the non-repetitiveness is measured can vary from 3 amino acids to about 200 amino acids, about from 6 to about 50 amino acids, or from about 9 to about 14 amino acids. “Repetitiveness” used in the context of cleotide sequences refers to the degree of internal homology in the sequence such as, for example, the ncy of identical nucleotide sequences of a given length. Repetitiveness can, for example, be measured by analyzing the frequency of identical sequences.

A “vector” is a nucleic acid molecule, preferably self-replicating in an appropriate host, which transfers an inserted nucleic acid le into and/or between host cells. The term includes vectors that function primarily for insertion of DNA or RNA into a cell, replication of vectors that function primarily for the replication of DNA or RNA, and expression vectors that on for ription and/or translation of the DNA or RNA. Also included are vectors that provide more than one of the above functions. An “expression vector” is a polynucleotide which, when introduced into an appropriate host cell, can be transcribed and translated into a polypeptide(s). An “expression system” usually connotes a suitable host cell comprised of an expression vector that can function to yield a desired expression product.

“Serum degradation resistance,” as applied to a polypeptide, refers to the ability of the polypeptides to withstand degradation in blood or components f, which typically involves proteases in the serum or plasma. The serum degradation resistance can be measured by combining the protein with human (or mouse, rat, monkey, as appropriate) serum or plasma, typically for a range of days (e.g. 0.25, 0.5, l, 2, 4, 8, 16 days), typically at about 37°C. The samples for these time points can be run on a Western blot assay and the protein is detected with an antibody. The antibody can be to a tag in the n. If the protein shows a single band on the western, where the protein’s size is identical to that of the injected protein, then no degradation has occurred. In this exemplary method, the time point where 50% of the protein is degraded, as judged by Western blots or equivalent techniques, is the serum degradation half-life or “serum half-life” of the protein.

The term “t1/2 ” as used herein means the terminal half-life calculated as ln(2)/Kel. K61 is the terminal elimination rate constant calculated by linear sion of the terminal linear n of the log concentration vs. time curve. Half-life typically refers to the time required for half the quantity of an stered substance deposited in a living sm to be metabolized or eliminated by normal biological processes. The terms “mg”, “terminal half-life”, nation half-life” and “circulating fe” are used interchangeably herein.

] “Active clearance” means the mechanisms by which a protein is d from the ation other than by filtration or coagulation, and which includes l from the circulation mediated by cells, receptors, metabolism, or degradation of the protein.

“Apparent molecular weight factor” and “apparent molecular weight” are related terms referring to a measure of the relative increase or decrease in apparent lar weight ted by a particular amino acid sequence. The apparent molecular weight is determined using size exclusion chromatography (SEC) or similar methods by ing to globular protein standards, and is measured in “apparent kD” units. The apparent molecular weight factor is the ratio between the apparent molecular weight and the actual molecular weight; the latter predicted by adding, based on amino acid composition, the calculated molecular weight of each type of amino acid in the composition or by estimation from ison to molecular weight standards in an SDS ophoresis gel.

The terms “hydrodynamic radius” or “Stokes radius” is the effective radius (R}1 in nm) of a molecule in a solution measured by assuming that it is a body moving through the solution and resisted by the solution’s viscosity. In the embodiments of the invention, the hydrodynamic radius measurements of the XTEN fusion proteins correlate with the ‘apparent molecular weight factor’, which is a more intuitive measure. The “hydrodynamic radius” of a protein affects its rate of diffusion in aqueous solution as well as its ability to e in gels of macromolecules. The hydrodynamic radius of a protein is determined by its molecular weight as well as by its structure, including shape and compactness. Methods for determining the hydrodynamic radius are well known in the art, such as by the use of size exclusion chromatography (SEC), as described in US. Patent Nos. 6,406,632 and 7,294,513. Most proteins have globular structure, which is the most compact dimensional structure a protein can have with the smallest hydrodynamic radius. Some proteins adopt a random and open, unstructured, or ‘linear’ conformation and as a result have a much larger hydrodynamic radius ed to typical globular proteins of similar molecular .

“Physiological conditions” refers to a set of conditions in a living host as well as in vitro conditions, including temperature, salt concentration, pH, that mimic those conditions of a living subject.

A host of physiologically relevant ions for use in in vitro assays have been established. Generally, a physiological buffer contains a physiological concentration of salt and is adjusted to a neutral pH ranging from about 6.5 to about 7.8, and preferably from about 7.0 to about 7.5. A variety of physiological buffers are listed in Sambrook et al. (2001). Physiologically relevant temperature ranges from about 250C to about 380C, and preferably from about 350C to about 370C.

A “reactive group” is a chemical ure that can be coupled to a second reactive group.

Examples for ve groups are amino , carboxyl groups, sulfhydryl groups, hydroxyl groups, aldehyde groups, azide groups. Some reactive groups can be activated to facilitate coupling with a second reactive group. Non-limiting es for activation are the reaction of a carboxyl group with carbodiimide, the conversion of a carboxyl group into an activated ester, or the conversion of a carboxyl group into an azide function.

“Controlled release agent”, “slow release , “depot formulation” and “sustained e agent” are used interchangeably to refer to an agent capable of extending the duration of release of a polypeptide of the invention relative to the duration of release when the polypeptide is administered in the absence of agent. ent embodiments of the present invention may have ent release rates, resulting in different therapeutic amounts.

The terms “antigen”, “target n” and “immunogen” are used interchangeably herein to refer to the structure or binding determinant that an antibody fragment or an antibody fragment-based therapeutic binds to or has specificity against.

The term “payload” as used herein refers to a protein or peptide sequence that has biological or therapeutic activity; the rpart to the pharmacophore of small molecules. Examples of payloads include, but are not limited to, coagulation factors, cytokines, enzymes, hormones, and blood and grth factors.

The term “antagonist”, as used herein, includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native polypeptide disclosed herein. Methods for identifying antagonists of a polypeptide may comprise ting a native polypeptide with a candidate antagonist molecule and measuring a detectable change in one or more biological activities ly associated with the native polypeptide. In the t of the present invention, antagonists may include proteins, nucleic acids, carbohydrates, dies or any other molecules that decrease the effect of a biologically active protein.

The term “agonist” is used in the broadest sense and includes any le that mimics a biological activity of a native ptide disclosed herein. Suitable agonist molecules specifically include agonist antibodies or antibody fragments, fragments or amino acid sequence variants of native polypeptides, peptides, small organic molecules, etc. Methods for identifying agonists of a native ptide may comprise contacting a native polypeptide with a ate agonist molecule and measuring a detectable change in one or more biological activities normally ated with the native polypeptide.

As used herein, ” or ing,” or “palliating” or “ameliorating” are used interchangeably and mean administering a drug or a biologic to e a therapeutic benefit, to cure or reduce the severity of an existing condition, or to achieve a prophylactic benefit, prevent or reduce the likelihood of onset or severity the occurrence of a condition. By therapeutic benefit is meant eradication or amelioration of the underlying condition being treated or one or more of the physiological symptoms associated with the underlying condition such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying condition.

A “therapeutic effect” or “therapeutic benefit,” as used herein, refers to a physiologic effect, including but not limited to the mitigation, amelioration, or prevention of disease in humans or other animals, or to otherwise enhance physical or mental wellbeing of humans or animals, resulting from administration of a fusion protein of the invention other than the ability to induce the production of an antibody t an antigenic epitope possessed by the biologically active protein. For prophylactic benefit, the compositions may be stered to a t at risk of developing a particular e, condition or symptom of the disease (e. g., a bleed in a diagnosed hemophilia A subject), or to a subject reporting one or more of the physiological ms of a disease, even though a diagnosis of this disease may not have been made.

The terms “therapeutically effective amount” and “therapeutically effective dose”, as used herein, refer to an amount of a drug or a biologically active protein, either alone or as a part of a fusion protein composition, that is capable of having any detectable, beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition when administered in one or repeated doses to a subject. Such effect need not be absolute to be beneficial. ination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

The term “therapeutically effective dose n”, as used herein, refers to a schedule for consecutively administered multiple doses (i.e., at least two or more) of a biologically active protein, either alone or as a part of a fusion protein ition, wherein the doses are given in therapeutically effective amounts to result in sustained beneficial effect on any symptom, aspect, ed ter or characteristics of a e state or condition.

I). GENERAL TECHNIQUES The ce of the present invention employs, unless otherwise indicated, tional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See Sambrook, J. et al., “Molecular Cloning: A Laboratory Manual,” 3ml edition, Cold Spring Harbor Laboratory Press, 2001; “Current protocols in molecular biology”, F. M. Ausubel, et al. eds.,1987; the series “Methods in logy,” Academic Press, San Diego, CA.; “PCR 2: a practical ch”, M.J. MacPherson, B.D.

Hames and GR. Taylor eds., Oxford University Press, 1995; “Antibodies, a laboratory manual” Harlow, E. and Lane, D. eds., Cold Spring Harbor Laboratory,1988; “Goodman & Gilman’s The Pharmacological Basis of Therapeutics,” 11th Edition, McGraw—Hill, 2005; and Freshney, R.I., “Culture of Animal Cells: A Manual of Basic Technique,” 4th edition, John Wiley & Sons, Somerset, NJ, 2000, the contents of which are incorporated in their entirety herein by reference.

II). COAGULATION FACTOR VIII The present invention relates, in part, to compositions comprising factor VIII coagulation factor (CF) linked to one or more extended recombinant proteins (XTEN), resulting in a CFXTEN fusion protein composition. As used herein, “CF” refers to factor VIII ) or mimetics, sequence variants and truncated versions of FVIII, as described below.

“Factor VIII” or “FVIII” or “FVIII n” means a blood coagulation factor protein and species (including human, porcine, canine, rat or murine FVIII proteins) and sequence variants thereof that includes, but is not limited to the 2351 amino acid single-chain sor protein (with a 19-amino acid hydrophobic signal e), the mature 2332 amino acid factor VIII cofactor protein of approximately 270-330 kDa with the domain structure A1-A2-B-A3-C1-C2, as well as the nonenzymatic “active” or cofactor form of FVIII (FVIIIa) that is a circulating heterodimer of two chains that form as a result of proteolytic cleavage after R1648 of a heavy chain form composed of A1 -A2-B (in the range of 90-220 kD) of amino acids 1-1648 (numbered ve to the mature FVIII form) and a light chain A3- C1-C2 of 80 kDa of amino acids 1649-2232, each of which is depicted schematically in Further, and as used herein, each of A1, A2 and the A3 domain encompasses acidic spacer regions; a1, a2, and a3 acidic regions, respectively. Thus, it will be understood that CFXTEN constructs described as having A1, A2, A3, B, C1 and C2 domains include the al, a2 and a3 acidic regions. As used herein, r VIII” or “FVIII” or “FVIII polypeptide” also includes variant forms, ing proteins with substitutions, additions and/or deletions so long as the variant retains a desired ical activity such as procoagulant activity. Myriad functional FVIII variants have been constructed and can be used as recombinant FVIII proteins as bed . See PCT Publication Nos. WO 69164 A2, A2, A2, or A2, all of which are incorporated herein by reference in their entireties. A great many functional FVIII variants are known. In addition, hundreds of nonfunctional mutations in FVIII have been identified in hemophilia patients. See, e.g., Cutler et al., Hum. Mutat. 19:274-8 (2002), incorporated herein by reference in its ty. In addition, comparisons n FVIII from humans and other species have identified conserved residues that are likely to be required for function. See, e. g., Cameron et al., . Haemost. -22 (1998) and US 6,251,632, incorporated herein by reference in their entireties.

In one embodiment, the human factor VIII domains are defined by the following amino acid residues: A1, residues Ala1-Arg372; A2, residues Ser373-Arg740; B, residues -Arg1648; A3, residues Ser1649-Asn2019; C1, residues Lys2020-Asn2172; C2, residues Ser2l73-Tyr2332. The A3-C1- C2 sequence includes residues 9-Tyr2332. In another embodiment, residues Arg336-Arg372 is usually referred to as the al region, and the Arg3 72 is cleaved by thrombin. In certain embodiments, the a2 region is part of the A1 domain. In another embodiment, residues Glul649-Arg1689, is referred to as the a3 acidic region. In certain embodiments, the a3 acidic region is a part of the A3 domain. In another embodiment, a native FVIII protein has the following formula: A1-a1-A2-a2-B-a3-A3-C1-C2, where A1, A2, and A3 are the structurally-related ”A domains," B is the ”B domain,” C1 and C2 are the structurally- related ”C domains," and a1, a2 and a3 are acidic spacer regions. In the foregoing formula and referring to the primary amino acid sequence position in , the A1 domain of human FVIII extends from Alal to about Arg336, the al spacer region extends from about Met337 to about , the A2 domain extends from about Ser3 73 to about Tyr7l9, the a2 spacer region extends from about Glu720 to about , the B domain extends from about Ser741 to about Arg 1648, the a3 spacer region extends from about Glul649 to about Arg1689, the A3 domain extends from about Ser1690 to about Asn2019, the C1 domain extends from about Lys2020 to about 2, and the C2 domain s from about Ser2l73 to Tyr2332 (Saenko et al., 2005, J Thromb Hemostasis, 1, 922-930). Other than specific proteolytic cleavage sites, designation of the locations of the boundaries between the domains and regions of FVIII can vary in different literature references. The boundaries noted herein are therefore designated as approximate by use of the term “about.” Such factor VIII include truncated sequences such as B-domain deleted “BDD” sequences in which a portion or the majority of the B domain sequence is deleted (such as BDD sequences disclosed or referenced in US Pat Nos. 439 and 7,632,921). An example of a BDD FVIII is REFACTO® or ® (recombinant BDD FVIII), which comprises a first ptide corresponding to amino acids 1 to 743 of , fused to a second polypeptide ponding to amino acids 1638 to 2332 of . Exemplary BDD FVIII constructs which can be used to e recombinant proteins of the invention include, but are not limited to FVIII with a deletion of amino acids corresponding to amino acids 747-1638 of mature human FVIII () (Hoeben R.C., et al. J. Biol. Chem. 265 (13): 7318- 7323 (1990), orated herein by nce in its entirety), and FVIII with a deletion of amino acids corresponding to amino acids 771—1666 or amino acids 868-1562 of mature human FVIII () (Meulien P., et al. Protein Eng. 2(4): 301-6 (1988), incorporated herein by reference in its entirety).

] In addition, sequences that include heterologous amino acid insertions or substitutions (such as ic acid substituted for valine at position 75), or single chain FVIII (scFVIII) in which the heavy and light chains are ntly connected by a linker. As used herein, “FVIII” shall be any functional form of factor VIII molecule with the typical characteristics of blood ation factor VIII capable of correcting human factor VIII deficiencies when administered to such a subject, e.g., a subject with hemophilia A. FVIII or sequence variants have been isolated, characterized, and cloned, as described in US. Patent or Application Nos. 4,757,006; 4,965,199; 5,004,804; 5,198,349, 5,250,421; 5,919,766; 6,228,620; 6,818,439; 7,138,505; 7,632,921; and 20100081615.

Human factor VIII is encoded by a single-copy gene residing at the tip of the long arm of the X chromosome (q28). It comprises nearly 186,000 base pairs (bp) and constitutes approximately 0.1% of the X-chromosome (White, G.C. and Shoemaker, C.B., Blood (1989) 73:1-12). The human FVIII amino acid sequence was deduced from cDNA as shown in US. Pat. No. 4,965,199, which is incorporated herein by reference in its entirety. Native mature human FVIII derived from the cDNA sequence (i.e., without the secretory signal peptide but prior to other post-translational processing) is presented as FIG.

The DNA encoding the mature factor VIII mRNA is found in 26 separate exons ranging in size from 69 to 3,106 bp. The 25 intervening intron regions that separate the exons range in size from 207 to 32,400 bp. The complete gene consists of approximately 9 kb of exon and 177 kb of intron. The three repeat A domains have approximately 30% sequence homology. The B domain contains 19 of the approximately 25 predicted glycosylation sites, and the A3 domain is believed to contain a binding site for the von Willebrand factor. The tandem C domains follow the A3 domain and have approximately 37% homology to each other (White, G.C. and Shoemaker, C.B., Blood (1989) 73:1-12).

The B domain tes the A2 and A3 s of native factor FVIII in the newly synthesized precursor single-chain molecule. The precise boundaries of the B domain have been sly ed as extending from amino acids 712 to 1648 of the precursor ce (Wood et al., Nature (1984) 312:330-337) or amino acids 741—1648 (Pipe, SW, Haemophilia (2009) 7—1196 and US Pat. No. 7,560,107) or amino acids 740-1689 (Toole, JJ. Proc. Natl. Acad. Sci. USA (1986) 83:5939-5942). As used herein, ”B domain” means amino acids 48 of mature factor VIII. As used , “FVIII B domain on” or “FVIII BDD” means a FVIII sequence with any, a fragment of, or all of amino acids 741 to 1648 deleted. In one embodiment, FVIII BDD variants retain remnant amino acids of the B domain from the N-terminal end (“B1” as used herein) and C-terminal end (“B2” as used herein). In one FVIII BDD t, the B domain remnant amino acids are SFSQNPPVLKRHQR (SEQ ID NO: 1614). In one FVIII BDD variant, the B1 remnant is SFS and the B2 remnant is QNPPVLKRHQR (SEQ ID NO: 1615). In another FVIII BDD variant, the B1 t is SFSQN (SEQ ID NO: 1616) and the B2 remnant is PPVLKRHQR (SEQ ID NO: 1617). A ”B-domain—deleted factor VIII,” ”FVIII BDD,” or ”BDD FVIII” may have the full or partial deletions disclosed in US. Pat. Nos. 6,316,226, 6,346,513, 7,041,635, 5,789,203, 6,060,447, 886, 6,228,620, 5,972,885, 6,048,720, ,543,502, 5,610,278, 5,171,844, 5,112,950, 4,868,112, and 6,458,563, each ofwhich is incorporated herein by reference in its ty. In some ments, a B-domain—deleted factor VIII sequence of the present invention comprises any one of the deletions disclosed at col. 4, line 4 to col. 5, line 28 and examples 1-5 of US. Pat. No. 6,316,226 (also in US 6,346,513). In another embodiment, a B-domain deleted factor VIII is the 1638 B-domain deleted factor VIII (SQ version factor VIII) (e. g., factor VIII having a on from amino acid 744 to amino acid 1637, e. g., factor VIII having amino acids 1- 743 and amino acids 163 8-2332 of full-length factor VIII). In some embodiments, a B-domain-deleted factor VIII of the present invention has a deletion disclosed at col. 2, lines 26-51 and examples 5-8 of US. Patent No. 5,789,203 (also US 6,060,447, US 5,595,886, and US 6,228,620). In some embodiments, a B-domain-deleted factor VIII has a deletion described in col. 1, lines 25 to col. 2, line 40 of US Patent No. 5,972,885; col. 6, lines 1-22 and e 1 of US. Patent no. 6,048,720; col. 2, lines 17-46 of US.

Patent No. 5,543,502; col. 4, line 22 to col. 5, line 36 of US. Patent no. 5,171,844; col. 2, lines 55-68, figure 2, and example 1 ofU.S. Patent No. 950; col. 2, line 2 to col. 19, line 21 and table 2 ofU.S.

Patent No. 4,868,112; col. 2, line 1 to col. 3, line 19, col. 3, line 40 to col. 4, line 67, col. 7, line 43 to col. 8, line 26, and col. 11, line 5 to col. 13, line 39 ofU.S. Patent no. 7,041,635; or col. 4, lines 25-53, of US. Patent No. 6,458,563. In some embodiments, a B-domain-deleted factor VIII has a deletion of most of the B domain, but still contains terminal sequences of the B domain that are essential for in vivo proteolytic processing of the primary translation product into two polypeptide chain, as disclosed in W0 91/09122, which is incorporated herein by reference in its entirety. In some embodiments, a B-domain- deleted factor VIII is constructed with a on of amino acids 747-163 8, i. e., Virtually a complete deletion of the B domain. Hoeben R.C., et al. J. Biol. Chem. 265 (13): 7318-7323 (1990), incorporated herein by reference in its entirety. A B-domain-deleted factor VIII may also contain a deletion of amino acids 771—1666 or amino acids 868-1562 of factor VIII. Meulien P., et al. Protein Eng. 2(4): 301-6 (1988), incorporated herein by reference in its ty. onal B domain deletions that are part of the invention e: deletion of amino acids 982 through 1562 or 760 through 1639 (Toole et al., Proc.

Natl. Acad. Sci. U.S.A. (1986) 83, 5939-5942)), 797 through 1562 (Eaton, et al. Biochemistry (1986) :8343-8347)), 741 h 1646 (Kaufman (PCT published application No. WO 87/04187)), 747-1560 (Sarver, et al., DNA (1987) 6:553-564)), 741 though 1648 (Pasek (PCT application No.88/00831)), or 816 through 1598 or 741 through 1648 (Lagner (Behring Inst. Mitt. (1988) No 82:16-25, EP 295597)), each of which is orated herein by reference in its entirety. Each of the foregoing deletions may be made in any factor VIII sequence utilized in the embodiments of the present invention.

Proteins ed in clotting e factor I, factor II, factor III, factor IV, factor V, factor VI, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, n C, and tissue factor (collectively or individually “clotting protein(s)”). The interaction of the major clotting proteins in the intrinsic and extrinsic clotting pathways is showed in The ty of the clotting proteins are present in zymogen form, but when activated, exhibit a procoagulant protease ty in which they te another of the ng proteins, contributing to the intrinsic or extrinsic coagulation pathway and clot formation. In the intrinsic pathway of the coagulation cascade, FVIII associates with a complex of activated factor IX, factor X, calcium, and phospholipid. The factor VIII heterodimer has no enzymatic activity, but the heterodimer becomes active as a cofactor of the enzyme factor IXa after lytic activation by thrombin or factor Xa, with the actiVity of factor VIIIa characterized by its ability to form a membrane binding site for factors IXa and X in a conformation suitable for activation of the factor X by factor IXa. Upon cleavage by thrombin, activated FVIII (FVIIIa) dissociates from von Willebrand factor and binds to negatively charged phospholipid PL, and the resulting complex participates as a cofactor to factor IXa in the factor X activating (tenase) complex. Within the C2 domain and amino acid residues 1649 through 1689 in the A3 domain are von Willebrand factor (VWF) binding sites that act to complex with von Willebrand factor, the resulting circulating complex protects FVIII from rapid degradation in the blood (Weiss HJ, et al. Stabilization of factor VIII in plasma by the von Willebrand factor. Studies on ansfusion and dissociated factor VIII and in patients with von Willebrand's disease. J Clin Invest (1977) . ted factor VIII is a heterotrimer comprised of the A1 domain and the A2 domain and the light chain including domains C2. The activation of factor IX is achieved by a two-step removal of the activation peptide (Ala 146-Arg 180) from the molecule (Bajaj et al., Human factor IX and factor IXa, in METHODS IN ENZYMOLOGY. 1993). The first cleavage is made at the Arg 145-Ala 146 site by either factor XIa or factor VIIa/tissue factor. The second, and rate limiting cleavage is made at Arg 180-Val 181. The activation removes 35 residues. Activated human factor IX exists as a heterodimer of the C-terminal heavy chain (28 kDa) and an N—terminal light chain (18 kDa), which are held er by one de bridge attaching the enzyme to the Gla domain. Factor IXa in turn activates factor X in concert with activated factor VIII. Alternatively, factors IX and X can both be activated by factor VIIa complexed with lipidated tissue factor, generated Via the sic y. Factor Xa then participates in the final common pathway whereby prothrombin is converted to thrombin, and thrombin, in turn converts fibrinogen to fibrin to form the clot.

Defects in the coagulation process can lead to bleeding disorders (coagulopathies) in which the time taken for clot formation is prolonged. Such defects can be congenital or acquired. For example, hemophilia A and B are inherited diseases characterized by deficiencies in FVIII and FIX, respectively.

Stated ently, biologically active factor VIII ts the coagulation defect in plasma derived from individuals afflicted with hemophilia A. Recombinant FVIII has been shown to be effective and has been approved for the treatment of hemophilia A in adult and pediatric patients, and also is used to stop bleeding episodes or prevent bleeding associated with trauma and/or y. Current therapeutic uses of factor VIII can be matic in the treatment of indiViduals exhibiting a deficiency in factor VIII, as well as those indiViduals with Von Willebrand's disease. In addition, duals receiving factor VIII in replacement therapy frequently develop antibodies to these proteins that often reduce or eliminate the procoagulant actiVity of the bound FVIII. Continuing treatment is exceedingly lt because of the presence of these antibodies that reduce or negate the efficacy of the treatment.

In one aspect, the invention contemplates inclusion of FVIII sequences in the CFXTEN fusion n compositions that are cal to human FVIII, sequences that have homology to FVIII ces, sequences that are natural, such as from humans, non-human primates, mammals (including domestic animals), or truncated version of FVIII; all of which retain at least a portion of the procoagulant actiVity of native FVIII and that are useful for preventing, treating, mediating, or ameliorating ilia A or bleeding episodes related to trauma, surgery, or deficiency of coagulation factor VIII. Sequences with homology to FVIII may be found by standard homology searching techniques, such as NCBI BLAST, or in public databases such as Chemical Abstracts Services Databases (e. g., the CAS Registry), GenBank, The Universal n Resource (UniProt) and subscription provided databases such as GenSeq (e. g., Derwent).

In one embodiment, the FVIII incorporated into the subject CFXTEN itions is a recombinant polypeptide with a sequence corresponding to a FVIII protein found in nature. In another embodiment, the FVIII is a non-natural FVIII sequence variant, fragment, homolog, or a mimetic of a natural ce that retains at least a portion of the procoagulant activity of the corresponding native FVIII. In another embodiment, the FVIII is a truncated variant with all or a portion of the B domain deleted (“FVIII BDD”), which can be in either heterodimeric form or can remain as a single chain III”), the latter described in Meulien et al., Protein Eng. (1988) 2(4):301-306. Non-limiting examples of FVIII BDD are factor VIII sequences in which the amino acids are d n residue number 741 and residue number 1640 (numbered relative to native, mature FVIII), or between residue number 745 and residue number 1640, or n residue number 745 and residue number 1640, or between residue number 741 and residue number 1690, or between residue number 745 and residue number 1667, or between residue number 745 and residue number 1657, or between residue number 747 and residue number 1642, or between e number 751 and residue number 1667.

In another embodiment, heterologous sequences are incorporated into the FVIII, which may e XTEN, as described more fully below. Table 1 provides a non-limiting list of amino acid sequences of FVIII that are encompassed by the CFXTEN fusion proteins of the invention. In some embodiments, FVIII incorporated into CFXTEN fusion proteins include ns that have at least about 70% sequence identity, or alternatively 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% ce identity compared to an amino acid sequence of comparable length selected from Table 1.

Table 1: FVIII amino acid seguences Name Amino Acid Sequence ID (source) FVIII MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPK 1 precursor SFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNM polypeptide ASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKEN (human) GPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLF AVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKS VYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLL FCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFD DDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNG PQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASR PYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPR CLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENR SWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWY ILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHN SDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPS TRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLS DLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNE KLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQL DTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLF KGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVW QNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKE Name Amino Acid Sequence ID (source) GPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSV EGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEK KIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVL QDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQ QNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNE KEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPA DSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTY KKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQ GTEGAIKWNEANRPGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWK SQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCS QNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQK KTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPL YRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPR KNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLL VCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPT FHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVR KKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNK CQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDL HGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVD SSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDA QITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVT GVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVN SLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT mature DHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS (human) EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM SARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP KDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTD PWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAID SNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSST SNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSL SEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFK VSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDR MLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFL PESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEF TKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLP QIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTA HFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPL EETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHS IPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQG AKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKV ELLPKV RVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLN ACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSD QEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSS SPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHH MAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEF ALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGL RIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETV EMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASG Name Amino Acid Sequence ID (source) QYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSL YISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHP STLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKA RLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEF LISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVH QIALRMEVLGCEAQDLY FVIII MQVELYTCCFLCLLPFSLSATRKYYLGAVELSWDYMQSDLLSALHADTSFSSRVP (Canine) GSLPLTTSVTYRKTVFVEFTDDLFNIAKPRPPWMGLLGPTIQAEVYDTVVIVLKN MASHPVSLHAVGVSYWKASEGAEYEDQTSQKEKEDDNVIPGESHTYVWQVLKE NGPMASDPPCLTYSYFSHVDLVKDLNSGLIGALLVCKEGSLAKERTQTLQEFVLL FAVFDEGKSWHSETNASLTQAEAQHELHTINGYVNRSLPGLTVCHKRSVYWHVI GMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTFLMDLGQFLLFCHIPSH QHDGMEAYVKVDSCPEEPQLRMKNNEDKDYDDGLYDSDMDVVSFDDDSSSPFI QIRSVAKKHPKTWVHYIAAEEEDWDYAPSGPTPNDRSHKNLYLNNGPQRIGKKY KKVRFVAYTDETFKTREAIQYESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGI NYVTPLHTGRLPKGVKHLKDMPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSS FINLERDLASGLIGPLLICYKESVDQRGNQMMSDKRNVILFSVFDENRSWYLTEN MQRFLPNADVVQPHDPEFQLSNIMHSINGYVFDNLQLSVCLHEVAYWYILSVGA QTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWVLGCHNSDFR NRGMTALLKVSSCNRNIDDYYEDTYEDIPTPLLNENNVIKPRSFSQNSRHPSTKEK QLKATTTPENDIEKIDLQSGERTQLIKAQSVSSSDLLMLLGQNPTPRGLFLSDLREA TDRADDHSRGAIERNKGPPEVASLRPELRHSEDREFTPEPELQLRLNENLGTNTTV ELKKLDLKISSSSDSLMTSPTIPSDKLAAATEKTGSLGPPNMSVHFNSHLGTIVFGN NSSHLIQSGVPLELSEEDNDSKLLEAPLMNIQESSLRENVLSMESNRLFKEERIRGP ASLIKDNALFKVNISSVKTNRAPVNLTTNRKTRVAIPTLLIENSTSVWQDIMLERN TEFKEVTSLIHNETFMDRNTTALGLNHVSNKTTLSKNVEMAHQKKEDPVPLRAE NPDLSSSKIPFLPDWIKTHGKNSLSSEQRPSPKQLTSLGSEKSVKDQNFLSEEKVVV GEDEFTKDTELQEIFPNNKSIFFANLANVQENDTYNQEKKSPEEIERKEKLTQENV TMIGTKNFLKNLFLLSTKQNVAGLEEQPYTPILQDTRSLNDSPHSEGIHM REEANLEGLGNQTNQMVERFPSTTRMSSNASQHVITQRGKRSLKQPRLS QGEIKFERKVIANDTSTQWSKNMNYLAQGTLTQIEYNEKEKRAITQSPLSDCSMR MNDSALPVAKESASPSVRHTDLTKIPSQHNSSHLPASACNYTFRERTSGV QEGSHFLQEAKRNNLSLAFVTLGITEGQGKFSSLGKSATNQPMYKKLENTVLLQP GLSETSDKVELLSQVHVDQEDSFPTKTSNDSPGHLDLMGKIFLQKTQGPVKMNK VPFLKWATESSEKIPSKLLGVLAWDNHYDTQIPSEEWKSQKKSQTNTAF KRKDTILPLGPCENNDSTAAINEGQDKPQREAMWAKQGEPGRLCSQNPPVSKHH QREITVTTLQPEEDKFEYDDTFSIEMKREDFDIYGDYENQGLRSFQKKTRHYFIAA VERLWDYGMSRSPHILRNRAQSGDVQQFKKVVFQEFTDGSFTQPLYRGELNEHL GLLGPYIRAEVEDNIVVTFKNQASRPYSFYSSLISYDEDEGQGAEPRRKFVNPNET KIYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLICRSNTLNPA HGRQVTVQEFALVFTIFDETKSWYFTENLERNCRAPCNVQKEDPTLKENFRFHAI NGYVKDTLPGLVMAQDQKVRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMA VYNLYPGVFETVEMLPSQVGIWRIECLIGEHLQAGMSTLFLVYSKKCQTPLGMAS GHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKDPFSWIKVDLLAPMIIHGI MTQGARQKFSSLYVSQFIIMYSLDGNKWHSYRGNSTGTLMVFFGNVDSSGIKHNI AQYIRLHPTHYSIRSTLRMELLGCDFNSCSMPLGMESKAISDAQITASSYLS SMLATWSPSQARLHLQGRTNAWRPQANNPKEWLQVDFRKTMKVTGITTQGVKS LLISMYVKEFLISSSQDGHNWTLFLQNGKVKVFQGNRDSSTPVRNRLEPPLVARY VR LHPQSWAHHIALRLEVLGCDTQQPA FVIII (Pig) ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT DHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL Name Amino Acid Sequence ID (source) IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTD PWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAID SNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSST SNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSL SEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFK VSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDR MLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFL PESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEF TKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLP QIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTA EEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPL EETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHS IPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQG SLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKV ELLPKV RVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLN ACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSD QEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSS SPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHH MAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEF ALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGL VMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETV EMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASG PKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSL YISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHP THYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKA RLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEF LISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVH QIALRMEVLGCEAQDLY FVIII AIRRYYLGAVELSWNYIQSDLLSVLHTDSRFLPRMSTSFPFNTSIMYKKTVFVEYK (Mouse) DQLFNIAKPRPPWMGLLGPTIWTEVHDTVVITLKNMASHPVSLHAVGVSYWKAS EGDEYEDQTSQMEKEDDKVFPGESHTYVWQVLKENGPMASDPPCLTYSYMSHV DLVKDLNSGLIGALLVCKEGSLSKERTQMLYQFVLLFAVFDEGKSWHSETNDSY TQSMDSASARDWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEIHSIF LEGHTFFVRNHRQASLEISPITFLTAQTLLIDLGQFLLFCHISSHKHDGMEAYVKV DSCPEESQWQKKNNNEEMEDYDDDLYSEMDMFTLDYDSSPFIQIRSVAKKYPKT WIHYISAEEEDWDYAPSVPTSDNGSYKSQYLSNGPHRIGRKYKKVRFIAYTDETF KTRETIQHESGLLGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVSPLHARRLPR GIKHVKDLPIHPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFINPERDLASGLIGP LLICYKESVDQRGNQMMSDKRNVILFSIFDENQSWYITENMQRFLPNAAKTQPQD PGFQASNIMHSINGYVFDSLELTVCLHEVAYWHILSVGAQTDFLSIFFSGYTFKHK MVYEDTLTLFPFSGETVFMSMENPGLWVLGCHNSDFRKRGMTALLKVSSCDKST SDYYEEIYEDIPTQLVNENNVIDPRSFFQNTNHPNTRKKKFKDSTIPKNDMEKIEPQ FEEIAEMLKVQSVSVSDMLMLLGQSHPTPHGLFLSDGQEAIYEAIHDDHSPNAIDS NEGPSKVTQLRPESHHSEKIVFTPQPGLQLRSNKSLETTIEVKWKKLGLQVSSLPS NLMTTTILSDNLKATFEKTDSSGFPDMPVHSSSKLSTTAFGKKAYSLVGSHVPLN ASEENSDSNILDSTLMYSQESLPRDNILSIENDRLLREKRFHGIALLTKDNTLFKDN NKTYNHSTTNEKLHTESPTSIENSTTDLQDAILKVNSEIQEVTALIHDGT TYLRLNHMLNRTTSTKNKDIFHRKDEDPIPQDEENTIMPFSKMLFLSESS NWFKKTNGNNSLNSEQEHSPKQLVYLMFKKYVKNQSFLSEKNKVTVEQDGFTK NIGLKDMAFPHNMSIFLTTLSNVHENGRHNQEKNIQEEIEKEALIEEKVVLPQVHE ATGSKNFLKDILILGTRQNISLYEVHVPVLQNITSINNSTNTVQIHMEHFFKRRKDK ETNSEGLVNKTREMVKNYPSQKNITTQRSKRALGQFRLSTQWLKTINCSTQCIIKQ IDHSKEMKKFITKSSLSDSSVIKSTTQTNSSDSHIVKTSAFPPIDLKRSPFQNKFSHV QASSYIYDFKTKSSRIQESNNFLKETKINNPSLAILPWNMFIDQGKFTSPGKSNTNS VTYKKRENIIFLKPTLPEESGKIELLPQVSIQEEEILPTETSHGSPGHLNLMKEVFLQ Name Amino Acid Sequence ID KIQGPTKWNKAKRHGESIKGKTESSKNTRSKLLNHHAWDYHYAAQIPKDMWKS KEKSPEIISIKQEDTILSLRPHGNSHSIGANEKQNWPQRETTWVKQGQTQRTCSQIP PVLKRHQRELSAFQSEQEATDYDDAITIETIEDFDIYSEDIKQGPRSFQQKTRHYFI AAVERLWDYGMSTSHVLRNRYQSDNVPQFKKVVFQEFTDGSFSQPLYRGELNEH LGLLGPYIRAEVEDNIMVTFKNQASRPYSFYSSLISYKEDQRGEEPRRNFVKPNET KIYFWKVQHHMAPTEDEFDCKAWAYFSDVDLERDMHSGLIGPLLICHANTLNPA HGRQVSVQEFALLFTIFDETKSWYFTENVKRNCKTPCNFQMEDPTLKENYRFHA1 NGYVMDTLPGLVMAQDQRIRWYLLSMGNNENIQSIHFSGHVFTVRKKEEYKMA VYNLYPGVFETLEMIPSRAGIWRVECLIGEHLQAGMSTLFLVYSKQCQIPLGMAS GSIRDFQITASGHYGQWAPNLARLHYSGSINAWSTKEPFSWIKVDLLAPMIVHGIK TQGARQKFSSLYISQFIIMYSLDGKKWLSYQGNSTGTLMVFFGNVDSSGIKHNSF NPPIIARYIRLHPTHSSIRSTLRMELMGCDLNSCSIPLGMESKVISDTQITASSYFTN MFATWSPSQARLHLQGRTNAWRPQVNDPKQWLQVDLQKTMKVTGIITQGVKSL FTSMFVKEFLISSSQDGHHWTQILYNGKVKVFQGNQDSSTPMMNSLDPPLLTRYL RIHPQIWEHQIALRLEILGCEAQQQY FVIII BDD MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPK variant SFPFNTSVVYKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNM (US Pat ASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKEN No. GPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLF 763292 1 , AVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKS SEQ ID VYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLL NO: 3) FCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFD DDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNG PQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASR PYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPR CLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENR SWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWY QTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHN SDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL KRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYF IAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNE HLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPN ETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTL NPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRF IMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYK MALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLG MASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMII HGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIK HNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASS YFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQ GVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPL IHPQSWVHQIALRMEVLGCEAQDLY FVIII LGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT BDD-Z VHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDY EMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLR NRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVT FRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKD Name Amino Acid Sequence ID (source) WAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIF DETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQD QRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSK AGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQW APKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFII KKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIR STLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQ GRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQ DGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALR MEVLGCEAQDLY FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT BDD-3 VHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS (G1648) EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE AIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQGEITRTTLQSDQEEIDY DDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLR NRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVT FRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKD EFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIF DETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQD LLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSK AGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQW APKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFII MYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIR STLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQ GRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQ DGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALR MEVLGCEAQDLY FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT BDD-4 VHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK DTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK EDSYEDISAYLLSKNNAIEPRSFSQQSPRSFQKKTRHYFIAAVERLWDY GMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIR AEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKV QHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVT VQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMD TLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPG VFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQ ITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQ KFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYI Name Amino Acid Sequence ID (source) RLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWS PSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMY VKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQS LRMEVLGCEAQDLY FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT 10 BDD-5 VHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS DQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL ASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK QSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFT DGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEED QRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDV HSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAP CNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIH TVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMS TLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKE PFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGT LMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLG SDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVD FQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGN QDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT 11 BDD-6 DHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTD TISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNR AQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFR NQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEF DCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDE TENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQR IRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAG IWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAP KLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMY SLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTL RMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRS NAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGH QWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEV LGCEAQDLY FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT 12 BDD-7 VHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV Name Amino Acid Sequence ID (source) DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQSPRSFQKKTRHYFIAAVERLWDYG MSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRA EVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQ HHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTV QEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTL PGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVF ETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQIT ASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKF SSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRL HPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPS KARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVK EFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSW VHQIALRMEVLGCEAQDLY FVIII MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPK 13 BDD-8 SVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNM precursor ASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKEN (US Pat. GPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLF No. AVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKS 6818439 VYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLL SEQ ID FCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFD NO: 47) DDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNG PQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASR PYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPR CLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENR SWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWY ILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHN SDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL KRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYF IAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNE HLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPN ETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTL NPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRF HAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYK MALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLG MASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMII HGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIK HNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASS YFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQ GVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPL IHPQSWVHQIALRMEVLGCEAQDLY FVIII LGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT 14 BDD-9 DHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS mature EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV (US Pat. DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM No. QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL 6818439) VRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP Name Amino Acid Sequence ID (source) KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDY DDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLR SVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVT FRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKD EFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIF DETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQD QRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSK AGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQW APKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFII MYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIR STLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQ GRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQ DGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALR MEVLGCEAQDLY FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT 15 BDD- 1 0 DHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS DQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK DTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQAEITRTTLQSDQEEIDY DDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLR NRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVT FRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKD EFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIF DETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQD QRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSK ECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQW APKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFII MYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIR STLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQ GRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQ DGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALR MEVLGCEAQDLY FVIII ATRATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLF 16 BDD- 1 1 VEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSY WKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSY LSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETK NSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEV GHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAY VKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKK HPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMA YTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYS RRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDL ASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPA PEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSG YTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVS SCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQAEITRTTLQSDQ Name Amino Acid Sequence ID DTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSS PHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVED NIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHM APTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFA LFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLV MAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVE MLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQ YGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYI SQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTH YSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARL HLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLIS SSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIA LRMEVLGCEAQDLY FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT 17 BDD- 12 DHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK DTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQAEITRTTLQSDQEEIDY DDTISVEMKKEDFDIFDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLR NRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVT FRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKD WAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIF DETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQD QRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSK AGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQW APKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFII MYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIR STLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQ GRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQ DGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALR MEVLGCEAQDLY FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT 18 BDD- 1 3 DHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKAS EGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHV DLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLM QDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFL EGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKV DSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDE TFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLP KGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL CYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFK HKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDK NTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDY DDTISVEMKKEDFDIFDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLR NRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVT RPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKD EFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIF DETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQD (3:222) Amino Acid Sequence ID QRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSK AGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQW APKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFII MYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIR STLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQ GRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQ DGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALR MEVLGCEAQDLY The present invention also contemplates CFXTEN sing FVIII with various amino acid deletions, insertions and substitutions made in the FVIII sequences of Table 1 that retain procoagulant activity. Examples of conservative substitutions for amino acids in polypeptide sequences are shown in Table 2. In embodiments of the CFXTEN in which the sequence identity of the FVIII is less than 100% compared to a specific sequence disclosed herein, the invention contemplates substitution of any of the other 19 natural L-amino acids for a given amino acid residue of the given FVIII, which may be at any position within the sequence of the FVIII, including adjacent amino acid residues. If any one substitution results in an undesirable change in procoagulant activity, then one of the alternative amino acids can be employed and the construct protein evaluated by the methods described herein (e. g., the assays of Table 49), or using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in US. Pat. No. 5,364,934, the content of which is incorporated by reference in its entirety, or using methods generally known in the art. In a red substitution, the FVIII component of the CFXTEN embodiments is d by replacing the R1648 residue (numbered relative to the native mature form of FVIII) with glycine or alanine to prevent proteolytic processing to the heterodimer form. In another substitution, the FVIII component of the CFXTEN embodiments is modified by replacing the Y1680 residue (numbered relative to the native mature form of FVIII) with phenylalanine.

In another embodiment, the FVIII ent of the CFXTEN ments is modified by ing the Y1680 e (numbered relative to the native mature form of FVIII) with phenylalanine and the R1648 residue (numbered ve to the native mature form of FVIII) with glycine or alanine.

In one embodiment, the FVIII of the fusion n composition has one or more amino acid substitutions ed to reduce the binding of FVIII inhibitors at epitopes recognized by the antibodies of Table 9, including but not limited to tutions at Lys(377), Lys(466), Lys(3 80), Ser(488), Arg(489), Arg(490), Leu(491), Lys(493), Lys(496), His(497), Lys(499), 2), Lys(523), Lys(556), Met (2199), Phe(2200), Leu(2252), Val(2223), and Lys(2227). In addition, variants can include, for instance, polypeptides wherein one or more amino acid es are added or deleted at or near the N— or C-terminus of the full-length native amino acid sequence or of a domain of a FVIII so long as the variant s some if not all of the procoagulant ty of the native peptide. The resulting FVIII sequences that retain at least a portion (e. g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least 95% or more) of the procoagulant ty in comparison to native circulating FVIII are considered useful for the fusion protein compositions of this invention. Examples of FVIII variants are known in the art, including those described in US Patent and Application Nos. 6,316,226; 6,818,439; 7,632,921; 20080227691, which are incorporated herein by reference. In one embodiment, a PV111 sequence variant has an aspartic acid substituted for valine at amino acid position 75 (numbered relative to the native mature form of FVIII).

Table 2: Exemplary conservative amino acid substitutions Original e ary Substitutions Ala (A) val; leu; ile Arg (R) lys; gln; asn Asn (N) gin; his; lys; arg Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp Gly (G) Pro His (H) asn: gin: lys; arg lle (l) leu; val; met; ala; phe: norleucine Leu (L) norleucine: ile: val; met; ala: phe Lys (K) arg: gin: asn Met (M) leu; phe; ile Phe (F) leu: val: ile; ala Pro (P) Gly Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr(Y) Trp: phe: thr: ser Val (V) lle; leu; met; phe; ala; norleucine III). EXTENDED RECOMBINANT POLYPEPTIDES In one aspect, the invention provides XTEN polypeptide compositions that are useful as fusion protein partner(s) to link to and/or incorporate Within a FVIII polypeptide, resulting in a CFXTEN fusion protein. XTEN are lly polypeptides With non-naturally ing, substantially non-repetitive sequences having a low degree of or no secondary or tertiary structure under physiologic conditions.

XTEN typically have from about 36 to about 3000 amino acids of Which the majority or the entirety are small hydrophilic amino acids. As used herein, “XTEN” specifically excludes Whole antibodies or dy fragments (e. g. single-chain antibodies and PC fragments). XTEN polypeptides have utility as a fusion n partners in that they serve various roles, conferring certain desirable cokinetic, physicochemical, pharmacologic, and pharmaceutical properties When linked to a FVlll protein to a create a CFXTEN fusion protein. Such CFXTEN fusion protein compositions have enhanced properties compared to the corresponding FVIH not linked to XTEN, making them useful in the ent of certain conditions related to FVHI deficiencies or ng disorders, as more fully described below.

The ion criteria for the XTEN to be fused to the FVIII proteins used to create the inventive fusion proteins compositions generally relate to attributes of physical/chemical properties and mational structure of the XTEN that is, in turn, used to confer enhanced ceutical, pharmacologic, and pharmacokinetic properties to the FVIII fusion proteins compositions. The unstructured characteristic and physical/chemical ties of the XTEN result, in part, from the overall amino acid composition disproportionately limited to 4-6 hydrophilic amino acids, the linking of the amino acids in a fiable non-repetitive design, and the length of the XTEN polypeptide. In an advantageous feature common to XTEN but uncommon to polypeptides, the properties ofXTEN disclosed herein are not tied to absolute y amino acid sequences, as evidenced by the ity of the exemplary sequences of Table 4 that, within varying ranges of , possess similar properties, many of which are documented in the Examples. The XTEN of the present invention may exhibit one or more, or all of the following advantageous properties: unstructured conformation, conformational flexibility, enhanced aqueous solubility, high degree of protease resistance, low genicity, low binding to mammalian receptors, a defined degree of charge, and increased ynamic (or Stokes) radii; properties that can make them ularly useful as fusion protein partners. Non-limiting examples of the enhanced ties that XTEN confer on the fusion proteins comprising FVIII fused to XTEN, compared to FVIH not linked to XTEN, include increases in the overall solubility and/or metabolic stability, reduced susceptibility to proteolysis, reduced genicity, reduced rate of absorption when administered subcutaneously or intramuscularly, reduced binding to FVIII nce receptors, reduced reactivity to anti-payload antibodies, ed interactions with substrate, and/or enhanced pharmacokinetic properties when administered to a subject. The enhanced pharmacokinetic properties of the CFXTEN compositions compared to FVIII not linked to XTEN include longer terminal half-life (e. g., two-fold, three-fold, four-fold or more), sed area under the curve (AUC) (e. g., 25%, 50%, 100% or more), lower volume of distribution, and enhanced tion after subcutaneous or intramuscular injection (an advantage compared to commercially-available forms of FVIII that must be administered intravenously). In addition, it is believed that the CFXTEN compositions comprising cleavage sequences (described more fully, below) permit sustained release of biologically active FVIH, such that the administered CFXTEN acts as a depot. It is specifically contemplated that the inventive CFXTEN fusion proteins can exhibit one or more or any combination of the improved properties disclosed herein. As a result of these enhanced properties, it is believed that CFXTEN compositions permit less frequent dosing ed to FVIH not linked to XTEN when administered at comparable dosages. Such CFXTEN fusion protein compositions have utility to treat certain factor VIII-related conditions, as described herein.

A variety of s and assays are known in the art for determining the physical/chemical properties of proteins such as the CFXTEN compositions comprising XTEN. Such properties include but are not limited to secondary or tertiary structure, solubility, protein aggregation, stability, absolute and apparent lar weight, purity and uniformity, melting properties, contamination and water content.

Methods to assay these properties include analytical centrifugation, EPR, HPLC-ion exchange, HPLC- size exclusion, HPLC-reverse phase, light scattering, capillary electrophoresis, circular dichroism, ential scanning calorimetry, fluorescence, HPLC-ion exchange, HPLC-size exclusion, IR, NMR, Raman oscopy, refractometry, and UV/Visible spectroscopy. Additional methods are disclosed in Arnau, er al., Prot Expr and Purif (2006) 48, 1-13.

The XTEN component(s) of the CFXTEN are designed to behave like denatured peptide sequences under physiological conditions, despite the extended length of the polymer. “Denatured” describes the state of a peptide in solution that is characterized by a large conformational freedom of the peptide ne. Most peptides and proteins adopt a red conformation in the ce of high concentrations of denaturants or at ed temperature. es in denatured conformation have, for example, characteristic circular dichroism (CD) spectra and are characterized by a lack of long-range interactions as determined by NMR. “Denatured conformation” and “unstructured conformation” are used synonymously herein. In some embodiments, the invention provides XTEN sequences that, under physiologic conditions, are largely devoid of secondary structure. In other cases, the XTEN ces are substantially devoid of secondary structure under physiologic conditions such that the XTEN can adopt random coil conformation. “Largely devoid,” as used in this context, means that at least 50% of the XTEN amino acid residues of the XTEN sequence do not contribute to secondary structure as ed or determined by the means described herein. “Substantially devoid,” as used in this context, means that at least about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or at least about 99% of the XTEN amino acid residues of the XTEN sequence do not contribute to secondary structure, as measured or determined by the methods described herein.

A variety of methods have been established in the art to n the presence or e of secondary and tertiary structures in a given polypeptide. In particular, secondary structure can be measured spectrophotometrically, e. g., by circular dichroism spectroscopy in the “far-UV” spectral region (190-250 nm). Secondary ure elements, such as alpha-helix and beta-sheet, each give rise to a characteristic shape and magnitude of CD spectra, as does the lack of these structure elements.

Secondary structure can also be predicted for a polypeptide sequence via certain computer programs or algorithms, such as the well-known Chou-Fasman algorithm (Chou, P. Y., et al. (1974) Biochemistry, 13: 222-45) and the Gamier-Osguthorpe-Robson (“GOR”) algorithm (Gamier J, Gibrat JF, Robson B. (1996), GOR method for predicting protein secondary structure from amino acid sequence. Methods 1266:540-553), as described in US Patent Application Publication No. 20030228309A1. For a given sequence, the algorithms can predict r there exists some or no secondary structure at all, expressed as the total and/or percentage of residues of the sequence that form, for example, alpha-helices or beta-sheets or the tage of residues of the sequence predicted to result in random coil formation (which lacks secondary structure).

In one embodiment, the XTEN ces used in the subject fusion protein compositions have an alpha-helix percentage ranging from 0% to less than about 5% as determined by the Chou-Fasman algorithm. In another embodiment, the XTEN ces of the fusion protein compositions have a beta- sheet tage ranging from 0% to less than about 5% as determined by the Chou-Fasman algorithm.

In some embodiments, the XTEN sequences of the fusion protein compositions have an alpha-helix percentage ranging from 0% to less than about 5% and a beta-sheet percentage ranging from 0% to less than about 5% as determined by the Chou-Fasman algorithm. In some embodiments, the XTEN sequences of the fusion protein compositions have an alpha-helix percentage less than about 2% and a beta-sheet percentage less than about 2%. The XTEN ces of the fusion protein compositions have a high degree of random coil tage, as determined by the GOR algorithm. In some embodiments, an XTEN sequence have at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and most preferably at least about 99% random coil, as determined by the GOR algorithm. In some embodiments, the XTEN sequences of the fusion protein compositions have an alpha- heliX percentage ranging from 0% to less than about 5% and a beta-sheet percentage ranging from 0% to less than about 5% as determined by the Chou-Fasman algorithm and at least about 90% random coil, as determined by the GOR algorithm. In other embodiments, the XTEN sequences of the fiJsion protein compositions have an alpha-helix percentage less than about 2% and a beta-sheet percentage less than about 2% at least about 90% random coil, as determined by the GOR thm. 1. Non-repetitive Sequences It is plated that the XTEN sequences of the CFXTEN embodiments are ntially non-repetitive. In general, repetitive amino acid sequences have a tendency to aggregate or form higher order structures, as ified by natural repetitive sequences such as collagens and leucine zippers.

These repetitive amino acids may also tend to form contacts resulting in crystalline or pseudocrystaline structures. In contrast, the low tendency of non-repetitive sequences to aggregate s the design of long-sequence XTENs with a relatively low frequency of charged amino acids that would otherwise be likely to aggregate if the sequences were repetitive. The non-repetitiveness of a subject XTEN can be observed by assessing one or more of the following features. In one embodiment, a “substantially non- repetitive” XTEN sequence has about 36, or at least 72, or at least 96, or at least 144, or at least 288, or at least 400, or at least 500, or at least 600, or at least 700, or at least 800, or at least 864, or at least 900, or at least 1000, or at least 2000, to about 3000 or more amino acid residues, or has a length ranging from about 36 to about 3000, about 100 to about 500, about 500 to about 1000, about 1000 to about 3000 amino acids and residues, in which no three uous amino acids in the sequence are identical amino acid types unless the amino acid is serine, in which case no more than three contiguous amino acids are serine residues. In r embodiment, as described more fully below, a “substantially non-repetitive” XTEN sequence comprises motifs of 9 to 14 amino acid residues n the motifs consist of 4 to 6 types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the sequence of any two contiguous amino acid residues in any one motif is not repeated more than twice in the sequence motif.

The degree of repetitiveness of a polypeptide or a gene can be ed by computer programs or algorithms or by other means known in the art. According to the current invention, thms to be used in ating the degree of repetitiveness of a particular polypeptide, such as an XTEN, are disclosed herein, and examples of ces analyzed by algorithms are provided (see Examples, .

In one aspect, the repetitiveness of a polypeptide of a predetermined length can be calculated (hereinafter “subsequence score”) according to the formula given by Equation 1: Subsequence score Em}. C $33.31: g‘ , . I wherein: m = (amino acid length of polypeptide) — (amino acid length of uence) + 1; and Count,- = cumulative number of occurrences of each unique subsequence within sequence,- ] An algorithm termed “SegScore” was developed to apply the foregoing equation to quantitate repetitiveness of polypeptides, such as an XTEN, providing the subsequence score wherein ces of a predetermined amino acid length “n” are analyzed for repetitiveness by determining the number of times (a ”) a unique subsequence of length “s” appears in the set length, divided by the absolute number of subsequences within the predetermined length of the ce. depicts a logic flowchart of the SegScore algorithm, while ys a schematic of how a subsequence score is derived for a fictitious XTEN with 11 amino acids and a subsequence length of 3 amino acid residues.

For example, a predetermined polypeptide length of 200 amino acid residues has 192 overlapping 9- amino acid subsequences and 198 3-mer subsequences, but the subsequence score of any given polypeptide will depend on the absolute number of unique subsequences and how frequently each unique subsequence (meaning a different amino acid sequence) appears in the predetermined length of the sequence.

In the context of the present invention, “subsequence score” means the sum of occurrences of each unique 3-mer frame across 200 consecutive amino acids of the cumulative XTEN polypeptide divided by the absolute number of unique 3-mer subsequences within the 200 amino acid sequence.

Examples of such uence scores derived from 200 consecutive amino acids of tive and etitive polypeptides are presented in Example 45. In one embodiment, the invention provides a CFXTEN comprising one XTEN in which the XTEN has a subsequence score less than 12, more ably less than 10, more ably less than 9, more preferably less than 8, more preferably less than 7, more preferably less than 6, and most preferably less than 5. In another embodiment, the invention provides CFXTEN comprising at least two to about six XTEN in which 200 amino acids of the XTEN have a subsequence score of less than 10, more preferably less than 9, more preferably less than 8, more preferably less than 7, more preferably less than 6, and most ably less than 5. In the embodiments of the CFXTEN fusion protein compositions described herein, an XTEN component of a fusion protein with a subsequence score of 10 or less (i.e., 9, 8, 7, etc.) is also substantially non- repetitive.

It is believed that the non-repetitive characteristic ofXTEN of the present invention together with the particular types of amino acids that predominate in the XTEN, rather than the absolute primary sequence, s many of the enhanced physicochemical and biological properties of the CFXTEN fusion proteins. These enhanced properties include a higher degree of expression of the fusion protein in the host cell, greater genetic stability of the gene encoding XTEN, a greater degree of solubility, less tendency to aggregate, and enhanced pharmacokinetics of the resulting CFXTEN compared to fusion proteins comprising polypeptides having repetitive ces. These enhanced ties permit more efficient manufacturing, lower cost of goods, and facilitate the formulation of XTEN-comprising pharmaceutical ations containing extremely high protein concentrations, in some cases ing 100 mg/ml. Furthermore, the XTEN polypeptide sequences of the embodiments are designed to have a low degree of internal repetitiveness in order to reduce or substantially eliminate immunogenicity when administered to a mammal. Polypeptide sequences composed of short, repeated motifs largely limited to only three amino acids, such as glycine, serine and ate, may result in relatively high antibody titers when administered to a mammal despite the absence of predicted T-cell epitopes in these sequences.

This may be caused by the repetitive nature of polypeptides, as it has been shown that immunogens with repeated epitopes, ing protein ates, cross-linked immunogens, and tive carbohydrates are highly immunogenic and can, for example, result in the cross-linking of B-cell ors causing B- cell tion. (Johansson, J., et al. (2007) Vaccine, 25 :1676-82 ; Yankai, Z., et al. (2006) Biochem Biophys Res Commun, 345 :1365-71 ; Hsu, C. T., et al. (2000) Cancer Res, 60:3701-5); Bachmann MF, et al. Eur J Immunol. (1995) 25(12):3445-3451). 2. Exemplary Sequence Motifs The present invention encompasses XTEN used as fusion partners that se multiple units of shorter sequences, or motifs, in which the amino acid sequences of the motifs are non-repetitive. The non-repetitive property is met despite the use of a “building block” approach using a library of sequence motifs that are multimerized to create the XTEN sequences. Thus, while an XTEN ce may consist of multiple units of as few as four different types of sequence motifs, because the motifs themselves generally consist of non-repetitive amino acid sequences, the overall XTEN ce is ed to render the sequence substantially non-repetitive.

In one embodiment, an XTEN has a substantially non-repetitive sequence of greater than about 36 to about 3000, or about 100 to about 2000, or about 144 to about 1000 amino acid residues, or even longer wherein at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or about 100% of the XTEN sequence consists of non-overlapping ce motifs, and wherein each of the motifs has about 9 to 36 amino acid residues. In other embodiments, at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or about 100% of the XTEN sequence consists of non-overlapping sequence motifs wherein each of the motifs has 9 to 14 amino acid residues. In still other embodiments, at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or about 100% of the XTEN sequence consists of non-overlapping sequence motifs wherein each of the motifs has 12 amino acid residues. In these embodiments, it is preferred that the sequence motifs are composed of substantially (e. g., 90% or more) or exclusively small hydrophilic amino acids, such that the overall sequence has an unstructured, flexible characteristic. Examples of amino acids that are included in XTEN are, e. g., arginine, , threonine, alanine, asparagine, glutamine, aspartate, glutamate, serine, and glycine. As a result of testing variables such as codon optimization, assembly polynucleotides encoding sequence motifs, sion of protein, charge bution and solubility of expressed protein, and secondary and ry structure, it was discovered that XTEN compositions with the enhanced characteristics disclosed herein mainly or exclusively e glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues wherein the sequences are designed to be substantially non-repetitive. In one embodiment, XTEN sequences have predominately four to six types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), ate (E) or proline (P) that are ed in a substantially non-repetitive sequence that is r than about 36 to about 3000, or about 100 to about 2000, or about 144 to about 1000 amino acid residues in length. In some embodiment, an XTEN sequence is made of 4, 5, or 6 types of amino acids selected from the group consisting of glycine (G), e (A), serine (S), threonine (T), glutamate (E) or proline (P). In some embodiments, XTEN have sequences of greater than about 36 to about 1000, or about 100 to about 2000, or about 400 to about 3000 amino acid residues wherein at least about 80% of the sequence consists of non-overlapping sequence motifs wherein each of the motifs has 9 to 36 amino acid residues and wherein at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or 100% of each of the motifs consists of 4 to 6 types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the content of any one amino acid type in the full-length XTEN does not exceed 30%. In other embodiments, at least about 90% of the XTEN sequence consists of non-overlapping sequence motifs wherein each of the motifs has 9 to 36 amino acid residues n the motifs consist of 4 to 6 types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the content of any one amino acid type in the full-length XTEN does not exceed 40%, or about 30%, or 25%, or about 17%. In other embodiments, at least about 90% of the XTEN sequence consists of non-overlapping sequence motifs wherein each of the motifs has 12 amino acid residues consisting of 4 to 6 types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the content of any one amino acid type in the full-length XTEN does not exceed 40%, or 30%, or about 25%. In yet other embodiments, at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% of the XTEN sequence consists of non-overlapping sequence motifs wherein each of the motifs has 12 amino acid residues consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P).

In still other embodiments, XTENs comprise ntially non-repetitive sequences of r than about 36 to about 3000 amino acid residues n at least about 80%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of the sequence consists of non-overlapping sequence motifs of 9 to 14 amino acid residues wherein the motifs consist of 4 to 6 types of amino acids selected from e (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the sequence of any two contiguous amino acid residues in any one motif is not repeated more than twice in the sequence motif.

In other embodiments, at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of an XTEN sequence consists of non-overlapping sequence motifs of 12 amino acid residues wherein the motifs t of four to six types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and e (P), and wherein the sequence of any two contiguous amino acid residues in any one sequence motif is not repeated more than twice in the sequence motif. In other embodiments, at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of an XTEN sequence consists of non-overlapping sequence motifs of 12 amino acid residues n the motifs consist of e (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the sequence of any two uous amino acid residues in any one sequence motif is not repeated more than twice in the sequence motif In yet other ments, XTENs consist of 12 amino acid sequence motifs wherein the amino acids are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the sequence of any two contiguous amino acid residues in any one sequence motif is not repeated more than twice in the sequence motif, and wherein the content of any one amino acid type in the full-length XTEN does not exceed 30%. The foregoing embodiments are examples of substantially non-repetitive XTEN sequences. onal examples are ed below.

In some embodiments, the invention provides CFXTEN compositions comprising one, or two, or three, or four, five, six or more non-repetitive XTEN sequence(s) of about 36 to about 1000 amino acid residues, or cumulatively about 100 to about 3000 amino acid residues wherein at least about 80%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% to about 100% of the sequence consists of multiple units of four or more non-overlapping ce motifs selected from the amino acid ces of Table 3, n the overall sequence remains substantially non-repetitive. In some embodiments, the XTEN comprises non-overlapping sequence motifs in which about 80%, or at least about 85%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% or about 100% of the sequence ts of multiple units of non- overlapping sequences selected from a single motif family selected from Table 3, resulting in a family sequence. As used herein, “family” means that the XTEN has motifs selected only from a single motif category from Table 3; i.e., AD, AE, AF, AG, AM, AQ, BC, or BD XTEN, and that any other amino acids in the XTEN not from a family motif are selected to achieve a needed property, such as to permit incorporation of a restriction site by the encoding nucleotides, incorporation of a cleavage sequence, or to achieve a better linkage to a FVIII ation factor component of the CFXTEN. In some embodiments ofXTEN families, an XTEN sequence comprises multiple units of non-overlapping ce motifs of the AD motif family, or of the AE motif , or of the AF motif family, or of the AG motif family, or of the AM motif family, or of the AQ motif family, or of the BC family, or of the BD family, with the resulting XTEN ting the range of homology described above. In other embodiments, the XTEN comprises multiple units of motif sequences from two or more of the motif families of Table 3. These sequences can be selected to achieve desired physical/chemical characteristics, including such properties as net charge, hydrophilicity, lack of secondary structure, or lack of repetitiveness that are conferred by the amino acid composition of the , described more fully below. In the embodiments above described in this paragraph, the motifs orated into the XTEN can be selected and assembled using the methods described herein to achieve an XTEN of about 36 to about 3000 amino acid residues.

Table 3: XTEN Seguence Motifs of 12 Amino Acids and Motif Families AD GESPGGSSGSES 19 AD GSEGSSGPGESS 20 AD GSSESGSSEGGP 21 AD GSGGEPSESGSS 22 AE, AM GSPAGSPTSTEE 23 AE, AM, AQ GSEPATSGSETP 24 AE, AM, AQ GTSESATPESGP 25 AE, AM, AQ GTSTEPSEGSAP 26 AF, AM GSTSESPSGTAP 27 AF, AM GTSTPESGSASP 28 AF, AM GTSPSGESSTAP 29 AF, AM GSTSSTAESPGP 30 AG, AM GTPGSGTASSSP 31 AG, AM GSSTPSGATGSP 32 AG, AM GSSPSASTGTGP 33 AG, AM GASPGTSSTGSP 34 AQ GEPAGSPTSTSE 35 AQ GTGEPSSTPASE 36 AQ GSGPSTESAPTE 37 AQ GPSETA 38 AQ GPSETSTSEPGA 39 AQ GSPSEPTEGTSA 40 BC GSGASEPTSTEP 41 BC GSEPATSGTEPS 42 BC GTSEPSTSEPGA 43 BC GTSTEPSEPGSA 44 BD GSTAGSETSTEA 45 BD GSETATSGSETA 46 BD GTSESATSESGA 47 BD GTSTEASEGSAS 48 permutations, results in a “family ce” In some embodiments ofXTEN families, an XTEN sequence comprises multiple units of non- overlapping sequence motifs of the AD motif family, the AE motif family, or the AF motif family, or the AG motif family, or the AM motif family, or the AQ motif family, or the BC family, or the BD family, with the resulting XTEN exhibiting the range of homology bed above. In other embodiments, the XTEN comprises le units of motif sequences from two or more of the motif families of Table 3, selected to achieve desired physicochemical characteristics, including such properties as net charge, lack of secondary structure, or lack of repetitiveness that may be red by the amino acid composition of the motifs, described more fully below. In the embodiments hereinabove described in this paragraph, the motifs or ns of the motifs incorporated into the XTEN can be selected and assembled using the methods bed herein to achieve an XTEN of about 36, about 42, about 72, about 144, about 288, about 576, about 864, about 1000, about 2000 to about 3000 amino acid residues, or any intermediate length. Non-limiting examples ofXTEN family sequences useful for incorporation into the subject CFXTEN are presented in Table 4. It is intended that a specified sequence mentioned relative to Table 4 has that sequence set forth in Table 4, While a generalized reference to an AEl44 sequence, for example, is intended to encompass any AE sequence having 144 amino acid residues; e. g., AEl44_1A, AEl44_2A, etc., or a generalized reference to an AG144 sequence, for example, is intended to encompass any AG sequence having 144 amino acid residues, e. g., AG144_1, AG144_2, AG144_A, AG144_B, AG144_C, etc.

Table 4: XTEN Polypeptides XTEN Amino Acid Sequence IDQ Name AE42 GAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPASS 49 AE42_1 TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGS 50 AE42_2 PAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSG 51 AE42_3 SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSP 52 AG42_1 GAPSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGPSGP 53 AG42_2 SGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASP 54 AG42_3 SPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGA 55 AG42_4 SASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG 56 AE48 SPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGS 57 AM48 SPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGS 58 AE 1 44 GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGS 59 EPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEP ATSGSETPGTSTEPSEGSAP AEl44_ SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS 60 1A SAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSES ATPESGPGTSTEPSEGSAPG AEl44_ TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS 61 2A TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSES ATPESGPGTSESATPESGPG AEl44_ TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS 62 2B TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSES ATPESGPGTSESATPESGPG AEl44_ SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTS 63 3A TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAG SPTSTEEGTSTEPSEGSAPG AE 1 44_ SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTS 64 3B TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAG SPTSTEEGTSTEPSEGSAPG AEl44_ TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS 65 4A TEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSES ATPESGPGTSTEPSEGSAPG AEl44_ TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS 66 4B SAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSES ATPESGPGTSTEPSEGSAPG XTEN Amino Acid Sequence ID Name AE144_ TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS 67 5A SAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAG EGSPAGSPTSTEEG AE144_ TSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSE 68 6B PATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSES ATPESGPGTSTEPSEGSAPG AF144 GTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSGTAPGS 69 TSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSPS GESSTAPGTSPSGESSTAP AG144_ SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSA 70 STGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSAST GTGPGSSPSASTGTGPGASP AG144_ PGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGP 71 GASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPG ASPGTSSTGSPGTPGSGTASSS AG144_ SSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPG 72 SSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGA SPGTSSTGSPGASPGTSSTGSP AG144_ GTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG 73 SSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGA SPGTSSTGSPGASPGTSSTGSP AG144_ TASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPG 74 TPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSS TPSGATGSPGASPGTSSTGSP AG144_ GSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPG 75 SSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSS TPSGATGSPGASPGTSSTGSP AG144_ GTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPG 76 ASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGA SPGTSSTGSPGASPGTSSTGSP AG144_ GTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPG 77 ASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTP GSGTASSSPGSSTPSGATGSP AE288_ GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT 78 STEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPA GSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESA TPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE GSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP AE288_ GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGT 79 STEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPA GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPAT SGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPT STEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP AG288_ PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSP 80 GSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPG SSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSS PSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASP GTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGS AG288_ GSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPG 81 STGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGA SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSST PSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPG TSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSP AF504 SSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPG 82 SXPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGA SPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPG SGTASSSPGSSTPSGATGSPGSXPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPS GATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTS STGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSST XTEN Amino Acid Sequence ID Name GSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTG SPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGP GASPGTSSTGSP AF54O GSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGSTSSTAESPGPGT 83 STPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPS GESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPS GTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESP GPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASP SGSASPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSSTAESPGPGT STPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSE PGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGSTSSTAESPGPGTSPSGE SSTAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAP AD576 GSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPG 84 SEGGPGSSESGSSEGGPGESPGGSSGSESGSEGSSGPGESSGSSESGSSEGGPGSS ESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSGG EPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGSEGSSGPGESSGESPGG GSGGEPSESGSSGSGGEPSESGSSGSGGEPSESGSSGSSESGSSEGGPGESPGGSS GSESGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGSSESGSSEG GPGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGG PGSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSGGEPSESGSS GSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGESPGGSSGSESGSEGSSGPGESSG SSESGSSEGGPGSEGSSGPGESS AE576 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGT 85 STEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSE SATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP SEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSE GSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPES GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGT SESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST EPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESA TPESGPGTSTEPSEGSAP AF576 GSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGSTSSTAESPGPGT 86 STPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPS GESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPS GTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESP GPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASP GTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSSTAESPGPGT STPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSE SPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGSTSSTAESPGPGTSPSGE STSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGSTSSTAESPGPGTSTPESGS ASPGTSTPESGSASP AGS76 PGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSP 87 GSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPG ASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGA SPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASP GTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSA STGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTS STGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTA SSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGT GPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTG PGSSPSASTGTGPGASPGTSSTGS AE624 MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTSTEE 88 GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGT SESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTST EPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESA TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSE GSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE TPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP XTEN Amino Acid Sequence ID Name GTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGS EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSE SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP SEGSAP AD836 SSEGGPGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGESPGGSSGSESG 89 ESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGES PGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSES GSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGG SSGSESGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGSEGSSGP GESSGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGSGGEPSESGSSGSGGEPSES GSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGS ESGSEGSSGPGESSGSEGSSGPGESSGSGGEPSESGSSGSSESGSSEGGPGSSESGSSEGGP GESPGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSEGSSGPGSSES GSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSEGSSGPGESSGSEGSSGPGESSGSGGEP GSGGEPSESGSSGESPGGSSGSESGESPGGSSGSESGSGGEPSESGSSGSEGSSGP GESSGESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSES GSSGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGS ESGSGGEPSESGSSGESPGGSSGSESGSGGEPSESGSS AE864 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGT 90 STEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSE SATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP SEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSE GSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPES GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGT SESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST EPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESA TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG SETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE TPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGP GTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGT STEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP AF864 PSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGT 91 STPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGTSPS PGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGSTSSTA ESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGS ASPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPG PGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPGPGSTSSTAESPGPG ESPGPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGPXXXGAS ASGAPSTXXXXSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSES PSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAE SPGPGTSPSGESSTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESST APGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASP GSTSSTAESPGPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGT SPSGESSTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGSTSS TAESPGPGSTSSTAESPGPGTSPSGESSTAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSG ATGSP AG864_ GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPG 92 SSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGA SPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPG SGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPS GATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTS STGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSST GSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTG SPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGP GASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSS TPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPS ASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSA XTEN Amino Acid Sequence ID Name STGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTS STGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSST AM875 GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGS 93 TSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSE SATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEP GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATP ESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSE TPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAP SEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGA SASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSE SPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATS GSETPGTSESATPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESS TAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSAS PGSTSESPSGTAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPG SSPSASTGTGPGASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSS TPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTE PSEGSAP AE912 MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTSTEE 94 TPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGT ESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTST EPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESA TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSE GSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE TPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP GTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGS EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSE SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATP ESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGT STEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP AM923 MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTSTEPSEGSAP 95 GSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGS TSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSESATPESGPGSPA GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGS PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSE GSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTST EEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAP GSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGT SESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSP SGESSTAPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESA TPESGPGSEPATSGSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSG SETPGSEPATSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGT APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGP GASPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGS SPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP AM1318 GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGS 96 TSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSE GPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEP SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATP ESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSE TPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTSTEPSEGSAP GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGP EPTGPAPSGGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSPA GSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSST AESPGPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGES STAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSESATPES XTEN Amino Acid Sequence ID Name GPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSPSGESSTAP GTSPSGESSTAPGTSPSGESSTAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGS SPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGAS PGTSSTGSPGASASGAPSTGGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSESA GTSTEPSEGSAPGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGASPGTSS TGSPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSESATPESGPGSEPATSGSE TPGTSTEPSEGSAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSPAGSPTSTEE GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGS STPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGSTSESPSGTAPGTSPSGESSTAPGSTS GPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSPAGSPTSTEEGSPAG SPTSTEEGTSTEPSEGSAP BC 864 GTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGS 97 EPATSGTEPSGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGTST EPSEPGSAGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGSEPAT GSEPATSGTEPSGTSEPSTSEPGAGSGASEPTSTEPGTSEPSTSEPGAGSEPATSG EPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGSGASEPTSTEPGSEPATSGTE PSGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPS GSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGS GASEPTSTEPGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGSGASEPTSTEPGTST EPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGTSTEP SEPGSAGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSE STEPSEPGSAGTSTEPSEPGSAGTSEPSTSEPGAGSGASEPTSTEPGTSTEPSEPG SAGTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPS GSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGTSEPSTSEPGAGSEPATSGTEPSGS GASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSA BD864 GSETATSGSETAGTSESATSESGAGSTAGSETSTEAGTSESATSESGAGSETATSGSETA 98 GSETATSGSETAGTSTEASEGSASGTSTEASEGSASGTSESATSESGAGSETATSGSETA GTSTEASEGSASGSTAGSETSTEAGTSESATSESGAGTSESATSESGAGSETATSGSETA GTSESATSESGAGTSTEASEGSASGSETATSGSETAGSETATSGSETAGTSTEASEGSAS GSTAGSETSTEAGTSESATSESGAGTSTEASEGSASGSETATSGSETAGSTAGSETSTEA GSTAGSETSTEAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETA GTSESATSESGAGTSESATSESGAGSETATSGSETAGSETATSGSETAGTSTEASEGSAS GSTAGSETSTEAGSETATSGSETAGTSESATSESGAGSTAGSETSTEAGSTAGSETSTEA GSTAGSETSTEAGTSTEASEGSASGSTAGSETSTEAGSTAGSETSTEAGTSTEASEGSAS GSTAGSETSTEAGSETATSGSETAGTSTEASEGSASGTSESATSESGAGSETATSGSETA GTSESATSESGAGTSESATSESGAGSETATSGSETAGTSESATSESGAGSETATSGSETA GTSTEASEGSASGTSTEASEGSASGSTAGSETSTEAGSTAGSETSTEAGSETATSGSETA GTSESATSESGAGTSESATSESGAGSETATSGSETAGSETATSGSETAGSETATSGSETA GTSTEASEGSASGTSESATSESGAGSETATSGSETAGSETATSGSETAGTSESATSESGA GTSESATSESGAGSETATSGSETA AE948 GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGS 99 PAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSEP ATSGSETPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPAT GTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSEPATSG SETPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPES GPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSEPATSGSETP GTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGT SESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSE SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEP SEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPT STEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSE TPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEE GSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGT STEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSE SATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSESA TPESGPGTSESATPESGP AE1044 GSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGT 100 STEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSE SATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSTEP XTEN Amino Acid Sequence ID Name SEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP ESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGS ATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAP GTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGT SESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTST EPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGS GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP ESGPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPES GPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEE TPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGT STEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGSEP ATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP SEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT STEEGTSESATPESGPGTSESATPESGPGTST AE1140 GSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGS 101 EPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSE SATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGS PTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP ESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES GPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGS PAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPA GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEP SEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPT STEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSE SATPESGPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAP GTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGS STEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEP ATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEP SEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPT STEEGTSTEPSEGSAPGSPAGSPTSTEEGSPA AE1236 GSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGT 102 STEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEP ATSGSETPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGS PTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPT STEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE TPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP GSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGT SESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTST EPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESA TPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATP PAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSE TPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP GSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGT SESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTST EPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESA TPESGPGSEPATSGSETPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSG SETPGTSTEPSEGSAPGTSTEPSEGSAPGSEP AE1332 GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGT 103 STEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSPA GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGSEPAT SGSETPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSG SETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPES GPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAP GTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGS EPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTST EPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSPAGS PTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATP ESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSE XTEN Amino Acid Sequence ID Name TPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGS EPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTST APGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPAT SGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATP ESGPGTSESATPESGPGTSTEPSEGSAPGTST AE1428 GSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGT 104 STEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSPA GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPAT GTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSE GSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGS EPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETP GSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGS EPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSE SATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSEPAT SGSETPGTSESATPESGPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSPAGSPT STEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPES GPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETP GTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGT SESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSE SATPESGPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEP SEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSE GSAPGSPAGSPTSTEEGTSESATPESGPGSPA AE1524 GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGS 105 PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGTST EPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPAT SGSETPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPT STEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPES GPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEE GSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGS EPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEP ATSGSETPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSPAGS PTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATP ESGPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPTST EEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSESATPESGP GTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGT SESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSPA GSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESA TPESGPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGTSTEPSE GSAPGTSTEPSEGSAPGTSESATPESGPGSPA AE1620 GSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGT 106 SESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTST EPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSESA TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATP ESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPES GPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEE GTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGT STEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEP ATSGSETPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEP GSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSE GSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTST EEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGP GSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGS EPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSE SATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESA TPESGPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSE GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST AE1716 GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGS 107 PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSE XTEN Amino Acid ce ID Name SATPESGPGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSESA TPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSE GSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGS APGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEE GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGS PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSPAGSPTSTEEGTST EPSEGSAPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSEPAT SGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT STEEGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGTSESATPES GPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE GSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGS PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPA EEGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGTSTEP SEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSG SETPGSPAGSPTSTEEGTSESATPESGPGTSE AE1812 GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGT 108 STEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSE SATPESGPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESA TPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSE GSAPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS APGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETP GSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS PAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTST EPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESA TPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSTEPSE GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES GPGSEPATSGSETPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGP GTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGT SESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESA TPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATP PAGSPTSTEEGTSTEPSEGSAPGSEP AE1908 GSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGS 109 PAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGTST EPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGTSTEP SEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATP ESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS APGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSESATPESGP GTSESATPESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGT STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSE GPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTSTEP SEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGTSTEPSE GSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGS APGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETP GTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGS PAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTST EPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEP SEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATP ESGPGSPAGSPTSTEEGTSESATPESGPGSEP AE2004 GTSTEPSEGSAPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGS 110 A PAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTST EPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSTEP SEGSAPGTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGSEPATSG SETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPES GPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEE GTSESATPESGPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGS PAGSPTSTEEGTSESATPESGPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSE GPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEP SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGTSESATP XTEN Amino Acid Sequence ID Name ESGPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPES GPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEE TPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGS PAGSPTSTEEGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEP ATSGSETPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEP SEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSPAGSPT STEEGTSTEPSEGSAPGTSESATPESGPGTSE AG948 GSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPG 111 TPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGSS TPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASP SPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGASPG TSSTGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSG ATGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGASPGTSS TGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTAS SSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTG PGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGP GSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPG SSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSS PSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTP SGATGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSG TASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGA TGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTAS SSPGSSTPSGATGSPGSSTPSGATGSP AG1044 GTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPG 112 TPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGSS PSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGTPG SGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGASPG TSSTGSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTS STGSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGAT PGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTG SGTASSSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSP GASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPG TPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSS TPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGASP GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGSSTPS GATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSAS TGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSS TGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGAT GSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGT GPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSST AG1140 GASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPG 113 SSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSS TPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPS ASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPS GATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSTPSG ATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGA SPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSST GSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATG SPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGP GASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG TPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSS PSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPG SGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPS GSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSTPSG ATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSAST GTGPGTPGSGTASSSPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGAT GSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSST AG1236 GSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPG 114 ASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTP XTEN Amino Acid Sequence ID Name GSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPS ASTGTGPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSSPSA STGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSG ATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGA SPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSST GSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATG SPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSP GSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPG TGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGA SPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGSSP SASTGTGPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPG TSSTGSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSG TASSSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGT ASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTA SSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASP AG1332 GSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPG 115 SSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSS TPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTP SGATGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSA STGTGPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTS STGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGAT GSPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATG SPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSP GSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPG TPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSS TPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSTP SGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPS GATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTS STGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSST TPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATG SPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGP GSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPG AG1428 TASSSPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPG 116 TPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSS TPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASP GTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGS PGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSAS TGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTA SSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGT GPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTG PGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSP GASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPG SSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGA SPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGASP GTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGS GTASSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSG ATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSPSAST GTGPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGAT GSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGASP AG1524 GSSTPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPG 117 TPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGTP GSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGASP GTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGS GTASSSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSG ATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTA SSSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASS SPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGP GSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPG TPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSS XTEN Amino Acid Sequence ID Name TPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGSSPS PGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSG TASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGA TGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGAT GSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTG SPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSP GASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGTPG AG1620 GSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPG 118 ASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSS PSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSTP SGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGASPGT SSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAST GTGPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSST GSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGT GPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGS PGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSP GASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPG TPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSS TPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPS ASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSA STGTGPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGSSTPSG ATGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSPSAST GTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTAS SSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSST AG1716 GASPGTSSTGSPGSSPSASTGTGPGSSTPSGATGSPGSSPSASTGTGPGTPGSGTASSSPG 119 SSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSS PSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPG SPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSTPS GATGSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGSSPSASTGTGPGTPGSGT TPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSST GSPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASS SPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSPSASTGTGP GTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPG TPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGPGSSPSASTGTGPGTP GSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGASP GTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGS GTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSG ATGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSPSAST GTGPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSST GSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTG SPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPG AG1812 GSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPG 120 TGTGPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSS PSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSSTP SGATGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGTPGSG TASSSPGSSPSASTGTGPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGTPGSGT ASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGAT GSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATG SPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSP GASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPG ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGSS TPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPG SGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPG PGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGTPGSG TASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGA TGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGAT GSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTG SPGTPGSGTASSSPGASPGTSSTGSPGSSTPSGATGSPGASP AG1908 GSSPSASTGTGPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSPSASTGTGPG 121 XTEN Amino Acid Sequence ID Name SSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGTPGSGTASSSPGA SPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGSSP SASTGTGPGASPGTSSTGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGS GTASSSPGTPGSGTASSSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSAS TGTGPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAST GTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSST GSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASS SPGASPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGSSPSASTGTGPGSSPSASTGTGP GSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPG SSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSS TPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGTPG SGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPS GATGSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGSSPSAS TGTGPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSPSAST STPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGAT TPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSP AG2004 GSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPG 122 A SSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGA SPGTSSTGSPGSSTPSGATGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSST PSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPG TSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGSSTPSGATGSPGASPGTS STGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTA SSSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASS SPGASPGTSSTGSPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSP GASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSTPSGATGSPG SSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGSS PSASTGTGPGTPGSGTASSSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASP SPGTPGSGTASSSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGTPGS GTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGSSPSASTGTGPGSSTPSG ATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGATGSPGASPGTSS TGSPGTPGSGTASSSPGTPGSGTASSSPGSSPSASTGTGPGASPGTSSTGSPGSSTPSGAT GSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG SGTASSSPGSSPSASTGTGPGSSPSASTGTGPGASP AE72B SPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSE 123 PATSGSETPG AE72C TSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTS 124 TEPSEGSAPG AE108A TEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA 125 PGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTS AE108B GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGS 126 EPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAP AE144A STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE 127 GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGS PTSTEEGSPAGSPTSTEEGS AE144B SEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSP 128 AGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAG SPTSTEEGTSTEPSEGSAPG AE180A TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTS 129 TEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSET PGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS AE2 1 6A PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPE 130 SGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESG PGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPG TSTEPSEGSAPGSEPATSGSETPGTSESAT AE252A ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES 131 GPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAP GTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTST EPSE XTEN Amino Acid Sequence ID Name AEZ88A TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG 132 SETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE TPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGP GTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGT STEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESA AE324A PESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEG 133 SAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESG PGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG SPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTS ESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTE PGSEPATS AE360A PESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTS 134 TEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESG PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPG GSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTS ESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAG SPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT AE396A PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTS 135 TEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESG PGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSP AGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPA TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS AE432A EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPE 136 SGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTE EGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG SEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSP AGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAG EGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATS GSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS AE468A EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE 137 SGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPG TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS ESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESAT PESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEG SAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT AE504A EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTS 138 TEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSET PGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTS ESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPS EGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGS ETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESG PGTSTEPS AE540A TPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE 139 STEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS APGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGS PAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGS PTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPT STEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSE TPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP AE576A TPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP 140 ESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS APGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP XTEN Amino Acid Sequence ID Name GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS PAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSE SATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEP SEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPT STEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPES GPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP GSEPATSGSETPGTSESA AE612A GSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTS 141 TEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEG TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAG SPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESAT PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPE PATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSA PGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPG SPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT AE648A PESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEG 142 SAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSET PGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPG TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSE PATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSES ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPS EGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPE SGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESG PGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPG TSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTS TEPSEGSAPGSEPATSGSETPGTSESAT AE684A EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEG 143 SAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG PGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSP TSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPE PATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA SPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEG SPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSE PATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPA AE720A TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPS 144 EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEG SAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG PGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSP SPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPE SGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEG SPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSE PATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTE AE756A TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPS 145 EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEG SAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG PGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSP XTEN Amino Acid Sequence ID Name TSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPE PATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA SPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEG SPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSE PATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPA TSGSETPGTSES AE792A EGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPE 146 SGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA PGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPG TSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSE PATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTE PSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESAT PESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEG SAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESG PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPG TSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTS ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSES ATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPS EGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS AE828A PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPE 147 SGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSA PGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTS ESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGS ETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESG PGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAG EGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESAT PESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEG SAPGSEPATSGSETPGTSESAT AG72A GPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGS 148 GTASS AG72B GATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPG 149 ASSSP AG72C SPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSST 150 PSGATGSPGA AG108A SASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPG 151 TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASP AG108B PGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSP 152 GSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSS AG144A PGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGP 153 GASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPG ASPGTSSTGSPGTPGSGTASSS AG144B PSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPS 154 ASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSA STGTGPGASPGTSSTGSPGASP AG180A TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS 155 TGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSS TGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGS AGZ 1 6A TGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSS 156 TGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSST GSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG SPGSSPSASTGTGPGSSPSASTGTGPGSSTPSG AG252A TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS 157 TGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSS TGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTAS XTEN Amino Acid Sequence ID Name SSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGS PGASPG AGZ88A TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS 158 TGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSS TGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTAS SSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGS PGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGS AG324A TSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS 159 STGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTA SSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTG SPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSP GASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPG TPGSGTASSSPGSSTP AG360A TSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS 160 STGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGAT GSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGT GPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTG TSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSP GSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPG AG396A GSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGT 161 ASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTA SSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATG SPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSP GSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPG SSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSS TPSGATGSPGSSTPSGATGSPGASPGT AG432A GATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSG 162 ATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTA SPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASS SPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSP GSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPG ATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSS TPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTP AG468A TSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS 163 STGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT TPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTG SPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSP GTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPG SSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTP GSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASP GTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPG AG504A TSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS 164 STGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT GSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTG SPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSP GTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPG SSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTP GSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASP GTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGS GTASSSPGSSTP AG540A TSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTS 165 STGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTA SSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTG SPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSP GASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPG TPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGA SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASP GTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSA XTEN Amino Acid Sequence ID Name STGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPG AG576A TSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSAS 166 TGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGA TGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGAT GSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASS SPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSP GATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPG SSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSS TPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTP SGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPS GATGSPGSSTPSGATGSPGASPG AG612A STGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT 167 GSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTG GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSP GASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG ASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSS TPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASP GTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPG TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSG TASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSS TGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTS AG648A GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSG 168 ATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSS TGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSST GSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTG SPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGP GASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSS TPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPS ASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSA STGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTS STGSPGSSPSASTGTGPGTPGSGTASSSPGSSTP AG684A TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSG 169 ATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTA SSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASS SPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSP GASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPG SSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGA SPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPG SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSA STGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSAST GTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTG TGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATG SPGASPG AG720A TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSG 170 SSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSS SPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSST GSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTG GTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSP GSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPG ASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGA SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPG SGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPG PGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTS STGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTG TGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPG AG756A TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS 171 TGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSS TGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTAS XTEN Amino Acid Sequence ID Name SSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGS PGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSP GASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPG ASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTP GSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASP SPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPG TSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSG ATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAST GTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG TGPGASPGTSSTGSPGASPG AG792A TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS 172 TGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSS TGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTAS SSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGS PGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSP GASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPG ASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTP GSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASP GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPG TSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSG ATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAST GTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG TGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPG AG828A TSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS 173 TGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSS TGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTAS SSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGS PGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSP GASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPG ASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTP GSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASP GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPG TSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSG ATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAST GTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG TGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTG SPGSSPSASTGTGPGTPGSGTASSSPGSSTP AG288_ GTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPG 1699 DE ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGA SPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSST PSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPS PGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP In other embodiments, the CFXTEN composition comprises one or more non-repetitive XTEN sequences of lengths ranging from about 36 to about 3000 amino acid es, wherein at least about 80%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% to about 100% of the sequence consists of non- overlapping 36 amino acid ce motifs selected from one or more of the polypeptide sequences of Tables 13-17, either as a family sequence, or where motifs are selected from two or more es of motifs.

In those embodiments wherein the XTEN ent of the CFXTEN fusion n has less than 100% of its amino acids consisting of 4, 5, or 6 types of amino acid selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), or less than 100% of the sequence consisting of the sequence motifs from Table 3 or the XTEN sequences of Tables 4, and 13-17, the other amino acid residues of the XTEN are selected from any of the other 14 natural L-amino acids, but are preferentially selected from hydrophilic amino acids such that the XTEN sequence contains at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% hydrophilic amino acids. The XTEN amino acids that are not glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) are either interspersed throughout the XTEN sequence, are d within or between the sequence motifs, or are concentrated in one or more short stretches of the XTEN sequence, e. g., to create a linker between the XTEN and the FVIII components. In such cases where the XTEN component of the CFXTEN ses amino acids other than glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), it is preferred that less than about 2% or less than about 1% of the amino acids be hydrophobic residues such that the resulting sequences generally lack secondary structure, e. g., not having more than 2% alpha helices or 2% beta-sheets, as determined by the methods disclosed herein.

Hydrophobic residues that are less favored in construction ofXTEN include tryptophan, alanine, tyrosine, leucine, isoleucine, valine, and methionine. Additionally, one can design the XTEN sequences to contain less than 5% or less than 4% or less than 3% or less than 2% or less than 1% or none of the following amino acids: cysteine (to avoid disulfide formation and ion), methionine (to avoid oxidation), asparagine and glutamine (to avoid desamidation). Thus, in some embodiments, the XTEN component of the CFXTEN fusion n comprising other amino acids in addition to glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) have a ce with less than 5% of the residues buting to helices and heets as measured by the Chou-Fasman algorithm and have at least 90%, or at least about 95% or more random coil formation as ed by the GOR algorithm. 3. Length of Sequence In r , the invention provides XTEN of varying lengths for incorporation into CFXTEN compositions wherein the length of the XTEN sequence(s) are chosen based on the property or function to be achieved in the fusion protein. Depending on the intended property or function, the CFXTEN compositions comprise short or intermediate length XTEN located internal to the FVIH sequence or between FVHI domains and/or longer XTEN sequences that can serve as carriers, located in the fusion proteins as described herein. While not intended to be limiting, the XTEN or fragments of XTEN include short segments of about 6 to about 99 amino acid residues, intermediate lengths of about 100 to about 399 amino acid residues, and longer s of about 400 to about 1000 and up to about 3000 amino acid residues. Thus, the XTEN for incorporation into the subject CFXTEN encompass XTEN or fragments ofXTEN with s of about 6, or about 12, or about 36, or about 40, or about 42, or about 72 or about 96, or about 144, or about 288, or about 400, or about 500, or about 576, or about 600, or about 700, or about 800, or about 864, or about 900, or about 1000, or about 1500, or about 2000, or about 2500, or up to about 3000 amino acid es in length. Alternatively, the XTEN sequences can be about 6 to about 50, about 50 to about 100, about 100 to 150, about 150 to 250, about 250 to 400, about 400 to about 500, about 500 to about 900, about 900 to 1500, about 1500 to 2000, or about 2000 to about 3000 amino acid residues in . The e length of an XTEN incorporated into the subject CFXTEN can vary without adversely affecting the activity of a CFXTEN composition. In one embodiment, one or more of the XTEN used in the CFXTEN disclosed herein has 36 amino acids, 42 amino acids, 144 amino acids, 288 amino acids, 576 amino acids, or 864 amino acids in length and may be selected from one of the XTEN family sequences; i.e., AD, AE, AF, AG, AM, AQ, BC or BD. In another embodiment, two or more of the XTEN used in the CFXTEN sed herein has 36 amino acids, 42 amino acids, 144 amino acids, 288 amino acids, 576 amino acids, or 864 amino acids in length and may be selected from two of the XTEN family sequences; i.e., AD, AE, AF, AG, AM, AQ, BC or BD, with combinations of AE and AG family sequences preferred. In some embodiments, CFXTEN comprising one or more of the XTEN used herein n XTEN selected from any one of the sequences in Table 4, which may be linked to the FVIII component directly or Via spacer sequences disclosed herein.

] In particular CFXTEN configuration designs, where the XTEN serve as a e linker, or are inserted in external loops or unordered regions of the FVIII sequence to increase the bulk, flexibility, or hydrophilicity of the region, or are designed to interfere with clearance receptors for FVIII to enhance pharmacokinetic properties, or to interfere with binding of FVIII inhibitors or other anti-FVIII antibodies, or where a short or ediate length ofXTEN is used to facilitate tissue penetration or to vary the strength of interactions of the CFXTEN fusion protein with its target, or where it is desirable to bute the cumulative length ofXTEN in ts of short or intermediate length at multiple locations within the FVIII sequence, the invention contemplates CFXTEN compositions with one, two, three, four, five or more short or intermediate XTEN sequences inserted between or within one or more FVIII domains or within external loops, or at other sites in the FVIII sequence such as, but not limited to, locations at or al to the insertion sites identified in Table 5, Table 6, Table 7, Table 8, and Table 9 or as illustrated in FIGS. 8-9. In one ment of the foregoing, the CFXTEN fusion protein contains multiple XTEN segments, e. g., at least two, or at least three, or at least four, or at least five or more XTEN segments in which the XTEN segments can be cal or they can be different and wherein the CFXTEN retains at least 10%, 20%, 30%, 40%, 50%, 60%, 70% or more of the procoagulant activity of native FVIII when assayed by one of the assays disclosed herein. In other particular CFXTEN configuration s, where the XTEN serves as a carrier to increase the bulk of the fusion protein, or to vary the strength of interactions of the CFXTEN fusion protein with its target, or to enhance the pharmacokinetic properties of the fusion protein, the invention contemplates CFXTEN compositions with one or more intermediate or longer length XTEN sequences inserted at the C-terminus, within the B domain (or the al of the BDD sequence) between or within one or more FVIII domains, within external loops, or at other sites in the FVIII sequence such as, but not limited to, insertion sites identified in Table 5, Table 6, Table 7, Table 8, and Table 9 or as illustrated in FIGS. 8-9. However, it is believed that the incorporation of le XTEN of short to intermediate lengths into CFXTEN compositions confers enhanced properties on the fusion proteins compared to CFXTEN fusion proteins with the same number of amino acids in fewer but longer length XTEN, yet still results in compositions with procoagulant activity and extended half-life; the rationale of which is detailed herein regarding the derived radii of multiple XTEN.

In the embodiments wherein the CFXTEN fusion proteins comprise multiple XTEN sequences, the cumulative length of the total residues in the XTEN sequences is greater than about 100 to about 3000, or about 200 to about 2000, or about 400 to about 1000 amino acid residues and the XTEN can be identical or they can be different in ce, net charge, or in length. In one embodiment of CFXTEN comprising multiple XTEN, the individual XTEN sequences each exhibit at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% ce identity compared to a motif or an XTEN selected from Tables 3, 4, and 13-17 or a fragment thereof, when optimally aligned with a sequence of comparable length.

As described more fully below, methods are disclosed in which the CFXTEN are designed by selecting the length of the XTEN and its site of incorporation within the CFXTEN to confer a target half- life, retention of procoagulant actiVity, reduced binding to FVIII inhibitors or an enhanced physicochemical property (e.g., stability or solubility) of a CFXTEN fusion protein, encoding constructs are created and expressed and the recombinant CFXTEN fusion proteins are isolated and recovered. In general, XTEN cumulative s longer that about 400 residues incorporated into the CFXTEN compositions result in longer half-life compared to r cumulative lengths, e.g., r than about 280 residues. In one embodiment, CFXTEN fusion proteins designs are contemplated that comprise at least a single XTEN as a carrier, with a long sequence length of at least about 400, or at least about 600, or at least about 800, or at least about 900, or at least about 1000 or more amino acids. In r embodiment, multiple XTEN are incorporated into the fusion protein to achieve cumulative lengths of at least about 400, or at least about 600, or at least about 800, or at least about 900, or at least about 1000 or more amino acids, wherein the XTEN can be identical or they can be different in ce or length. As used herein, “cumulative length” is intended to encompass the total length, in amino acid es, when more than one XTEN is incorporated into the CFXTEN fusion n. Both of the foregoing embodiments are designed to confer increased ilability and/or increased terminal ife after administration to a t compared to CFXTEN comprising r cumulative XTEN lengths, yet still result in a procoagulant activity and hemostasis . When administered subcutaneously or intramuscularly, the Cmax is reduced but the area under the curve (AUC) is increased in comparison to a comparable dose of a CFXTEN with shorter cumulative length XTEN or FVIII not linked to XTEN, thereby buting to the ability to maintain effective levels of the CFXTEN composition for a longer period of time and permitting increased periods of 2, 4, 7, 10, 14 or 21 days between dosing, as described more fully below. Thus, the XTEN confers the property of a depot to the administered CFXTEN, in addition to the other physicochemical properties bed herein.

When XTEN are used as a carrier, the invention takes advantage of the ery that increasing the length of the non-repetitive, unstructured polypeptides enhances the ctured nature of the XTENs and correspondingly enhances the physical/chemical and pharmacokinetic properties of fusion proteins comprising the XTEN carrier. As described more fully in the Examples, tional increases in the length of the XTEN, even if created by a repeated order of single family sequence motifs (e. g., the four AE motifs of Table 3), result in a sequence With a higher percentage (e. g., 90% or more) of random coil formation, as determined by GOR algorithm, or reduced content of helices or beta- sheets (e. g., less than 2%), as determined by Chou-Fasman thm, ed to shorter XTEN lengths. In addition, increasing the length of the unstructured polypeptide fusion partner, as described in the Examples, results in a fusion protein With a disproportionate increase in terminal half-life (e.g., as much as 50, 100, 200 or more hours) compared to fusion proteins With unstructured polypeptide partners With r sequence lengths. The enhanced pharmacokinetic properties of the CFXTEN in ison to FVIII not linked to XTEN are described more fully, below.

In another , the invention provides s to create XTEN of short or intermediate lengths from longer ” XTEN sequences, Wherein the longer donor XTEN sequence is truncated at the inus, or the inus, or a fragment is created from the interior of a donor sequence, thereby resulting in a short or intermediate length XTEN. In non-limiting examples, as schematically depicted in A—C, an AG sequence of 864 amino acid residues can be truncated to yield an AG sequence With 144 residues, an AG sequence With 288 residues, an AG sequence With 576 residues, or other intermediate lengths, While the AE sequence of 864 residues (as depicted in D, E) can be ted to yield multiple AE sequences of 144 residues, an AE sequence With 288 or 576 residues or other r or intermediate lengths. It is specifically contemplated that such an approach can be ed with any of the XTEN embodiments described herein or with any of the sequences listed in Tables 4 or 13-17 to result in XTEN of a desired length. In preferred embodiments, the CFXTEN comprising multiple XTEN have XTEN exhibiting at least about 80%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% sequence identity to sequences selected from AE42_1, AE42_2, AE42_3, AG42_1, AG42_2, AG42_3, AG42_4, AE144_1A, AE144_2A, AE144_2B, AE144_3A, AE144_3B, AE144_4A, AE144_4B, 5A, AE144_6B, AG144_1, AG144_2, AG144_A, AG144_B, AG144_C, AG144_F, AG144_3, AG144_4, AE288_1, AE288_2, AG288_1, AG288_2, and AG288_DE. 4. Net charge In other embodiments, the unstructured characteristic of an XTEN polypeptide can be enhanced by incorporation of amino acid residues with a net charge and/or reduction of the overall percentage (e. g. less than 5%, or 4%, or 3%, or 2%, or 1%) of hydrophobic amino acids in the XTEN sequence. The overall net charge and net charge density is controlled by modifying the content of charged amino acids in the XTEN sequences, either positive or negative, With the net charge lly represented as the tage of amino acids in the polypeptide contributing to a charged state beyond those residues that are cancelled by a residue With an opposite charge. In some embodiments, the net charge density of the XTEN of the compositions may be above +0.1 or below -0.1 charges/residue. By “net charge density” of a protein or e herein is meant the net charge diVided by the total number of amino acids in the protein or propeptide. In other embodiments, the net charge of an XTEN can be about 0%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10% about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% or more. Based on the net charge, some XTENs have an isoelectric point (pI) of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, or even 6.5. In preferred embodiments, the XTEN will have an isoelectric point between 1.5 and 4.5 and carry a net negative charge under physiologic ions.

Since most tissues and surfaces in a human or animal have a net negative charge, in some embodiments the XTEN sequences are designed to have a net negative charge to minimize non-specific interactions between the XTEN containing compositions and various surfaces such as blood vessels, healthy tissues, or various receptors. Not to be bound by a particular theory, an XTEN can adopt open conformations due to electrostatic repulsion between individual amino acids of the XTEN polypeptide that dually carry a net ve charge and that are distributed across the sequence of the XTEN polypeptide. In some embodiments, the XTEN sequence is designed with at least 90% or 95% of the charged residues separated by other residues such as serine, alanine, threonine, e or glycine, which leads to a more uniform distribution of charge, better expression or purification behavior. Such a distribution of net negative charge in the ed sequence lengths ofXTEN can lead to an unstructured conformation that, in turn, can result in an effective increase in hydrodynamic radius. In preferred embodiments, the negative charge of the subject XTEN is conferred by incorporation of glutamic acid residues. lly, the glutamic residues are spaced uniformly across the XTEN sequence. In some cases, the XTEN can contain about 10-80, or about 15-60, or about 20-50 glutamic residues per 20kDa of XTEN that can result in an XTEN with charged residues that would have very r pKa, which can increase the charge homogeneity of the product and sharpen its isoelectric point, enhance the physicochemical properties of the resulting CFXTEN filsion protein for, and hence, simplifying purification procedures. For example, where an XTEN with a negative charge is desired, the XTEN can be selected solely from an AB family sequence, which has approximately a 17% net charge due to incorporated ic acid, or can include varying proportions of glutamic acid-containing motifs of Table 3 to provide the desired degree of net charge. Non-limiting examples of AE XTEN include, but are not limited to the 36, 42, 144, 288, 576, 624, 864, and 912 AB family sequences ofTables 4 and 14 or fragments thereof In one embodiment, an XTEN sequence of Tables 4, or 13-17 can be modified to include additional glutamic acid residues to achieve the desired net negative . Accordingly, in one embodiment the ion provides XTEN in which the XTEN sequences n about 1%, 2%, 4%, 8%, 10%, 15%, 17%, 20%, 25%, or even about 30% glutamic acid. In one embodiment, the ion plates incorporation of up to 5% aspartic acid residues into XTEN in addition to glutamic acid in order to achieve a net negative charge.

] In other embodiments, where no net charge is desired, the XTEN can be selected from, for example, AG XTEN ents, such as the AG motifs of Table 3, or those AM motifs of Table 3 that have no net charge. Non-limiting es of AG XTEN include, but are not limited to 36, 42, 144, 288, 576, and 864 AG family sequences of Tables 4 and 16, or fragments thereof In another embodiment, the XTEN can comprise varying proportions of AE and AG motifs (in order to have a net charge that is deemed optimal for a given use or to in a given physicochemical property.

Not to be bound by a ular theory, the XTEN of the CFXTEN compositions with the higher net charge are expected to have less non-specific interactions with various negatively-charged surfaces such as blood vessels, tissues, or various receptors, which would further contribute to reduced active clearance. Conversely, it is believed that the XTEN of the CFXTEN compositions with a low (or no) net charge would have a higher degree of interaction with surfaces that can iate the actiVity of the associated coagulation factor, given the known contribution of cell (e.g., platelets) and vascular surfaces to the coagulation process and the intensity of activation of coagulation factors (Zhou, R., et al., Biomaterials (2005) 26(16):2965-2973; London, F., et al. Biochemistry (2000) 39(32):9850—9858).

The XTEN of the compositions of the present invention generally have no or a low content of positively d amino acids. In some embodiments, the XTEN may have less than about 10% amino acid residues with a positive charge, or less than about 7%, or less than about 5%, or less than about 2%, or less than about 1% amino acid es with a ve charge. r, the invention contemplates constructs where a limited number of amino acids with a positive charge, such as lysine, are incorporated into XTEN to permit conjugation between the epsilon amine of the lysine and a reactive group on a peptide, a linker bridge, or a ve group on a drug or small molecule to be conjugated to the XTEN backbone. In one embodiment of the foregoing, the XTEN of the subject CFXTEN has between about 1 to about 100 lysine residues, or about 1 to about 70 lysine residues, or about 1 to about 50 lysine residues, or about 1 to about 30 lysine residues, or about 1 to about 20 lysine residues, or about 1 to about lysine residues, or about 1 to about 5 lysine residues, or atively only a single lysine residue.

Using the foregoing lysine-containing XTEN, fusion proteins can be constructed that comprise XTEN, a FVIII ation factor, plus a chemotherapeutic agent or other coagulation factor or cofactor useful in the ent of coagulopathy conditions, wherein the maximum number of molecules of the agent incorporated into the XTEN component is determined by the numbers of lysines or other amino acids with reactive side chains (e.g., cysteine) incorporated into the XTEN.

As hydrophobic amino acids impart structure to a polypeptide, the invention provides that the content of hydrophobic amino acids in the XTEN will typically be less than 5%, or less than 2%, or less than 1% hydrophobic amino acid content. In one embodiment, the amino acid content of methionine and tryptophan in the XTEN component of a CFXTEN fusion protein is typically less than 5%, or less than 2%, and most preferably less than 1%. In another embodiment, the XTEN of the t CFXTEN compositions will have a sequence that has less than 10% amino acid es with a positive charge, or less than about 7%, or less that about 5%, or less than about 2% amino acid residues with a positive charge, the sum of methionine and tryptophan residues will be less than 2%, and the sum of asparagine and glutamine residues will be less than 5% of the total XTEN sequence.

. Low genicity In another aspect, the XTEN sequences ed herein have a low degree of immunogenicity or are substantially non-immunogenic. Several s can contribute to the low immunogenicity of XTEN, e. g., the non-repetitive sequence, the unstructured conformation, the high degree of solubility, the low degree or lack of self-aggregation, the low degree or lack of proteolytic sites within the sequence, and the low degree or lack of epitopes in the XTEN sequence.

Conformational epitopes are formed by regions of the protein surface that are composed of multiple discontinuous amino acid ces of the protein antigen. The precise folding of the protein brings these sequences into a well-defined, stable spatial configurations, or epitopes, that can be recognized as gn” by the host humoral immune system, resulting in the production of antibodies to the protein or the tion of a cell-mediated immune response. In the latter case, the immune response to a protein in an individual is heavily influenced by T-cell epitope ition that is a function of the peptide binding specificity of that individual’s HLA—DR allotype. ment of a MHC Class II peptide complex by a cognate T-cell receptor on the surface of the T-cell, together with the cross-binding of certain other co-receptors such as the CD4 molecule, can induce an activated state within the T-cell. tion leads to the e of cytokines further activating other lymphocytes such as B cells to produce antibodies or ting T killer cells as a full cellular immune response.

The ability of a peptide to bind a given MHC Class II molecule for presentation on the surface of an APC (antigen presenting cell) is dependent on a number of factors; most notably its primary sequence. In one embodiment, a lower degree of immunogenicity is ed by designing XTEN sequences that resist antigen processing in antigen presenting cells, and/or choosing ces that do not bind MHC receptors well. The invention provides CFXTEN fusion proteins with ntially non- repetitive XTEN polypeptides designed to reduce binding with MHC II receptors, as well as avoiding formation of epitopes for T-cell receptor or dy binding, ing in a low degree of immunogenicity. Avoidance of immunogenicity can attribute to, at least in part, a result of the mational flexibility ofXTEN sequences; i.e., the lack of secondary ure due to the selection and order of amino acid residues. For example, of particular interest are sequences having a low tendency to adapt compactly folded conformations in aqueous solution or under physiologic ions that could result in conformational epitopes. The stration of fusion proteins comprising XTEN, using conventional therapeutic practices and dosing, would generally not result in the formation of neutralizing antibodies to the XTEN sequence, and also reduce the immunogenicity of the FVIII fusion partner in the CFXTEN compositions.

In one embodiment, the XTEN sequences utilized in the subject fusion proteins can be substantially free of epitopes recognized by human T cells. The elimination of such epitopes for the purpose of generating less genic proteins has been disclosed previously; see for example WO 98/52976, WO 02/079232, and WO 00/3317 which are incorporated by reference herein. Assays for human T cell epitopes have been described (Stickler, M., et al. (2003) JImmunol Methods, 281: 95-108).

Of particular interest are peptide sequences that can be erized without generating T cell epitopes or non-human sequences. This is achieved by testing direct repeats of these sequences for the presence of T-cell epitopes and for the occurrence of 6 to 15-mer and, in particular, 9-mer sequences that are not human, and then altering the design of the XTEN sequence to eliminate or disrupt the epitope sequence.

In some embodiments, the XTEN sequences are ntially munogenic by the restriction of the numbers of epitopes of the XTEN predicted to bind MHC receptors. With a ion in the numbers of epitopes capable of binding to MHC receptors, there is a concomitant reduction in the ial for T cell activation as well as T cell helper on, reduced B cell activation or upregulation and reduced antibody production. The low degree of predicted T-cell epitopes can be determined by epitope prediction algorithms such as, e. g., TEPITOPE (Stumiolo, T., et al. (1999) Nat Biotechnol, 17: 555-61), as shown in Example 46. The TEPITOPE score of a given peptide frame within a protein is the log of the Kd (dissociation constant, affinity, off-rate) of the binding of that peptide frame to multiple of the most common human MHC alleles, as disclosed in Stumiolo, T. et al. (1999) Nature Biotechnology 17:555). The score ranges over at least 20 logs, from about 10 to about -10 (corresponding to binding constraints of 10e10 Kd to 10e'10 Kd), and can be reduced by avoiding hydrophobic amino acids that serve as anchor residues during peptide display on MHC, such as M, I, L, V, F. In some embodiments, an XTEN ent incorporated into a CFXTEN does not have a predicted T-cell epitope at a TEPITOPE threshold score of about -5, or -6, or -7, or -8, or -9, or at a TEPITOPE score of -10. As used herein, a score of “-9” is a more stringent TEPITOPE threshold than a score of -5.

In another embodiment, the inventive XTEN sequences, including those incorporated into the t CFXTEN fusion proteins, are rendered substantially non-immunogenic by the restriction of known proteolytic sites from the sequence of the XTEN, reducing the processing ofXTEN into small es that can bind to MHC 11 receptors. In another embodiment, the XTEN sequence is rendered substantially non-immunogenic by the use a sequence that is substantially devoid of ary structure, ring resistance to many proteases due to the high entropy of the structure. Accordingly, the reduced TEPITOPE score and elimination of known proteolytic sites from the XTEN render the XTEN compositions, including the XTEN of the CFXTEN fusion protein compositions, substantially unable to be bound by mammalian receptors, ing those of the immune system or active clearance receptors that target FVIII. In one embodiment, an XTEN of a CFXTEN fusion protein can have >100 nM Kd binding to a ian receptor, or greater than 500 nM Kd, or greater than 1 uM Kd towards a mammalian cell surface receptor or circulating polypeptide or.

Additionally, the non-repetitive sequence and corresponding lack of epitopes ofXTEN limit the ability of B cells to bind to or be activated by XTEN. A repetitive sequence is recognized and can form multivalent contacts with even a few B cells and, as a uence of the linking of multiple T-cell independent receptors, can stimulate B cell proliferation and antibody production. In contrast, while an XTEN can make contacts with many different B cells over its extended ce, each individual B cell may only make one or a small number of contacts with an individual XTEN due to the lack of repetitiveness of the ce. Not being to be bound by any theory, XTENs typically have a much lower tendency to stimulate proliferation of B cells and thus an immune response. In one embodiment, the CFXTEN have reduced immunogenicity as compared to the corresponding FVIII that is not fused to an XTEN. In one embodiment, the administration of up to three parenteral doses of a CFXTEN to a mammal result in detectable anti-CFXTEN IgG at a serum dilution of 1:100 but not at a dilution of 1:1000. In another embodiment, the administration of up to three parenteral doses of a CFXTEN to a mammal result in detectable VIII IgG at a serum dilution of 1:100 but not at a dilution of 1:1000. In another ment, the stration of up to three parenteral doses of a CFXTEN to a mammal result in detectable anti-XTEN IgG at a serum dilution of 1:100 but not at a dilution of 1:1000. In the foregoing embodiments, the mammal can be a mouse, a rat, a rabbit, or a cynomolgus monkey.

An additional feature of XTENs with non-repetitive sequences relative to sequences with a high degree of repetitiveness is petitive XTENs form weaker contacts with antibodies. Antibodies are multivalent molecules. For instance, IgGs have two identical binding sites and Ing contain 10 identical g sites. Thus antibodies t tive sequences can form multivalent contacts with such repetitive sequences with high avidity, which can affect the potency and/or elimination of such repetitive sequences. In contrast, antibodies against non-repetitive XTENs may yield monovalent ctions, resulting in less likelihood of immune nce such that the CFXTEN compositions can remain in ation for an increased period of time. In addition, it is believed, as schematically portrayed in the e unstructured nature of XTEN provides steric shielding of FVIII regions proximal to the XTEN site of insertion and providing steric hindrance to binding by FVIII tors.

In another aspect, a subject XTEN useful as a fusion partner has a high hydrodynamic radius; a property that in some embodiments confers a corresponding increased apparent molecular weight to the CFXTEN fusion protein incorporating the XTEN, while in other embodiments enhances steric nce to FVIII inhibitors and to anti-FVIII antibodies, reducing their ability to bind to CFXTEN. As ed in Example 26, the linking ofXTEN to therapeutic protein sequences results in CFXTEN itions that can have increased hydrodynamic radii, increased apparent molecular weight, and increased apparent molecular weight factor compared to a eutic n not linked to an XTEN. For example, in therapeutic applications in which prolonged half-life is desired, compositions in which an XTEN with a high hydrodynamic radius is orated into a fusion protein comprising a therapeutic protein can effectively enlarge the hydrodynamic radius of the composition beyond the ular pore size of approximately 3-5 nm (corresponding to an apparent lar weight of about 70 kDa) (Caliceti. 2003.

Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates. Adv Drug Deliv Rev 55 :1261-1277), resulting in reduced renal clearance of ating proteins with a corresponding increase in terminal half-life and other enhanced pharmacokinetic properties. The hydrodynamic radius of a protein is conferred by its molecular weight as well as by its structure, including shape or compactness. Not to be bound by a particular theory, the XTEN can adopt open conformations due to electrostatic repulsion between individual charges of the peptide or the inherent flexibility imparted by the particular amino acids in the sequence that lack potential to confer secondary structure. The open, extended and unstructured conformation of the XTEN polypeptide can have a greater proportional hydrodynamic radius compared to polypeptides of a comparable sequence length and/or molecular weight that have secondary and/or tertiary structure, such as typical globular proteins.

Methods for determining the hydrodynamic radius are well known in the art, such as by the use of size exclusion chromatography (SEC), as described in US. Patent Nos. 6,406,632 and 7,294,513. e 26 trates that increases in XTEN length result in proportional increase in the hydrodynamic radius, apparent molecular weight, and/or apparent molecular weight factor, and thus permit the tailoring of CFXTEN to desired cut-off values of apparent molecular weights or hydrodynamic radii.

Accordingly, in certain embodiments, the CFXTEN fusion protein can be configured with an XTEN such that the fusion protein can have a hydrodynamic radius of at least about 5 nm, or at least about 8 nm, or at least about 10 nm, or about 12 nm, or about 15 nm, or about 20 nm, or about 30 nm or more. In the foregoing embodiments, the large hydrodynamic radius conferred by the XTEN in a CFXTEN fusion n can lead to d clearance of the resulting fusion protein, an increase in terminal half-life, and an increase in mean nce time.

Generally, the actual molecular weight of the mature form of FVIII component is about 265 kDa, while in the case of a FVIII BDD, it is about 165 kDa. The actual molecular weight of a CFXTEN fusion protein for comprising a FVIII BDD plus one or more XTEN ranges from about 200 to about 270 kDa, depending on the length of the XTEN components. As described in the Examples, when the molecular weights of the CFXTEN fusion proteins are d from size exclusion chromatography analyses, the open conformation of the XTEN due to the low degree of secondary structure results in an increase in the apparent molecular weight of the fusion proteins into which they are incorporated. In some embodiments, the CFXTEN comprising a FVIII and at least one or more XTEN exhibits an apparent molecular weight of at least about 400 kD, or at least about 500 kD, or at least about 700 kD, or at least about 1000 kD, or at least about 1400 kD, or at least about 1600 kD, or at least about 1800kD, or at least about 2000 kD. Accordingly, the CFXTEN fusion proteins comprising one or more XTEN exhibit an apparent lar weight that is about ld greater, or about 2-fold greater, or about 3- fold greater or about 4-fold greater, or about 8-fold greater, or about 10-fold greater, or about 12-fold r, or about 15-fold greater than the actual molecular weight of the fusion protein. In one embodiment, the isolated CFXTEN fusion protein of any of the embodiments disclosed herein exhibit an apparent molecular weight factor under physiologic conditions that is greater than about 1.3, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 10, or r than about 15.

In another embodiment, the CFXTEN fusion n has, under physiologic ions, an apparent molecular weight factor that is about 3 to about 20, or is about 5 to about 15, or is about 8 to about 12, or is about 9 to about 10 relative to the actual lar weight of the fusion protein. It is believed that the increased apparent molecular weight of the subject CFXTEN compositions enhances the pharmacokinetic properties of the fusion proteins by a combination of factors, which include reduced active nce, reduced binding by FVIII inhibitors, and d loss in capillary and venous bleeding.

IV). CFXTEN COMPOSITIONS The present invention provides compositions comprising fusion proteins having factor VIII linked to one or more XTEN sequences, wherein the fusion protein acts to replace or t the amount of existing FVIII in the intrinsic or contact activated coagulation pathway when administered into a subject. The invention addresses a long-felt need in increasing the terminal half-life of exogenously administered factor VIII to a subject in need thereof. One way to increase the circulation half-life of a therapeutic protein is to ensure that renal clearance or metabolism of the protein is reduced.

Another way to increase the terminal half-life is to reduce the active nce of the therapeutic protein, r mediated by receptors, active lism of the protein, or other nous mechanisms. Both may be achieved by conjugating the protein to a polymer, which, on one hand, is capable of conferring an increased molecular size (or hydrodynamic ) to the protein and, hence, reduced renal clearance, and, on the other hand, interferes with binding of the protein to clearance receptors or other proteins that contribute to metabolism or clearance. Thus, certain objects of the present invention include, but are not limited to, providing ed FVIII les with a longer ation or terminal half-life, decreasing the number or frequency of necessary administrations of FVIII compositions, retaining at least a portion of the actiVity ed to native coagulation factor VIII, and/or enhancing the ability to treat coagulation deficiencies and uncontrolled bleedings more efficiently, more effectively, more ically, and/or with greater safety compared to presently available factor VIII preparations.

] Accordingly, the present ion provides inant factor VIII fusion n compositions comprising an FVIII covalently linked to one or more extended inant polypeptides (“XTEN”), resulting in a CFXTEN fusion protein composition. The term “CFXTEN”, as used herein, is meant to encompass fusion polypeptides that comprise at least one payload region comprising a FVIII or a portion of a FVIII that is capable of procoagulant actiVity associated with a FVIII coagulation factor and at least one other region comprising one or more XTEN polypeptides that may be interspersed within the payload region and/or attached to the terminus. In one embodiment, the FVIII is native FVIII. In another embodiment, the FVIII is a sequence variant, fragment, homolog, or mimetic of a natural sequence that retains at least a portion of the procoagulant actiVity of native FVIII, as disclosed .

Non-limiting examples of FVIII suitable for inclusion in the compositions include the sequences of Table l or sequences having at least 80%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity to a sequence of Table 1. In a preferred embodiment, the FVIII is a B-domain deleted (BDD) FVIII ce variant, such as those BDD ces from Table l or other such sequences known in the art. In r preferred embodiment, the CFXTEN comprises a B-domain deleted (BDD) FVIII ce variant sed with the native 19 amino acid signal sequence, which is cleaved during the maturation of the protein.

The compositions of the invention include fusion proteins that are useful, when administered to a subject in need thereof, for mediating or preventing or ameliorating a condition associated with factor VIII deficiencies or defects in endogenously produced FVIII, or bleeding disorders associated with trauma, surgery, factor VIII deficiencies or defects. Of particular interest are CFXTEN fusion n compositions for which an increase in a pharmacokinetic parameter, increased solubility, increased stability, or some other enhanced pharmaceutical property compared to native FVIII is sought, or for which increasing the terminal half-life would improve efficacy, safety, or result in reduced dosing frequency and/or e patient management. The CFXTEN fusion proteins of the embodiments disclosed herein exhibit one or more or any combination of the improved properties and/or the embodiments as ed herein. In some embodiments, the CFXTEN fusion ition remains at a level above a threshold value of at least 0.01-0.05, or 0.05 to 0.1, or 0.1 to 0.4 IU/ml when administered to a subject, for a longer period of time when compared to a FVIII not linked to XTEN and administered at a comparable dose to a subject in need thereof (e. g., a subject such as a human or mouse or monkey with hemophilia A).

The FVIII of the subject compositions, ularly those disclosed in Table 1, together with their corresponding nucleic acid and amino acid sequences, are available in public databases such as Chemical cts Services Databases (e. g., the CAS Registry), GenBank, The Universal Protein Resource ot), subscription provided databases such as GenSeq (e.g., Derwent), as well as in the patent and primary ture. Polynucleotide sequences applicable for expressing the subject CFXTEN sequences may be a wild type polynucleotide sequence encoding a given FVIH (e. g., either full length or mature), or in some instances the sequence may be a variant of the wild type polynucleotide sequence (e. g., a polynucleotide which encodes the wild type biologically active protein, wherein the DNA sequence of the polynucleotide has been zed, for example, for expression in a particular species, or a polynucleotide encoding a variant of the wild type protein, such as a site directed mutant or an allelic variant. It is well within the ability of the skilled artisan to use a wild-type or consensus cDNA sequence or a codon-optimized variant of a FVIII to create CFXTEN constructs contemplated by the invention using s known in the art and/or in conjunction with the guidance and methods provided , and described more fully in the Examples.

In one embodiment, a CFXTEN fusion protein comprises a single FVIH le exhibiting at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%, or 100% sequence identity to a sequence of Table 1 linked to a single XTEN (e. g., an XTEN as described above) including, but not limited to sequences of the AE or AG family with 42, 144, 288, 576, or 864 amino acids, as set forth in Table 4. In another embodiment, the CFXTEN comprises a single FVIH linked to two XTEN, wherein the XTEN may be identical or they may be ent. In another embodiment, the CFXTEN fusion protein comprises a single FVIH molecule linked to one, two, three, four, five, six or more XTEN sequences, in which the FVIII is a sequence that has at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%, or 100% sequence identity compared to a protein sequence selected from Table 1, when lly aligned, and the one or more XTEN are each having at least about 80% sequence ty, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%, or 100% sequence identity compared to one or more sequences selected from any one of Tables 3, 4, and 13-17, when optimally aligned. In the foregoing embodiment, where the CFXTEN has two or more XTEN, the XTEN may be cal or they may be different sequences. In yet another embodiment, the CFXTEN fusion protein comprises a single FVIH exhibiting at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%, or 100% sequence identity compared to sequences of comparable length selected from Table 1, when optimally aligned, with the portions persed with and linked by three, four, five, six or more XTEN sequences that may be identical or may be different and wherein each has at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%, or 100% sequence identity compared to ces selected from any one of Tables 3, 4, and 13-17, or fragments thereof, when optimally aligned. In yet another embodiment, the ion provides a CFXTEN fusion protein comprising a ce with at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%, or 100% sequence identity to a sequence from Table 21, when optimally aligned. 1. CFXTEN Fusion Protein Configurations ] The invention provides CFXTEN fusion protein compositions with the CF and XTEN components linked in ic N— to inus configurations.

In one embodiment of the CFXTEN composition, the invention es a fusion protein of formula I: (XTEN)X-CF-(XTEN)y 1 wherein independently for each occurrence, CF is a factor VIII as defined herein, including sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity with ced from Table 1; X is either 0 or 1 and y is either 0 or 1 wherein x+y 31; and XTEN is an extended recombinant polypeptide as described herein, including, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 4. Accordingly, the CFXTEN fusion composition can have XTEN-CF, XTEN-CF-XTEN, or CF-XTEN configurations.

In another embodiment of the CFXTEN composition, the invention provides a fusion protein of formula II: (XTEN)X-(S)x-(CF)-(XTEN) y 11 wherein independently for each occurrence, CF is a factor VIII as defined herein, including sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 1; S is a spacer sequence having n 1 to about 50 amino acid residues that can optionally include a cleavage sequence or amino acids ible with restrictions sites; x is either 0 or 1 and y is either 0 or 1 wherein x+y 21; and XTEN is an extended recombinant ptide as described herein ing, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 4.

In r embodiment of the CFXTEN ition, the invention provides a recombinant factor VIII fusion protein, wherein the fusion protein is of formula III: (XTEN)X-(S)X-(CF)-(S)y-(XTEN)y 111 wherein ndently for each occurrence, CF is a factor VIII as defined herein, including sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequence set for in Table 1; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally e a cleavage sequence or amino acids compatible with restrictions sites; X is either 0 or 1 and y is either 0 or 1 wherein X+y 21; and XTEN is an extended recombinant polypeptide as described herein including, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence ty to sequences set forth in Table 4.

In another embodiment of the CFXTEN composition, the invention provides a recombinant factor VIII fusion protein of formula IV: (A1)-(XTEN)u-(A2)-(XTEN)V-(B)-(XTEN)W-(A3)-(XTEN)x-(C1)-(XTEN)y-(C2)-(XTEN)Z wherein independently for each occurrence, A1 is an A1 domain of FVIII; A2 is an A2 domain of FVIII; A3 is an A3 domain of FVIII; B is a B domain of FVIII which can be a fragment or a splice variant of the B ; C1 is a C1 domain of FVIII; C2 is a C2 domain of FVIII; V is either 0 or 1; w is either 0 or 1; X is either 0 or 1; y is either 0 or 1; y is either 0 or 1 with the proviso that u + V + X + y+z 31; and XTEN is an extended recombinant ptide as described herein including, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 4.

In another embodiment of the CFXTEN composition, the invention provides a recombinant factor VIII fusion protein of formula V: (XTENx-(sx -(A1)-(S)b-(XTEN)u-(S)b-(A2)-(S)c-(XTEN)V-(S)c-(B)-(S)d-(XTEN)w-(S)d-(A3)—(S)e- (XTEN)x-(S)e-(C1)-(s)r(XTEN)y-(S)r(cz)-(S)g-(XTEN)Z V wherein independently for each occurrence, A1 is an A1 domain of FVIII; A2 is an A2 domain of FVIII; A3 is an A3 domain of FVIII; B is a B domain of FVIII which can be a fragment or a splice variant of the B domain; C1 is a C1 domain of FVIII; C2 is a C2 domain of FVIII; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally e a cleavage sequence or amino acids ible with restrictions sites; a is either 0 or 1; b is either 0 or 1; c is either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is either 0 or 1; g is either 0 or 1; t is either 0 or 1; u is either 0 or 1; V is either 0 or 1; w is 0 or 1, X is either 0 or 1; y is either 0 or 1; z is either 0 or 1 with the proviso that t + u + V + w+ X + y + z 31; and XTEN is an eXtended recombinant polypeptide as described herein including, but not limited to sequences haVing at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 4. In another embodiment of formula V, the spacer ce is glycine or a sequence selected from Tables 11 and 12.

In another ment of the CFXTEN composition, the invention provides a recombinant factor VIII fusion protein of formula VI: (XTENX—(SX-(Al)—(S)b-(XTEN)V-(S)b-(A2)-(S)c-(XTEN)W-(S)c-(A3)—(S)d-(XTEN)x-(S)d-(C1)- (s)e-(XTEN)y-(8)6-(C2)—(S)r(XTEN)Z VI wherein independently for each occurrence, A1 is an A1 domain of FVIII; A2 is an A2 domain of FVIII; A3 is an A3 domain of FVIII; C1 is a C1 domain of FVIII; C2 is a C2 domain of FVIII; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence or amino acids compatible with restrictions sites; a is either 0 or 1; b is either 0 or 1; c is either 0 or 1; dis either 0 or 1; e is either 0 or 1; fis either 0 or 1; uis either 0 or 1; Vis either 0 or 1; Wis 0 or 1, X is either 0 or 1; y is either 0 or 1; z is either 0 or 1 with the proviso that u + V + w+ X + y + z 31; and XTEN is an extended recombinant polypeptide as described herein including, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity to sequences set forth in Table 4. In another embodiment of formula V, the spacer sequence is e or a sequence selected from Tables 11 and 12.

In another embodiment of the CFXTEN composition, the invention provides a recombinant factor VIII fusion protein of formula VII: XTEN)X-(CS)x-(S)x-(FVIII_1-745)-(S)y-(XTEN)y-(S)y-(FVIII_1635-2332)-(S)Z-(CS)Z- (XTEN)Z VII wherein independently for each occurrence, SP is a signal peptide, preferably with sequence MQIELSTCFFLCLLRFCFS (SEQ ID NO: 1611), CS is a cleavage sequence listed in Table 12, S is a spacer sequence having n 1 to about 50 amino acid residues that can optionally include amino acids compatible with restrictions sites, “FVIII_1-745” is residues 1-745 of Factor FVIII and “FVIII_l635-2332” is residues 332 of FVIII, X is either 0 or 1, y is either 0 or 1, and z is either 0 or 1, wherein x+y+z >2; and XTEN is an eXtended recombinant ptide as bed herein including, but not limited to ces having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% ce ty sequences set forth in Table 4. In one embodiment of formula VII, the spacer sequence is GPEGPS (SEQ ID NO: 1612). In another embodiment of formula V, the spacer sequence is glycine or a sequence selected from Tables 11 and 12.

In another embodiment of the CFXTEN composition, the invention provides a recombinant factor VIII fusion protein of formula VIII: (A1)—(S)a-(XTEN)V-(S)a-(A2)-(B1)—(S)b-(XTEN)w-(S)b-(B2)-(A3)—(S)c-(XTEN)x-(S)c-(C1)—(S)d- (XTEN)y-(S)d-(C2)-(S)e-(XTEN)Z VIII wherein independently for each occurrence, A1 is an A1 domain of FVIII; A2 is an A2 domain of FVIII; B1 is a fragment of the B domain that can have from residue 741 to 0 of FVIII or alternatively from about residue 741 to about residues 745 of FVIII; B2 is a fragment of the B domain that can have from residues 1635-1686 to 1689 of FVIII or alternatively from about residue 1640 to about residues 1689 of FVIII; A3 is an A3 domain of FVIII; C1 is a C1 domain of FVIII; C2 is a C2 domain of FVIII; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence or amino acids compatible With restrictions sites; a is either 0 or 1; b is either 0 or 1; c is either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is either 0 or 1; u is either 0 or 1; v is either 0 or 1; W is 0 or 1, X is either 0 or 1; y is either 0 or 1; z is either 0 or 1 With the proviso that u + v + w+ X + y + z 21; and XTEN is an extended recombinant polypeptide as described herein including, but not limited to sequences having at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% ce identity to sequences set forth in Table 4. In one embodiment of formula VIII, the spacer sequence is GPEGPS (SEQ ID NO: 1612). In another embodiment of formula V, the spacer sequence is e or a sequence selected from Tables 11 and 12.

In another embodiment of the CFXTEN composition, the invention es a recombinant factor VIII fusion protein of formula IX: (S)a'(XTEN)I'(S)b'(A1C)'(A2 N)-(S)c-(XTEN)u-(S)d-(A2c)-(BN)-(S)e-(XTEN)v-(S)r(Bc)-(A3N)- (S)g'(XTEN)W'(S)h'(A3C)'(C1N)'(S)i'(XTEN)x'(S)j'(C1C)'(C2N)'(S)k'(XTEN)y'(S)1'(C2C)'(S)m'(XTEN)z Wherein independently for each occurrence, AlN is a fragment of the A1 domain from at least residue number 1 (numbered relative to native, mature FVIII) to no more than residue number 371, A1C is a fragment of the A1 domain from at least residue number 2 to no more than residue number 372; A2N is a fragment of the A2 domain from at least residue number 373 to no more than residue number 739, Me is a fragment of the A2 domain from at least residue number 374 to no more than residue number 740; BN is a nt of the B domain from at least residue number 741 to no more than residue number 1647, BC is a fragment of the B domain from at least residue number 742 to no more than residue number 1648; A3N is a fragment of the A3 domain from at least residue number 1649 to no more than residue number 2019, A3C is a fragment of the A3 domain from at least residue number 1650 to no more than residue number 2019; ClN is a fragment of the C1 domain from at least residue number 2020 to no more than residue number 2171, C1C is a fragment of the C1 domain from at least residue number 2021 to no more than e number 2172; C2N is a fragment of the C2 domain from at least residue number 2173 to no more than residue number 2331, C2C is a fragment of the C2 domain from at least e number 2174 to no more than residue number 2332; S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence or amino acids compatible With ctions sites; a is either 0 or 1; b is either 0 or 1; c is either 0 or 1; d is either 0 or 1; e is either 0 or 1; f is either 0 or 1; g is either 0 or 1; h is either 0 or 1; i is either 0 or 1; j is either 0 or 1; k is either 0 or 1; l is either 0 or 1; mis either 0 or 1; t is either 0 or 1; uis either 0 or 1; vis either 0 or 1; Wis 0 or 1, X is either 0 or 1; y is either 0 or 1; z is either 0 or 1 With the proviso that t + u + v + w+ X + y + z 31; and XTEN is an ed recombinant polypeptide as bed herein including, but not limited to sequences having at least 90% identity to ces set forth in Table 4. In one embodiment of formula IX, the spacer sequence is GPEGPS (SEQ ID NO: 1612). In another embodiment of formula IX, the spacer sequence is glycine or a sequence selected from Tables 11 and 12.

] The ments of formulae IV-VIII encompass CFXTEN configurations wherein one or more XTEN of s ranging from about 6 amino acids to >_1000 amino acids (e. g., sequences selected from any one of Tables 3, 4, and 13-17 or nts thereof, or sequences exhibiting at least about 90- 99% or more sequence identity thereto) are inserted and linked between adjoining domains of the factor VIII or are linked to the N— or C-terminus of the FVIII. In other embodiments of formulae V-VIII, the invention further provides configurations wherein the XTEN are linked to FVIII domains Via spacer sequences which can optionally comprise amino acids compatible with restrictions sites or can include cleavage sequences (e. g., the sequences of Tables 11 and 12, described more fully below) such that the XTEN encoding sequence can be, in the case of a restriction site, integrated into a CFXTEN construct and, in the case of a cleavage sequence, the XTEN can be released from the fusion protein by the action of a protease appropriate for the cleavage sequence.

The embodiments of formulae VI -VIII differ from those of a V in that the FVIII component of formulae VI-VIII are only the in deleted forms (“FVIII BDD”) of factor VIII that retain short residual sequences of the B-domain, non-limiting examples of sequences of which are provided in Table 1, n one or more XTEN or fragments ofXTEN of lengths g from about 6 amino acids to 11000 amino acids (e. g., sequences selected from any one of Tables 3, 4, and 13-17) are inserted and linked between ing domains of the factor VIII and/or n the ts of the B domain residues, such as those of Table 8. The embodiment of formula IX generally differs from those of the other formulae in that the one or more XTEN are each inserted within domains of FVIII rather than between domains, and/or has an XTEN linked to the inus of the FVIII (or is linked Via a spacer sequence to the C-terminus of the FVIII).

] In some embodiments of a CFXTEN, the fusion protein comprises a B-domain deleted form of FVIII wherein the B-domain on starts from a first on at about amino acid residue number 745 and ends at a second position at amino acid residue number 1635 to about 1690 with reference to the full- length human factor VIII sequence and an XTEN links the first position and the second position of the B- domain deletion. In one embodiment of the foregoing, the first position and the second position of the B- domain deletion are selected from the ons of Table 8. In another embodiment of the foregoing, at least one XTEN links the first and second position wherein the at least one XTEN links factor VIII amino acid residue 745 and amino acid residue 1640, or amino acid residue 741 and amino acid residue 1640, or amino acid residue 741 and amino acid residue 1690, or amino acid residue 745 and amino acid residue 1667, or amino acid residue 745 and amino acid residue 1657, or amino acid residue 745 and amino acid residue 1657, or amino acid residue 747 and amino acid residue 1642, or amino acid residue 751 and amino acid residue 1667. In one embodiment of the CFXTEN, wherein the factor VIII comprises an XTEN linking a first position and a second position of a B-domain deletion described in the embodiments of this paragraph, the XTEN is a sequence haVing at least 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or 100% sequence identity compared to a sequence of comparable length selected from any one of Table 4, Table 13, Table 14, Table 15, Table 16, and Table 17, when optimally aligned, wherein the CFXTEN s at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% of the procoagulant activity of native FVIII.

The invention contemplates all possible permutations of ions ofXTEN between or within the domains of FVIII or at or proximal to the insertion points of Table 5, Table 6, Table 7, Table 8, and Table 9 or those illustrated in FIGS. 8-9, with optional linking of an additional XTEN to the N— or C- terminus of the FVIII, optionally linked via an additional cleavage sequence selected from Table 12, resulting in a CFXTEN composition; non-limiting examples of which are yed in FIGS. 5 and 12.

In one embodiment, the CFXTEN comprises a FVIII BDD sequence of Table 1 in which one or more XTEN that each has at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% or more sequence identity compared to a sequence from any one of Tables 3, 4, and 13-17 or fragments thereof are inserted between any two of the residual B domain amino acids of the FVIII BDD sequence, resulting in a single chain FVIII fusion protein, wherein the CFXTEN retains at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% of the procoagulant activity of native FVIII. In the foregoing embodiment, the CFXTEN can have an additional XTEN sequence of any one of Tables 4, and 13-17 linked to the N— or C-terminus of the fusion protein. In another ment, a CFXTEN comprises at least a first XTEN inserted at a site set forth in Table 8, wherein the CFXTEN retains at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% of the procoagulant activity of native FVIII. In one embodiment of a fusion n of formula VII, the CFXTEN comprises a FVIII BDD sequence of Table 1 in which two or more XTEN that each has at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% sequence identity ed to a sequence from any one of Tables 3, 4, and 13-17 or nts thereof are linked to a FVIII-BDD sequence in which at least one XTEN is ed from about 3 to about 20 amino acid residues to the C-terminus side of the FVIII cleavage site amino acid R740 and from about 3 to about 20 amino acid residues to the inus side of the FVIII cleavage site amino acid R1689 of the residual B domain amino acids of the FVIII BDD sequence, resulting in a single chain FVIII fusion protein, and one or two XTEN are linked by a cleavage sequence to the N— and/or C-terminus of the FVIII-BDD sequence, n the CFXTEN exhibits at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% of the procoagulant activity of native FVIII after release of the XTEN by cleavage of the cleavage sequences.

In one embodiment, the A3 domain comprises an a3 acidic region or a portion thereof. In r embodiment, at least one XTEN is inserted within the a3 acidic region or the n thereof, N- terminus of the a3 acidic region or the portion thereof, C-terminus of the a3 acidic region or the portion thereof, or a combination thereof. In certain embodiments, at least one XTEN is inserted within the C2 domain, N—terminus of C2 domain, C-terminus of C2 domain, or a combination thereof. In still other embodiments, the Factor VIII comprises all or portion of B domain. In yet other embodiments, at least one XTEN is inserted within all or a portion of B domain, N—terminus of B domain, C-terminus of B domain, or a ation thereof 2. CFXTEN Fusion Protein Configurations with Internal XTEN ] In another aspect, the invention provides CFXTEN configured with one or more XTEN sequences located al to the FVIII sequence. In one embodiment, invention provides CFXTEN configured with one or more XTEN sequences located internal to the FVIII sequence to confer ties such as, but not limited to, increased stability, increased resistance to proteases, increased resistance to clearance isms including but not limiting to interaction with clearance receptors or FVIII inhibitors, and increased hydrophilicity, compared to FVIII without the incorporated XTEN.

] The ion contemplates that different configurations or sequence ts of FVIII can be utilized as the platform into which one or more XTEN are inserted. These configurations include, but are not limited to, native FVIII, FVIII BDD, and single chain FVIII (scFVIII), and variants of those configurations. In the case of scFVIII, the invention provides CFXTEN that can be constructed by ing one or multiple amino acids of the processing site of FVIII. In one embodiment, the scFVIII utilized in the CFXTEN is created by replacing the R1648 in the FVIII sequence RHQREITR (SEQ ID NO: 1698) with glycine or alanine to prevent proteolytic processing to the heterodimer form. It is specifically contemplated that any of the CFXTEN embodiments disclosed herein with a 1648 FVIII residue can have a glycine or alanine substitution for the arginine at position 1648. In some embodiments, the invention provides CFXTEN comprising scFVIII wherein parts of the sequence surrounding the R1648 processing site are replaced with XTEN, as illustrated in FIGS. 10A and 10B. In one embodiment, at least about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97% or more of the B-domain is replaced with an XTEN sequence disclosed herein, including one or more of the R740, R1648, or R1689 ge sites. In another embodiment, the CFXTEN has the FVIII sequence of the B-domain between the FXIa cleavage sites at R740 and R1689 (with at least 1-5 adjacent in amino acids also retained n the cut site and the start of the XTEN to permit the protease to access the cut site) replaced with XTEN. In another embodiment, the CFXTEN has the FVIII sequence of the B-domain between the FXIa cleavage site at N745 and P1640 replaced with XTEN. In other embodiments, the ion es CFXTEN FVIII BDD sequence variants in which portions of the B-domain are deleted but only one of the FXI R740 or R1689 activation sites (and 1-5 adjacent amino acids of the B-domain) are left within the construct, wherein the XTEN remains attached at one end to either the light or heavy chain after cleavage by FXIa, as illustrated in and 5D. In one embodiment of the foregoing, the CFXTEN comprises a FVIII BDD sequence in which the amino acids n N745 to P1640 or between S743 to Q1638 or between P747 to V1642 or between N745 and Q1656 or between N745 and S1657 or between N745 and T1667 or between N745 and Q1686 or between R747 and V1642 or n T751 and T1667 are deleted and an XTEN sequence is linked between these amino acids, connecting the heavy and light chains, and can further comprise onal XTEN inserted either in external surface loops, between FVIII domains, or at the N— or C-termini of the FVIII BDD sequence, such as one or more insertion sites from Table 5, Table 6, Table 7, Table 8, and Table 9 or those illustrated in FIGS. 8-9. In another embodiment of the foregoing, the CFXTEN comprises a FVIII BDD sequence in which the amino acids between K713 to Q1686 or between residues 741 and 1648 are deleted and an XTEN linked between the two amino acids, and additional XTEN can be inserted either in surface loops, between FVIII domains, or at the N— or ini of the FVIII BDD sequence, including but not limited to one or more insertion sites from Table 5, Table 6, Table 7, Table 8, and Table 9 or those rated in FIGS. 8-9. In some embodiments such CFXTEN sequences can have one or more XTEN exhibiting at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% sequence identity to an XTEN sequence from any one of Tables 4 and 13-17.

The invention contemplates other CFXTEN with internal XTEN in various urations; schematics of exemplary configurations are rated in FIGS. 5 and 10. The regions suitable for XTEN insertion sites include the known domain boundaries of FVIII, exon boundaries, known surface (external) loops and solvent accessible surface area sites identified by X-ray crystallography analysis, and structure models derived from lar dynamic simulations of FVIII, regions with a low degree of order (assessed by programs described in FIGS. 7 legend), regions of low homology/lack of conservation across different species, and hilic regions. In r embodiment, XTEN insertion sites were selected based on FVIII putative clearance receptor binding sites. In another embodiment, CFXTEN comprises XTEN inserted at locations not within close proximity to mutations implicated in hemophilia A listed in the Haemophilia A Mutation, Search, Test and Resource Site (HAMSTeRS) database were eliminated (Kemball-Cook G, et al. The factor VIII Structure and Mutation Resource Site: HAMSTeRS n 4. Nucleic Acids Res. (1998) 26(1):216-219). In another ment, potential sites for XTEN insertion include residues within FVIII epitopes that are capable of being bound by anti-FVIII antibodies occurring in sensitized hemophiliacs and that do not otherwise serve as protein ctive sites. Regions and/or sites that are ered for ion as XTEN insertion sites include residues/regions of factor VIII that are important in various interactions including other clotting proteins, residues surrounding each arginine ting/inactivating ge site acted on by the proteases thrombin, factor Xa, activated protein C, es surrounding the signal peptide processing site (residue 1) if the construct contains the signal peptide, regions known to interact with other proteins such as FIXa, FX/FXa, in, ted protein C, protein S cofactor to Protein C, von Willebrand , sites known to interact with phospholipid cofactors in coagulation, residues involved in domain interactions, residues coordinating Ca++ or Cu++ ions, cysteine residues involved in S-S intramolecular bonds, documented amino acid ion and point mutation sites in FVIII produced in hemophilia A subjects affecting procoagulant activity, and mutation sites in FVIII made in a research lab that affect procoagulant activity. Sites considered for either insertion (to prolong half-life) or for exclusion (needed to remove spent FVIIIa or FXa) include regions known to interact with heparin sulfate proteoglycan (HSPG) or low-density lip oprotein receptor-related protein (LPR).

By analysis of the foregoing ia, as described in Example 34, different insertion sites or ranges of insertions sites across the FVIII BDD sequence have been identified and/or confirmed as candidates for insertion of XTEN, non-limiting examples of which are listed in Table 5, Table 6, Table 7, Table 8, and Table 9 and are shown schematically in FIGS. 8 and 9. In one embodiment, CFXTEN comprise XTEN insertions between the individual domains of FVIII, i.e., between the Al and A2, or between the A2 and the B, or between the B and the A3, or between the A3 and the C1, or between the Cl and the C2 s. In another embodiment, CFXTEN comprises XTEN inserted within the B domain or between remnant residues of the BDD sequence. In another ment, CFXTEN comprises XTEN inserted at known exon boundaries of the encoding FVIII gene as exons represent evolutionary ved sequence modules that have a high probability of oning in the context of other protein ces. In another embodiment, CFXTEN comprise XTEN inserted within surface loops identified by the x-ray structure of FVIII. In another embodiment, CFXTEN comprise XTEN inserted within regions of low order identified as having low or no ed electron density by X-ray structure analysis.

In another embodiment, CFXTEN comprise XTEN inserted within regions of low order, predicted by structure prediction algorithms such as, but not limited to FoldIndex, RONN, and Kyte & lle algorithms. In another embodiment, CFXTEN comprise XTEN inserted within sequence areas of high frequency of hydrophilic amino acids. In another embodiment, CFXTEN comprise XTEN inserted within epitopes e of being bound by naturally-occurring anti-FVIII antibodies in sensitized hemophiliacs.

In r embodiment, CFXTEN comprise XTEN inserted within sequence areas of low sequence conservation and/or differences in sequence segment length across FVIII sequences from different species. In another embodiment, CFXTEN comprise XTEN linked to the N—terminus and/or C-terminus.

In another embodiment, the ion provides CFXTEN configurations with inserted XTEN selected from two or more of the criteria from the embodiments listed above. In another ment, the invention provides CFXTEN configurations with at least one, alternatively at least two, alternatively at least three, alternatively at least four, alternatively at least five or more XTEN inserted into a factor VIII sequence wherein the points of insertion are at or proximal to the N— or C-terminus side of the at least one, two, three, four, or five, or six or more amino acids selected from the insertion residue amino acids of Table 5, Table 6, Table 7, Table 8, and Table 9 or those rated in FIGS. 8-9, or alternatively within one, or within two, or within three, or within four, or within five, or within six amino acids of the insertion residue amino acids from Table 5, Table 6, Table 7, Table 8, and Table 9, or within the various spans of the ion e amino acids schematically yed for an exemplary FVIII BDD sequence in As described above, the one or more intemally—located XTEN or a fragment ofXTEN can have a sequence length of 6 to 1000 or more amino acid residues. In some embodiments, n the CFXTEN have one or two or three or four or five or more XTEN sequences internal to the FVIII, the XTEN sequences can be identical or can be different. In one embodiment, each internally-located XTEN has at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to comparable lengths or nts ofXTEN or motifs selected from any one of Tables 3, 4, and 13-17, when optimally aligned. In another embodiment, the invention provides a CFXTEN configured with one or more XTEN inserted internal to a FVIII BDD sequence with at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to a sequence of Table 1, wherein the insertions are located at the insertion points or range of insertion points ted in Table 5, Table 6, Table 7, Table 8, and Table 9, or within the range of insertions as illustrated in It will be understood by those of skill in the art that an XTEN ed within the FVIII sequence at an insertion point of Table 5, Table 6, Table 7, Table 8, and Table 9 is linked by its N— and C-termini to flanking FVIH amino acids (or via a linking spacer or cleavage sequences, as described above), while an XTEN linked to the N— or C-terminus of FVIII would only be linked to a single FVIII amino acid (or to a linking spacer or cleavage ce amino acid, as described above). By way of example only, variations of CFXTEN with three internal XTEN could have: XTEN (as described herein) orated between FVHI BDD residues 741 and 1640, residues 18 and 19, and residues 1656 and 1657; or XTEN incorporated between FVHI BDD es 741 and 1640, residues 1900 and 1901, and at the C-terminus at residue 2332; or XTEN orated between FVHI BDD residues 26 and 27, residues 1656 and 1657, and residues 1900 and 1901; or XTEN incorporated between FVHI BDD residues 741 and 1640, residues 1900 and 1901, and at the C-terminus at residue 2332.

In evaluating the CFXTEN fusion proteins with XTEN inserted in the locations from Table 5, it was discovered that insertions in certain regions of the FVIII sequence resulted in CFXTEN with good expression and retention of procoagulant activity. Accordingly, in preferred embodiments, the invention provides CFXTEN fusion proteins red with one, or two, or three, or four, or five, or six or more XTEN, each having at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to an XTEN ed from any one of Tables 4, and 13-17 inserted internal or linked to a FVIII BDD sequence with at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% ce identity compared to a sequence of Table 1, wherein the insertions are located at an insertion point within one, or two, or three, or four, or five, or six or more ranges set forth in Table 7. 1n the foregoing embodiments, the CFXTEN fusion proteins with the XTEN insertions retain at least about %, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the gulant activity compared to the corresponding FVIII not linked to XTEN.

In evaluating the CFXTEN fusion proteins with XTEN inserted in one or more locations from Table 5, it was singly discovered that a high percentage of fusion proteins with the XTEN insertions retained procoagulant activity, as described in Example 25. Accordingly, the invention provides CFXTEN fusion ns configured with one, two, three, four, five, six or more XTEN wherein the resulting fusion protein exhibits at least about 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 90% or more of the procoagulant activity compared to the corresponding FVIII not linked to XTEN when assayed by a coagulation assay described herein. In a preferred embodiment, the invention provides CFXTEN fusion proteins comprising one, or two, or three, or four, or five, or six or more XTEN, each having at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to an XTEN selected from any one of Tables 4, and 13-17 linked to a FVIII BDD ce with at least about 80% sequence ty, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to a sequence of Table 1, wherein the ions are located at one or more insertion points selected from Table 5, Table 6, Table 7, Table 8, and Table 9, and wherein the resulting fusion protein exhibits at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70% or more procoagulant activity ed to the ponding FVIII not linked to XTEN, when assayed in vitro by an assay described herein (e. g., a chromogenic assay). As the subject CFXTEN fusion ns typically exhibit sed terminal half-life compared to native FVHI, it will be appreciated by one of skill in the art that a CFXTEN with lower procoagulant activity ve to an equimolar amount of native FVIH would nevertheless be acceptable when administered as a therapeutic composition to a subject in need therof. In another embodiment, the CFXTEN fusion proteins comprising one, or two, or three, or four, or five or more XTEN, each having at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity ed to an XTEN selected from any one of Tables 4, and 13-17 linked to a FVIII BDD sequence with at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to a sequence of Table 1, n the insertions are located at one or more insertion points or the range of insertion points ed from Table , Table 6, Table 7, Table 8, and Table 9, wherein the resulting fusion protein exhibits at least about 0.5 IU/ml, or at least about 0.75 lU/ml, or at least about 1.0 lU/ml, or at least about 1.5 lU/ml, or at least about 2.0 IU/ml, or at least about 2.5 lU/ml, or at least about 3 lU/ml, or at least about 4 lU/ml, or at least about 5 lU/ml, or at least about 7 lU/ml, or at least about 10 lU/ml, or at least about 20 lU/ml, or at least about 30 lU/ml FVIII activity when expressed in cell culture medium and assayed in a chromogenic assay, wherein the culture and sion are according to methods described herein; e.g., the methods of Example 25.

It is believed that the discovery of the insertions sites wherein the FVIH retains at least a portion of its procoagulant activity would also permit the insertion of other peptides and polypeptides with either ctured or structured characteristics that are associated with the prolongation of half-life when fused to a FVIII protein in one or more of those same sites. Non-limiting examples include albumin, albumin fragments, Fc fragments of immunoglobulins, the B subunit of the C-terminal peptide (CTP) of human chorionic gonadotropin, a HAP sequence, a transferrin, the PAS polypeptides of US.

Pat Application No. 92130, polyglycine linkers, rine linkers, es and short polypeptides of 6-40 amino acids of two types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) with varying degrees of secondary structure from less than 50% to greater than 50%, amongst others, would be suitable for insertion in the identified active insertions sites of FVIII.

In the fusion protein ments described herein, the CFXTEN fusion protein can fithher comprise one or more cleavage sequence from Table 12 or other sequences known in the art, the cleavage sequence being located between or within 6 amino acid residues of the intersection of the FVIII and the XTEN sequences, which may include two cleavage sequences in a given internal XTEN sequence. In one embodiment, the CFXTEN comprising cleavage ces has two identical cleavage sequences, each located at or near the tive ends of one or more internal XTEN such that the XTEN is released from the fusion protein when cleaved by the protease that binds to and cleaves that sequence.

The sequences that can be cleaved are described more fully below and exemplary sequences are provided in Table 12.

Table 5: Insertion locations for XTEN linked to the FVIII BDD ce FVIH BDD XTEN Insertion Insertion FVH? P01nt. . Downstream Domaln Resume Sequence 1 0 (N-terminus) ATR A1 2 3 R RYY A1 3 17 M QSD A1 4 18 Q SDL A1 22 G ELP A1 6 24 L PVD A1 7 26 V DAR A1 8 28 A RFP A1 9 32 P RVP A1 3 8 F PFN A1 11 40 F NTS A1 12 41 N TSV A1 13 60 N IAK A1 14 61 I AKP A1 65 R PPW A1 16 81 Y DTV A1 17 111 G AEY A1 18 116 D QTS A1 19 119 S QRE A1 120 Q REK A1 21 128 V FPG A1 22 129 F PGG A1 23 130 P GGS A1 24 182 G SLA A1 185 A KEK A1 26 188 K TQT A1 27 205 G KSW A1 28 210 S ETK A1 29 211 E TKN A1 216 L MQD A1 31 220 R DAA A1 32 222 A ASA A1 FVHI BDD XTEN Insertion Insertion FVH? N0. . . ream Domaln Pomt ReSIdue —Seb 33 223 A SAR A1 34 224 s ARA A1 230 K MHT A1 36 243 p GL1 A1 37 244 G L1G A1 3 8 250 R KSV A1 39 318 D GME A1 40 333 P QLR A1 42 334 Q LRM A1 43 336 R MKN a1 44 339 N NEE a1 45 345 D YDD a1 46 357 V VRF a1 47 367 S FIQ a1 48 370 s RPY a1 49 375 A KKH A2 50 376 K KHP A2 51 378 H PKT A2 52 399 V LAP A2 53 403 D DRS A2 54 405 R SYK A2 55 409 S QYL A2 56 416 P QRI A2 57 434 E TFK A2 58 43 8 T REA A2 59 441 A IQH A2 60 442 1 QHE A2 61 463 I IFK A2 62 487 Y SRR A2 63 490 R LPK A2 64 492 P KGV A2 65 493 K GVK A2 66 494 G VKH A2 67 500 D FPI A2 68 506 G EIF A2 69 518 E DGP A2 70 556 K ESV A2 71 565 Q IMS A2 72 566 I MSD A2 73 598 P AGV A2 74 599 A GVQ A2 75 603 L EDP A2 76 616 s ING A2 77 686 G LWI A2 78 713 K NTG A2 79 719 Y EDS A2 80 730 L LSK A2 81 733 K NNA A2 82 745 N PPV B 83 1640 P PVL B 84 1652 R TTL B 85 1656 Q SDQ A3 86 1685 N QSP A3 FVHI BDD XTEN ion Insertion FVH? N0. . . Downstream Domaln P01nt ReSIdue Sequence 87 171 1 M SSS A3 88 1713 S SPH A3 89 1720 N RAQ A3 90 1724 S GSV A3 91 1725 G SVP A3 92 1726 S VPQ A3 93 1741 G SFT A3 94 1744 T QPL A3 95 1749 R GEL A3 96 1773 V TFR A3 97 1792 Y EED A3 98 1793 E EDQ A3 99 1796 Q RQG A3 100 1798 Q GAE A3 101 1799 G AEP A3 102 1802 P RKN A3 103 1803 R KNF A3 104 1807 V KPN A3 105 1808 K PNE A3 106 1827 K DEF A3 107 1844 E KDV A3 108 1861 N TLN A3 109 1863 L NPA A3 1 10 1896 E RNC A3 1 1 1 1900 R APC A3 1 12 1904 N IQM A3 1 13 1905 I QME A3 1 14 1910 P TFK A3 1 15 1920 A ING A3 1 16 1937 D QRI A3 1 17 1981 G VFE A3 1 18 2019 N KCQ A3 1 19 2020 K CQT C1 120 2044 G QWA C1 121 2068 F SW1 C1 122 2073 V DLL C1 123 2090 R QKF C1 124 2092 K FSS C1 125 2093 F SSL C1 126 21 1 1 K WQT C1 127 21 15 Y RGN C1 128 2120 T GTL C1 129 2125 V FFG C1 130 2171 L NSC C1 131 2173 S CSM C2 132 2188 A QIT C2 133 2223 V NNP C2 134 2224 N NPK C2 135 2227 K EWL C2 136 2268 G HQW C2 137 2277 N GKV C2 13 8 2278 G KVK C2 139 2290 F TPV C2 140 2332 Y C terminus of FVIII CT Indicates an ion point for XTEN based on the amino acid number of mature full-length human FVHI, wherein the insertion could be either on the N- or C-terminal side of the indicated amino acid Downstream sequence in FVIII BDD with 746-1639 deletion Table 6. agy insertion locations for XTEN linked to a FVIII polypeptide FVHI BDD XTEN Insertion FVIII D1§t3“°6.fr°m Insertion Point. . . Downstream . insertion Resume Domain Se residue. uence 9 32 P RVP A1 -3, --6 31 220 R DAA A1 - 34 224 S ARA A1 +5 43 336 R MKN a1 -1, --6 44 339 N NEE a1 -4, --5 52 399 V LAP A2 -6, ——3 56 416 P QRI A2 +6 75 603 L EDP A2 _6, --6 85 1656 Q SDQ B —3, --6 87 1711 M SSS A3 -6, --1 91 1725 G SVP A3 --6 1 13 1905 I QME A3 --6 114 1910 P TFK A3 -5, --6 Distance from insertion residue refers to the relative number of amino acids away from the N-terminus (negative numbers) or C-terminus (positive numbers) of the ated insertion residue (residue “0”) where an insertion may be made. The designation “-X” refers to an insertion site which is X amino acids away on the N-terminal side of the ated ion e. Similarly, the designation “+X” refers to an insertion site which is X amino acids away on the C-terminal side of the designated insertion residue.

For example, “-1, +2” indicates that the insertion is made at the N-terminus or C-terminus of amino acid residues denoted -1, 0, +1 or +2.

Table 7. Further exemplagy insertion locations for XTEN linked to a FVIII polypeptide XTEN Insertion First Insertion FVIII Domain Point Range Residue 3 18-32 Q A1 8 40 F A1 18 21 1-224 E A1 27 336-403 R A1, A2 43 599 A A2 47 745-1640 N B 50 728 Q B, A3 57 1796-1804 R A3 65 1900-1912 R A3 81 2171-2332 L C1,C2 indicates range of insertion sites numbered relative to the amino acid number of mature human FVIII Table 8. Exemplary XTEN ion locations within B-domain deleted variants of a FVIII polypeptide XTEN Insertion First Insertion Second Insertion Point Range Residue Residue 740-1640 R P 740-1690 R S 741-1648 S R 743-163 8 S Q 745-163 8 N Q 745-1640 N P 745-1656 N Q 745-1657 N S 745-1667 N T 745-1686 N Q 747-1642 R V 751-1667 T T indicates the amino acids linked Within the B-domain deleted variant and adjacent A3 domain, With the amino acids numbered relative to the amino acid number of mature human FVIII indicates the amino acids linked by an XTEN ed in the HI Table 9. Exemplary insertion locations for XTEN linked to a FVIII polypeptide resulting in gulant activity A F 111 BDD F III XTEN Insertlon N0. Insertion ReSidue Dywnmam Danain Point ——_____%gm_— 2 3 R RYY A1 4 18 Q SDL A1 22 G ELP A1 7 26 V DAR A1 11 40 F NTS A1 18 116 D QTS A1 19 119 S QRE A1 26 188 K TQT A1 29 211 E TKN A1 216 L MQD A1 31 220 R DAA A1 34 224 S ARA A1 230 K MHT A1 40 333 P QLR A1 43 336 R MKN a1 44 339 N NEE a1 52 399 V LAP A2 53 403 D DRS A2 55 409 s QYL A2 56 416 P QRI A2 60 442 l QHE A2 62 487 Y SRR A2 63 490 R LPK A2 66 494 G VKH A2 69 518 E DGP A2 74 599 A GVQ A2 75 603 L EDP A2 . FVIII BDD FVIII XTEN Insertion No. Insertion Residue Downstream Domain Pomt Sequence 78 713 K NTG A2 82 745 N PPV B 85 1656 Q SDQ A3 87 1711 M SSS A3 89 1720 N RAQ A3 91 1725 G SVP A3 99 1796 Q RQG A3 102 1802 P RKN A3 110 1896 E RNC A3 111 1900 R APC A3 112 1904 N IQM A3 113 1905 I QME A3 114 1910 P TFK A3 121 2068 F SWI C1 130 2171 L NSC C1 135 2227 K EWL C2 137 2277 N GKV C2 140 2332 Y C us of FVIII C2 Downstream sequence in FVIII BDD with 746-1639 deletion In another aspect, the ion provides libraries of components and methods to create the libraries d from nucleotides encoding FVIII segments, XTEN, and FVIII segments linked to XTEN that are useful in the preparation of genes encoding the t CFXTEN. In a first step, a library of genes encoding FVIII and XTEN ed into the various single sites at or within 1-6 amino acids of an insertion site identified in Table 5 or rated in FIGS. 8-9 are created, expressed, and the CFXTEN red and evaluated for activity and pharmacokinetics as illustrated in . Those CFXTEN showing enhanced properties are then used to create genes encoding a FVIII segment and the insertion site plus an XTEN, with components from each enhanced insertion represented in the library, as illustrated in . In one embodiment, the library components are assembled using standard recombinant techniques in combinatorial fashion, as rated in , resulting in permutations of CFXTEN with multiple internal and N- and C-terminus XTEN, that can include the insertion sites of or proximal to those Table 5, Table 6, Table 7, Table 8 and Table 9, or as illustrated in FIGS. 8-9. The resulting constructs would then be evaluated for activity and enhanced pharmacokinetics, and those candidates resulting in CFXTEN with enhanced properties, e.g., reduced active clearance, ance to proteases, d immunogenicity, and enhance pharmacokinetics, compared to FVIII not linked to XTEN, are evaluated further. 3. XTEN Permissive Loops As described in detail elsewhere herein and as illustrated in FIGS.33-3 6, the inventors have recognized that each FVIII “A” domain comprise at least two “XTEN permissive loops” into which XTEN sequences can be inserted without eliminating procoagulant activity of the inant protein, or the ability of the recombinant proteins to be expressed in Vivo or in Vitro in a host cell. The inventors have identified the XTEN permissive loops as regions with, among other attributes, high surface or solvent exposure and high mational flexibility. The Al domain comprises an XTEN permissive loop-l (Al -1) region and an XTEN permissive loop-2 (Al -2) region, the A2 domain ses an XTEN permissive loop-l (A2-l) region and an XTEN permissive loop-2 (A2-2) region, the A3 domain comprises an XTEN permissive loop-l (A3-l) region and an XTEN permissive loop-2 (A3-2) ..

In n aspects a recombinant FVIII protein as bed above comprises at least one XTEN sequence inserted into at least one of the XTEN permissive loops Al-l, A1-2, A2-1, A2-2, A3-1, or A3- 2, wherein the recombinant FVIII protein has procoagulant activity and can be expressed in Vivo or in Vitro in a host cell. In certain aspects a recombinant FVIII protein as described above comprises at least two XTEN sequences ed into FVIII, e. g., into two ent XTEN permissive loops Al-l, A1-2, A2-l, A2-2, A3-l, or A3 -2, wherein the recombinant FVIII protein has procoagulant activity and can be expressed in Vivo or in Vitro in a host cell. Alternatively, a recombinant FVIII protein as described above can comprise two or more XTEN sequences inserted into a single XTEN permissive loop either with our without XTEN sequences inserted into other XTEN permissive loops, wherein the recombinant FVIII protein has procoagulant actiVity and can be expressed in Vivo or in Vitro in a host cell. In certain aspects a recombinant FVIII protein as described above can comprise at least one XTEN sequence inserted into at least one of the XTEN permissive loops as described above, and can r comprise one or more XTEN sequences inserted into a3, wherein the recombinant FVIII protein has procoagulant actiVity and can be sed in Vivo or in Vitro in a host cell. In certain aspects, a recombinant FVIII protein of the invention can comprise three, four, five, six or more XTEN sequences inserted into one or more XTEN permissive loops or into a3, wherein the recombinant FVIII protein has procoagulant actiVity and can be expressed in Vivo or in Vitro in a host cell.

In certain aspects a recombinant FVIII protein as described above comprises at least one XTEN sequence inserted into a3, wherein the recombinant FVIII protein has procoagulant actiVity and can be expressed in Vivo or in Vitro in a host cell. In certain aspects a recombinant FVIII n of the invention comprises at least one XTEN sequence inserted into a3, and r comprises one or more XTEN sequences ed into one or more XTEN permissive loops as described above, wherein the recombinant FVIII protein has procoagulant actiVity and can be expressed in Vivo or in Vitro in a host cell.

The inventors have recognized that a recombinant FVIII protein of the invention comprises at least two XTEN permissive loops in each of the FVIII A domain regions which allows for insertion of an XTEN sequence while haVing procoagulant actiVity and still being able to be expressed in Vivo or in Vitro by a host cell. Various crystal structures of FVIII have been determined, of varying degrees of resolution. These structures of FVIII and FVIIIa, determined by X-ray crystallography and lar c simulation, were used to generate models of accessible surface area and conformational flexibility for FVIII. For example, the crystal structure of human FVIII has been determined by Shen et al. Blood 111: 1240-1247 (2008) and Ngo et al. Structure 16: 597-606 . The data for these structures is available from the Protein Data Bank (pdb.org) under ion Numbers 2R7E and 3CDZ, respectively.

The predicted secondary structure of the heavy and light chains of human FVIII according to the Shen et al. crystal structure is reproduced in FIGS. 37A and 37B. The s beta strands predicted from the Shen et al. crystal structure are numbered consecutively in FIGS. 8A and 8B. In n embodiments, the XTEN permissive loops A1-1, A1-2, A2-1, A2-2, A3-1, and A3 -2 are contained within surface-exposed, flexible loop structures in the A domains of FVIII. A1-1 is located between beta strand 1 and beta strand 2, A1-2 is located between beta strand 11 and beta strand 12, A2-1 is located between beta strand 22 and beta strand 23, A2-2 is located between beta strand 32 and beta strand 33, A3-1 is located between beta strand 38 and beta strand 39 and A3-2 is located between beta strand 45 and beta strand 46, ing to the ary structure of mature FVIII stored as Accession Number 2R7E of the PDB database (PDB:2R7E) and as shown in FIGS. 8A and 8B. The secondary structure of PDB Accession Number 2R7E shown in FIGS. 8A and 8B corresponds to the standardized secondary structure assignment according to the DSSP program (Kabsch and Sander, Biopolymers, 22:2577-2637 (1983)).

The DSSP secondary structure of the mature FVIII stored as PDB Accession Number 2R7E can be accessed at the DSSP database, available at the world wide web site swift.cmbi.ru.n1/gV/dssp/ (last accessed February 9, 2012) (Joosten et al., 39(Suppl. 1): D411-D419 (2010)).

In certain aspects, a surface-exposed, flexible loop structure comprising A1-1 ponds to a region in native mature human FVIII from about amino acid 15 to about amino acid 45 of . In certain aspects, A1-1 ponds to a region in native mature human FVIII from about amino acid 18 to about amino acid 41 of . In certain aspects, the surface-exposed, flexible loop ure comprising A1-2 corresponds to a region in native mature human FVIII from about amino acid 201 to about amino acid 232 of . In certain aspects, A1-2 corresponds to a region in native mature human FVIII from about amino acid 218 to about amino acid 229 of . In certain aspects, the surface-exposed, e loop structure comprising A2-1 corresponds to a region in native mature human FVIII from about amino acid 395 to about amino acid 421 of . In n aspects, A2-1 corresponds to a region in native mature human FVIII from about amino acid 397 to about amino acid 418 of . In certain aspects, the surface-exposed, e loop structure sing A2-2 corresponds to a region in native mature human FVIII from about amino acid 577 to about amino acid 635 of . In certain aspects, A2-2 corresponds to a region in native mature human FVIII from about amino acid 595 to about amino acid 607 of . In certain aspects, the surface-exposed, flexible loop structure comprising A3-1 corresponds to a region in native mature human FVIII from about amino acid 1705 to about amino acid 1732 of . In certain s, A3-1 corresponds to a region in native mature human FVIII from about amino acid 1711 to about amino acid 1725 of .

In certain aspects, the surface-exposed, flexible loop structure sing A3-2 corresponds to a region in native mature human FVIII from about amino acid 1884 to about amino acid 1917 of In certain aspects, A3 -2 corresponds to a region in native mature human FVIII from about amino acid 1899 to about amino acid 1911 of .

In certain aspects a recombinant FVIII protein of the ion comprises one or more XTEN sequences inserted into one or more XTEN permissive loops of FVIII, or into the a3 region, wherein the recombinant FVIII n has procoagulant activity and can be expressed in Vivo or in Vitro in a host cell. XTEN sequences to be inserted include those that increase the in Vivo half-life or the in Vivo or in Vitro stability of FVIII.

In certain aspects, a recombinant FVIII protein of the invention comprises an XTEN sequences inserted immediately downstream of one or more amino acids corresponding to one or more amino acids in mature native human FVIII ing, but not limited to: amino acid 18 of , amino acid 26 of , amino acid 40 of , amino acid 220 of , amino acid 224 of , amino acid 399 of , amino acid 403 of , amino acid 599 of , amino acid 603 of , amino acid 1711 of , amino acid 1720 of , amino acid 1725 of , amino acid 1900 of , amino acid 1905 of , amino acid 1910 of , or any combination thereof, including corresponding insertions in EDD-variants of FVIII described herein.

In certain aspects, a recombinant FVIII protein of the invention comprises at least one XTEN sequence inserted into the a3 region of FVIII, either alone or in combination with one or more XTEN sequences being inserted into the XTEN permissive loops of the A domains (e. g., Al-l, A1-2, A2-1, A2- 2, A3-1, or A3 -2 as described above), wherein the inant FVIII protein has procoagulant actiVity and can be expressed in Vivo or in Vitro in a host cell. In n aspects, at least one XTEN sequence is inserted into the a3 region immediately downstream of an amino acid which corresponds to amino acid 1656 of . In certain aspects, a recombinant FVIII protein of the invention comprises an XTEN sequence inserted into the a3 region as described, and further includes one or more XTEN sequences inserted immediately downstream of one or more amino acids ponding to one or more amino acids in mature native human FVIII including, but not d to: amino acid 18 of , amino acid 26 of , amino acid 40 of , amino acid 220 of , amino acid 224 of , amino acid 399 of , amino acid 403 of , amino acid 599 of , amino acid 603 of , amino acid 1711 of , amino acid 1720 of , amino acid 1725 of , amino acid 1900 of , amino acid 1905 of , amino acid 1910 of , or any combination thereof.

It will be understood by one of skill in the art that the foregoing aspects of permissive loops of a native FVIII protein into which a heterologous protein can be inserted are also applicable to the B- domain deleted FVIII variants described herein; e. g., sequences set forth in Table 1. In practicing the present invention, it will be understood that a BDD-FVIII ce of Table 1 can be substituted for the inant FVIII protein of the various embodiments bed above, and it is believed that the resulting ucts will rly retain procoagulant actiVity. 4. Interference with FVIII binding agents It is an object of the present invention to provide procoagulant CFXTEN fusion protein compositions for use in human patients suffering from coagulopathies, such as haemophilia A, who have native or acquired antibodies, inhibitors, or other proteins or molecules that bind to FVIII that affect the actiVity or half-life of CFXTEN fusion proteins, wherein the CFXTEN retain a greater amount of procoagulant actiVity compared to the corresponding FVIII not linked to XTEN. As used herein, “FVIII binding agent” means any molecule capable of g to native FVIII or to a recombinant factor VIII fusion protein of the ion comprising factor VIII or a fragment thereof, whether native, derived, or produced recombinantly. It is specifically contemplated that FVIII binding agent includes anti-FVIII antibodies and FVIII inhibitors, amongst other proteins capable of ically binding to FVIII. In one aspect, the ion provides procoagulant CFXTEN fiJsion proteins that exhibit reduced binding to an anti-FVIII antibody or FVIII inhibitor that interferes with the procoagulant ty of FVIII. As used herein, “anti-FVIII antibody” or “anti-factor VIII antibody” means an antibody capable of binding FVIII or a FVIII component of a CFXTEN of the invention, said antibody including but not limited to the antibodies of Table 10 or polyclonal antibody from a hemophilia A patient with FVIII inhibitors. The term antibody includes monoclonal antibodies, polyclonal antibodies, antibody fragments and antibody fragment . As used herein, “FVIII inhibitor” or “anti-FVIII inhibitor antibody” means an dy capable of binding FVIII or a FVIII component of a CFXTEN of the invention and that reduces by any means the procoagulant actiVity of FVIII or the FVIII component of a CFXTEN. In r aspect, the invention provides CFXTEN fusion proteins that retain procoagulant actiVity in the presence of a FVIII inhibitor. In another aspect, the invention provides CFXTEN fusion proteins sing FVIII that exhibit increased terminal half-life in the presence of a FVIII binding agent compared to the FVIII not linked to XTEN.

The majority of inhibitory antibodies to human factor VIII act by binding to epitopes located in the A2 domain or the C2 domain of factor VIII, disrupting specific functions ated with these domains, (US. Patent No. 6,770,744; Fulcher et al. Localization of human factor FVIII inhibitor epitopes to two polypeptide fragments. Proc. Natl. Acad. Sci. USA (1985) 82:7728-7732; Scandella et al. Epitope mapping of human factor VIII inhibitor antibodies by deletion analysis of fVIII fragments expressed in Escherichia coli. Proc. Natl. Acad. Sci. USA (1988) 85:6152-6156). While 68% percent of inhibitory antibodies are reported to be directed t the A2 and/or C2 domain, 3% act against the A1 domain and 46% against the a3 acidic region (LaVigne-Lissalde, G., et al. Characteristics, mechanisms of action, and e mapping of anti-factor VIII antibodies. Clin ReV Allergy Immunol (2009) 37:67-79). For example, certain heavy chain-specific tors react with the 18.3-kD amino-terminal segment of the A2 domain (Scandella D, et al. 198 8); Lollar P et al. Inhibition of human factor VIIIa by anti-A2 subunit antibodies. J Clin Invest 1994;93:2497). FVIII contains a phospholipid binding site in the C2 domain between amino acids 2302 and 2332, and there is also a von Willebrand factor binding site in the C2 domain that acts in conjunction with amino acids 1649-1689 in the A3 domain. The C2 domain also has epitopes that, when bound by inhibitors, block the tion of FVIII by thrombin or factor Xa.

Inhibitors binding cally to the light chain ize epitopes in the A3 domain or a major antigenic region in the C2 domain and can result in reduced procoagulant ty by preventing the g of FVIII to phospholipid or reducing the dissociation rate of FVIII from von Willebrand factor (Gilles JG, et al. Anti-factor VIII antibodies of hemophiliac patients are frequently ed towards nonfunctional determinants and do not exhibit isotypic restriction. Blood (1993) 82:2452; Shima M, et al. A factor VIII neutralizing onal antibody and a human inhibitor alloantibody recognizing epitopes in the C2 domain inhibit factor VIII g to von Willebrand factor and to phosphatidylserine. Thromb Haemost (1993) 69:240). Non-limiting examples of monoclonal FVIII tors are listed in Table 9. In patients with high-titer inhibitors, there is an increased risk of developing recurrent bleeding in particular joints, which may ultimately result in decreased quality of life, disability, or death from excessive blood loss (US. Pat. Application No. 20120065077; Zhang et al., Clinic. Rev. Allerg. Immunol., 37:114-124 (2009); Gouw and van den Berg, Semin. . Hemost., 35 :723-734 (2009)) While not intending to be bound by any particular theory, it is believed that the unstructured characteristic of the XTEN incorporated into the CFXTEN fusion proteins permits the XTEN to adopt conformations that result in steric hindrance to inhibitors that would ise bind to FVIII epitopes.

As illustrated in as the incorporated XTEN assumes s random coil conformations, it spatially covers regions of the FVIII component of the fusion protein and sterically eres with the ability of an tor to bind to a FVIII epitope.

] In one embodiment, the invention es CFXTEN exhibiting procoagulant actiVity and reduced binding in the presence of an dy binding to the C2 domain of factor VIII ed to the corresponding factor VIII not linked to XTEN and/or to native FVIII. In another embodiment, the invention es CFXTEN exhibiting procoagulant activity and reduced binding in the presence of an antibody binding to the A2 domain of Factor VIII compared to the corresponding factor VIII not linked to XTEN or to native FVIII. In another embodiment, the invention es CFXTEN exhibiting procoagulant activity and reduced binding in the presence of antibodies g to the A2 and the C2 domain of Factor VIII, compared to the corresponding factor VIII not linked to XTEN or to native FVIII.

In one embodiment, the invention provides CFXTEN exhibiting procoagulant activity and reduced binding, compared to the corresponding FVIII not linked to XTEN, in the presence of an antibody selected from the group consisting of the antibodies of Table 10. In one embodiment, the CFXTEN fusion protein exhibits reduced binding to the antibody GMA8021. In another ment, the CFXTEN fusion protein exhibits reduced binding to the antibody GMA8008. In another embodiment, the CFXTEN fusion n exhibits reduced binding to the antibody ESH4. In r embodiment, the CFXTEN fusion protein exhibits reduced binding to the antibody ESH8. In another embodiment, the CFXTEN fusion protein exhibits reduced g to the antibody B02C11. In another embodiment, the CFXTEN fusion protein exhibits reduced binding and a greater degree of gulant activity, compared to the corresponding FVIII not linked to XTEN, in the presence of plasma from a hemophilia A subject with polyclonal antibody FVIII inhibitors, wherein the greater degree of procoagulant activity is determined by an in Vitro assay such as a Bethesda assay or other assay described herein.

The CFXTEN exhibiting reduced binding by FVIII inhibitors can have one, or two, or three, or four, or five, or six or more individual XTEN, embodiments of which are disclosed herein. In the foregoing embodiments of this paragraph, a CFXTEN exhibits at least 5%, or 10%, or 15%, or 20%, or %, or 40%, or 50%, or 60%, or 70% or less binding to the antibody when assessed in Vitro in an assay capable of assaying the binding of an dy to FVIII, such as assays described herein below or those known in the art. Alternatively, the reduced binding of the subject CFXTEN to the FVIII-binding antibodies can be assessed by retention of a higher degree of procoagulant activity in the presence of the antibody compared to FVIII not linked to XTEN, as described in the es. Thus, in the embodiments pertaining to reduced binding by FVIII inhibitors bed herein, a CFXTEN exhibits, when reacted with the anti-FVIH antibody, at least 5%, or 10%, or 15%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 100%, or 200%, or 300%, or 400%, or 500% or more activity in a coagulation assay (such as described herein below) compared to the corresponding FVIII not linked to XTEN and reacted with the antibody. In the foregoing, the VHI antibody can be an antibody from Table 9 or a circulating anti-FVHI antibody from a hemophilia A subject. In another embodiment, the invention provides CFXTEN in which the assayed fusion protein, when assayed utilizing the Bethesda assay and an anti-FVHI antibody selected from Table 10 or a polyclonal anti-FVHI antibody preparation such as, but not limited to, plasma from a hemophilia A subject with FVIII inhibitors, results in a Bethesda titer with at least about 2, 4, 6, 8, 10, 12, 15, 20, 30, 40, 50, 60, 70, 80, 100, or 200 fewer Bethesda units compared to a FVIII not linked to XTEN and assayed under able conditions. In another ment, the invention provides CFXTEN in which the d fusion protein results in less than 50%, or less than 40%, or less than 30%, or less than 25%, or less than 20%, or less than 15%, or less than 14%, or less than 13%, or less than 12%, or less than 11%, or less than 10% of the Bethesda Units compared to a FVIII not linked to XTEN when assayed under comparable conditions utilizing the da assay and a polyclonal anti-FVHI antibody preparation such as, but not limited to, plasma from a hemophilia A subject with FVHI inhibitors.

Table 10: Anti-factor VIII antibodies Antibody Epitope Inhibitor Titer Reference Designation BU/mg C2 Domain US. 6,770,744 BOZCH 20000 Met2199/Phe2200 Blood (2007) 110:4234—4242 C2 Domain NMC VIII-5 US. 6,770,744 Glu2181-Val2243 ESH2 Light Chain ADI Light Chain US. 6,770,744 ESH4 39 332 Blood (2007) 34—4242 C2 Domain US. 6,770,744 ESH8 10000 2248-2285 Blood {2007} 110:4234—4242 RHDS (LMBP C1 Domain US Pat. Appllcatlon 20090263380 6165CB; . US Pat. Application 20090263380 LE2E9 C1 Domam Blood (2000) 95:156-163 154 C2 Domain 1300 Blood (2007) 34—4242 F85 C2 Domain 6 Blood (2007) 34—4242 131 00 C2 Domain 5 Blood 2007 110:4234—4242 E137 C2 Domain 6 Blood (2007) 110:4234—4242 189 C2 Domain 1900 Blood (2007) 110:4234—4242 1117 C2 Domain 1800 Blood (2007) 110:4234—4242 ll 09 Meg9137;111:23200 1500 Blood (2007) 110:4234—4242 1135 C2 Domain 930 Blood (2007) 110:4234—4242 3C6 C2 Domain 71 Blood (2007) 110:4234—4242 31312 (3131:5113? 2600 Blood (2007) 110:4234—4242 Antibody Epitope Inhibitor Titer Reference Designation BU/mg 13102 C2 Domain 3800 Blood 2007 110:4234—4242 3G6 C2 Domain 25000 Blood (2007) 110:4234—4242 2—77 C2 Domain 25000 Blood (2007) 110:4234—4242 B45 C2 Domain 21000 Blood (2007) 110:4234—4242 B9 C2 Domain 31000 Blood 2007 110:4234—4242 1311 C2 Domain 3300 Blood 2007 110:4234—4242 B75 C2 Domain Indeterminate Blood (2007) 34—4242 3105 Va13221371i11212n227 08 Blood (2007) 110:4234—4242 F77 C2 Domain 26000 Blood 2007 110:4234—4242 13178 C2 Domain 18000 Blood (2007) 110:4234—4242 F67 C2 Domain 21000 Blood (2007) 34—4242 (399 Va13221371i11212n227 15000 Blood (2007) 110:4234—4242 G86 C2 Domain 4300 Blood (2007) 110:4234—4242 114 C2 Domain 44000 Blood (2007) 110:4234—4242 155 C2 Domain 10000 Blood (2007) 34—4242 2»—--1 17 C2 Domain >0.4 Blood 2007 110:4234—4242 A2 domain GMAOIZ GVIA 497-510; 584-593 GVIA8001 A3 Domain 156 GVIA GVIA8002 A1 Domain <1 GVIA GVIA8003 C2 Domain GVIA GVIA8004 A1 Domain GVIA GVIA8005 A1A3/A1 Domain GVIA GVIA8006 c2 Domain GVIA GVIA8008 C2 Domain 1047 GVIA GVIA8009 A2 Domain 7923 GVIA GVIA8010 LC Domain GVIA GVIA8011 C1 Domain 97 GVIA GVIA8012 A1A3 Domain 204 GVIA GVIA8013 A3C2 Domain 30 GVIA GVIA8014 C2 Domain 7799 GVIA GVIA8015 A2 Domain 17079 GVIA 16 A2 Domain <1 GVIA GVIA8017 A2 Domain 334 GVIA GVIA8018 LC Domain 242 GVIA GVIA8019 CR-LC Domain GVIA GVIA8020 A1A3 Domain 196 GVIA GVIA8021 A2 Domain 33928 GVIA 4A4 A2 Domain 40000 231mm) Haemost (2009) 7.658- 3136 C2 Domain 41 Blood (2007) 110:4234—4242 American Diagnostica Inc. intemet site, URL located on the World Wide Web at americandiagnostica.c0m/html/Pr0duct_Detail.asp?idCategorF5&idSubCategor3F104&idpr0=ESH-8 as it existed on January 12, 2012 Green Mountain Antibodies t site, URL located on the World Wide Web at greenmoab.com/product_details/16316/215 82.html as it d on January 12, 2012 Assays For Inhibitor and Antibody Binding The fusion proteins of the invention may be assayed to confirm reduced g by FVIII inhibitors using methods known in the art. The assays that can be used include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassays, ELISA, ich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion itin reactions, immunodiffusion assays, agglutination assays, immunoradiometric assays, fluorescent immunoassays, ng assays, factor VIII inhibitor assays to name but a few. Such assays are routine and well known in the art (see, e. g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary are described briefly below but are not ed by way of limitation.

The Bethesda assay and the Nijmegen modification of the Bethesda assay are factor VIII tor assays well-known as s to detect FVIII inhibitors (Kasper CK, et al. Proceedings: A more uniform measurement of factor VIII inhibitors. Thromb Diath Haemorrh. (1975) 34(2):612). r, the assays can be modified to assay binding of inhibitors to FVIII compositions using inhibitors such as polyclonal or monoclonal anti-FVIII antibodies, including the antibodies of Table 10, and methods such as described in Example 52. Briefly, the modified Bethesda assay involves mixing titered volumes of the test sample with an equal volume of an inhibitor at a set concentration. The mixtures are incubated for 2 hours at 37°C prior to analysis of the factor concentration by a coagulation assay such as a chromogenic assay. Similarly, a reference plasma with native factor VIII level is incubated that then assayed as the positive control. The nt is the titer resulting in 50% of the FVIII activity of the positive control, reported as Bethesda units. In the Nijimegen modification of the Bethesda assay, the assay samples are stabilized with imidazole buffer and the control sample is mixed with deficient plasma instead of buffer (Verbruggen B, et al. The Nijmegen modification of the Bethesda assay for factor VIII:C inhibitors: improved specificity and reliability. Thromb Haemost. (1995) 73(2):247-251).

Western blot is generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e. g., 8%—20% SDS-PAGE depending on the molecular weight of the antigen), erring the n sample from the rylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the ne in blocking solution (e. g., PBS with 3% BSA or t milk), washing the membrane in washing buffer (e. g., PBS-Tween 20), blocking the ne with primary antibody (the antibody of interest) diluted in blocking buffer, g the membrane in washing buffer, blocking the membrane with a secondary dy (which recognizes the y antibody, e. g., an anti-human antibody) conjugated to an enzymatic substrate (e. g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32 P or 125 I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e. g., Ausubel et al, eds, 1994, Current Protocols in Molecular y, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.

ELISA assays can detect antibodies to FVIII independent of their y to block the procoagulant activity of FVIII, and have been utilized for the detection of anti-FVIII developing in hemophilia A patients. In a tion of 131 patients with hemophilia A with inhibitors, the ELISA technique resulted in 97.7% sensitivity and 78.8% specificity, and had a high ve tive value (98.6%) n, P. G., et al. Evaluation of a novel ELISA screening test for detection of factor VIII inhibitory antibodies in haemophiliacs. Clin Lab Haematol (1999) 21 :125-128]. Other investigators have found a highly significant correlation between the Bethesda titer and the absorbance values in an ELISA assay for ing anti-FVIII Abs (Towfighi, F., et al. Comparative measurement of anti-factor VIII antibody by Bethesda assay and ELISA reveals restricted isotype profile and epitope specificity. Acta Haematol (2005) -90), with the added advantage of the ability to detect non-inhibitory anti-FVIII antibodies. Assay protocols comprise preparing the binding ligand, which may include a sample comprising either factor VIII polypeptide or the CFXTEN fusion protein, coating the well of a 96 well microtiter plate with the antibody, adding the ligand test sample and incubating, then adding a detection antibody and incubating prior to washing and adding a alkaline phosphatase- or dase-conjugated secondary antibody and incubating for an additional period before the addition of TMB substrate and sing for g by spectrophotometer at 450nm. In ELISAs the dy or inhibitor of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody or inhibitor of interest) conjugated to a detectable compound may be added to the well.

Further, instead of coating the well with the antibody, the ligand may be coated to the well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art (see, e. g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1). rd or modified ation assays are used to measure reduced binding of FVIII binding agents. In one exemplary method (further described in Example 28), the optimal concentration of a given FVIII inhibitor to utilize in the assay is first determined by a titration experiment using varying amounts of the inhibitory antibody incubated at 37°C for 2 hrs with the base vector expressing wild-type FVIII containing a His/Myc double tag. The FVIII activity is measured by the Coatest assay procedure described herein. The lowest concentration that results in optimal inhibition of FVIII activity is employed in the assay. In the assay, the FVIII inhibitor antibody at the optimal concentration is mixed with individual test samples and incubated at 37°C for 2 hrs. The resulting test samples are then collected and utilized in the Coatest activity assay, along with untreated aliquots of the CFXTEN and positive control in order to assess the residual and baseline FVIII ty for each test sample.

The invention provides s of making CFXTEN that t reduced binding to FVIII binding agents, ing FVIII inhibitors, and retention of procoagulant activity. In one ment, the method to make a CFXTEN with reduced g to FVIII inhibitors comprises the steps of selecting a FVIII sequence with at least 90% sequence identity to a sequence of Table 1, selecting one, two, three, four, five, or six or more XTEN each with at least 70%, or at least 80%, or at least 90%, or at least 95 - 99% sequence identity to XTEN sequences of comparable length from Table 4, creating expression ucts designed to locate said XTEN at or proximal to locations selected from Table 5, Table 6, Table 7, Table 8, and Table 9, expressing and recovering the resulting CFXTEN, and assaying the resulting fusion proteins in an assay described herein in order to confirm the d binding of the CFXTEN fusion protein. By the ive method, a CFXTEN ts at least 5% reduced, or at least % reduced, or at least 15% reduced, or at least 20% reduced, or at least 25% reduced, or at least 40% reduced, or at least 50% reduced, or at least 60% reduced, or at least 70% d, or at least 80% reduced binding to a FVIII binding agent including, but not limited to the dies of Table 10 or anti- FVIII antibodies from a hemophilia A subj ect, and retains at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70% procoagulant actiVity compared to the corresponding FVIII not linked to XTEN.

Up to 8-10% of hemophilia A patients have antibodies that bind FVIII without affecting its procoagulant properties; they are not, therefore categorized as FVIII inhibitors. However, the binding of antibodies to FVIII is believed to lead to immune complexes that are cleared by the innate immune response or are more tible to proteolytic degradation chkine MD. Circulating immune complexes containing anti-VIII antibodies in multi-transfused patients with hilia A. Clin Exp Immunol. (1980) 39(2):315-320). Accordingly, it is an object of the invention to provide CFXTEN fusion ns comprising one or more XTEN that exhibit reduced binding of antibodies to FVIII that are not inhibitors, wherein the degradation or clearance of the CFXTEN is reduced at least 5%, or 10%, or 15%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70% or less compared to a corresponding FVIII not linked to XTEN or to native FVIII bound by such antibodies. The reduced binding of antibodies to CFXTEN compared to FVIII not linked to XTEN or to native FVIII can be assayed by in Vitro and in vivo methods. In Vitro methods include the aforementioned ELISA and Western blot methods. The reduced degradation or clearance of CFXTEN can be assessed in vivo by use of animal models or in human clinical trials. In one type of trial, factor VIII or CFXTEN are administered separately, preferably by intravenous infusion, to cohorts of patients haVing factor VIII deficiency who have antibodies that promote degradation or clearance of therapeutic human factor VIII. The dosage of the administered test article is in a range between 5 and 50 IU/kg body weight, preferably 10-45 IU/kg, and most preferably 40 IU/kg body weight. Approximately 1 hour after each stration, the recovery of factor VIII or CFXTEN from blood samples is measured in a functional age or chromogenic coagulation assay to assess actiVity and by ELISA, HPLC, or similar assay to qualify the amount of intact factor VIII equivalent. s are taken again approximately 5-10 hours after infusion, and recovery is measured. Total ry and the rate of disappearance of factor VIII from the samples is predictive of the antibody titer, and the comparison of results from the factor VIII and CFXTEN indicates the degree of reduced clearance and/or degradation of the CFXTEN. In one ment, the CFXTEN fusion n exhibits at least 5% reduced, or at least 10% reduced, or at least 15% reduced, or at least 20% reduced, or at least 25% reduced, or at least 40% reduced, or at least 50% reduced, or at least 60% reduced, or at least 70% reduced, or at least 80% reduced binding to an anti-FVIII antibody that promotes nce but does not otherwise inhibit the procoagulant ty of intact native FVIII. In another embodiment, the CFXTEN fusion n exhibits at least 5% d, or at least 10% d, or at least 15% reduced, or at least 20% reduced, or at least 25% reduced, or at least 40% reduced, or at least 50% reduced, or at least 60% reduced, or at least 70% reduced, or at least 80% reduced binding to an anti-FVIII antibody that promotes the degradation of FVIII. In the foregoing embodiments of this paragraph, the reduced binding of the anti-FVIII antibody is atively characterized by an increased KD value of the FVIII antibody to the fusion protein ed to the FVIII of at least two-fold, or three- fold, or four-fold, or five-fold, or 10-fold, or 33-fold, or 100-fold, or 330-fold, or at least 1000-fold compared to the binding to the corresponding FVIII not linked to XTEN. In one embodiment, the CFXTEN fusion ns comprising one or more XTEN exhibiting reduced vity to an anti-FVIII antibody exhibits an sed terminal half-life when administered to a subject with anti-FVIII antibodies of at least 48 h, or at least 72 h, or at least 96 h, or at least 120 h, or at least 144 h, or at least 14 days, or at least 21 days compared to FVIII not linked to XTEN. In the foregoing embodiment, the subject can be a human hemophilia A subject or it can be a mouse hemophilia A subject with circulating anti-FVIII antibodies.

Another aspect of the present invention is the use of CFXTEN fusion protein for a specific therapy of a coagulopathy in a subject with a FVIII inhibitor. The invention provides a method of treating a subject with circulating FVIII inhibitor(s) comprising the step of administering a clotting- ive amount of a CFXTEN fusion protein to the subject wherein the fusion protein exhibits r procoagulant activity and/or clotting-effective concentrations of longer duration compared to either a corresponding factor VIII not linked to XTEN or compared to native factor VIII administered to the subject using a comparable amount and route of administration. In one embodiment of the method, the FVIII inhibitor in the subject is an anti-FVIII antibody. In another embodiment, the FVIII inhibitor is a neutralizing VIII antibody. In one embodiment, the FVIII inhibitor is an anti-FVIII antibody that binds to the A1 domain of FVIII. In another embodiment, the FVIII inhibitor is an anti-FVIII antibody that binds to the A2 domain of FVIII. In another embodiment, the FVIII inhibitor is an anti-FVIII antibody that binds to the A3 domain of FVIII. In another embodiment, the FVIII inhibitor is an anti- FVIII antibody that binds to the C1 domain of FVIII. In another embodiment, the FVIII inhibitor is an VIII antibody that binds to the C2 domain of FVIII. In another embodiment, the FVIII inhibitor is an anti-FVIII antibody that binds to both the C2 and A2 domain of FVIII. In another embodiment, the FVIII tor binds to a FVIII e capable of being bound by one or more antibodies of Table 10.

In r embodiment, the FVIII inhibitor is a polyclonal antibody from a hemophilia A subject with FVIII tor antibodies.

An obj ect of the present invention is the creation of CFXTEN with XTEN inserted to maximize the steric interference of FVIII binding agents that would otherwise bind to FVIII and lize procoagulant activity or result in the clearance or degradation of FVIII. Accordingly, in one approach the invention provides CFXTEN comprising one or more XTEN wherein the XTEN are inserted proximal to a binding site of a FVIII inhibitor or VIII antibody. In one embodiment, an XTEN is linked to the FVIII at a location selected from Table 5, Table 6, Table 7, Table 8, and Table 9 that is within about 50, or about 100, or about 150, or about 200, or about 250, or about 300 amino acids of a FVIII epitope that is bound by an antibody of Table 10. In another embodiment, the XTEN is linked to the FVIII within about 50, or about 100, or about 150, or about 200, or about 250, or about 300 amino acids of a FVIII epitope in the A2 or C2 domain that is bound by an antibody of Table 10. Accordingly, the invention provides CFXTEN fusion proteins comprising one or more XTEN wherein binding by FVIII inhibitors to the FVIII component of the fusion protein is reduced compared to the corresponding FVIII not linked to XTEN or to native FVIII and the CFXTEN retains procoagulant actiVity. In the foregoing embodiments hereinabove described in this paragraph, the fusion proteins can be assayed by the assays described herein below, the assays of the es, or other assays known in the art, and the inhibitors can be an antibody of Table 10, can be polyclonal anti-FVIII, or can be blood or plasma from a hemophilia A subject with FVIII inhibitors.

In another aspect, CFXTEN are designed to maximize the regions over which XTEN can adopt random coil conformations covering the fusion protein, thereby resulting in steric hindrance for anti- FVIII antibodies that would otherwise bind epitopes on the FVIII component of the fusion protein. It is believed that the incorporation of multiple XTEN into a CFXTEN provides a higher total hydrodynamic radius of the XTEN component ed to CFXTEN with fewer XTEN yet haVing imately the same total ofXTEN amino acids. Empirically, the hydrodynamic radius for a protein can be calculated based on size exclusion chromatography, and results of several fusion proteins using such methods are described in the Examples. Alternatively, the radius for XTEN ptides, such as those incorporated in the ments disclosed herein, can be approximated by mathematical ae because the limited types of amino acids utilized have known characteristics that can be quantified. In one embodiment, the maximum radius of a single XTEN polypeptide is calculated (hereinafter “XTEN Radius”) according to the a given by Equation II: XTEN Radius = («/XTEN length 0.2037) + 3.4627 11 In another embodiment, the sum of the maximum of the XTEN Radii for all XTEN segments in a CFXTEN is calculated (hereinafter “Sum XTEN ) according to the formula given by Equation III: Z XTEN Radiusi =1 Sum XTEN Radii = 111 wherein: n = the number ofXTEN segments and i is an iterator In r embodiment, the ratio of the SUM XTEN Radii of a CFXTEN comprising multiple XTEN to that of an XTEN Radius for a single XTEN of an equivalent length (in total amino acid residues to that of the ) is calculated (hereinafter “Ratio XTEN Radii”) ing to the formula given by Equation IV: [L1 XTEN Radiusi 1 [L1XTEN Lengthi * ) + 3.4627 Ratio XTEN Radii = ( IV wherein: n = the number ofXTEN segments and i is an iterator ] In applying the ons to the XTEN, it will be understood by one of skill in the art that the calculated values represent m values that could vary or be reduced depending on the host cell utilized for expression of the XTEN polypeptide. It is believed that while E. coli expression would result in XTEN that achieves the calculated values, expression in eukaryotic host cells in which XTEN may be glycosylated could result in a radius of the polypeptide less than the maximum calculated value. Such differences can be quantified by methods such as size exclusion chromatography, the methods of which are ed in the Examples.

In order to design CFTEN that maximize the area over which XTEN can adopt random coil conformations, it was discovered that CFXTEN designs with Ratio XTEN Radii above 2 provide greater coverage over the fusion protein than designs with values <2. Accordingly, in one embodiment the invention provides CFXTEN in which the Ratio XTEN Radii is at least 2.0, or 2.1, or 2.2, or 2.3, or 2.4, or 2.5, or 2.6, or 2.7, or 2.8, or 2.9, or 3.0, or 3.1, or 3.2, or 3.3, or 3.4, or 3.5 or greater. In some embodiments, the invention provides CFXTEN in which the Ratio XTEN Radii is at least 20-35 or greater comprise at least three XTEN with each XTEN having at least 42 to about 288 amino acids and wherein at least two of the XTEN are linked to the fusion n with no less than about 100, or about 200, or about 300, or about 400, or about 500 amino acids of separation between the two XTEN. In other embodiments, the invention provides CFXTEN in which the Ratio XTEN Radii is at least 20-35 or greater comprise at least four XTEN with each XTEN having at least 42 to about 288 amino acids and wherein at least three of the XTEN are linked to the fusion protein with no less than about 100, or about 200, or about 300, or about 400 amino acids of separation between any two of the three XTEN.

In another embodiment, the invention provides a CFXTEN in which the Ratio XTEN Radii is at least 20-35 or greater, the CFXTEN comprises at least three XTEN with each XTEN having at least 42 to about 288 amino acids and wherein at least two of the three of the XTEN linked to the fusion protein are separated by an amino acid sequence of at least 100, or about 200, or about 300 to about 400 amino acids, and the third XTEN is linked within the B domain (or fragment f) or within the C domain (or the terminus thereof). In another embodiment, the invention provides a CFXTEN in which the Ratio XTEN Radii is at least 20-35 or greater, the CFXTEN comprises at least four XTEN with each XTEN having at least 42 to about 288 amino acids and wherein at least three of the four of the XTEN linked to the fusion protein are separated by an amino acid sequence of at least 300 to about 400 amino acids and the fourth XTEN is linked within the B domain (or fragment thereof) or within the C domain (or the terminus thereof).

In yet other embodiments, the invention provides CFXTEN in which the Ratio XTEN Radii is at least 20-35 or r, the CFXTEN comprises at least five XTEN with four XTEN having at least 42 to about 144 amino acids n at least four of the XTEN are linked to the fusion protein with no less than about 100, 200, or about 300, or about 400 amino acids of separation between any two of the four XTEN and a fifth XTEN is linked Within the B domain (or fragment thereof) or Within the C domain (or the us thereof). In one embodiment, the invention provides a CFXTEN in Which the Ratio XTEN Radii is at least 20-35 or greater, the CFXTEN comprises at least five XTEN With four XTEN having at least 42 to about 144 amino acids Wherein at least three of the XTEN linked to the fusion protein are separated by an amino acid sequence of at least 300 to about 400 amino acids, the fourth XTEN is linked Within the B domain (or fragment thereof) and a fifth XTEN is linked Within the C domain (or the terminus thereof).

In one aspect, the invention provides CFXTEN in Which the Ratio XTEN Radii is at least 2.0, or 2.1, or 2.2, or 2.3, or 2.4, or 2.5, or 2.6, or 2.7, or 2.8, or 2.9, or 3.0, or 3.1, or 3.2, or 3.3, or 3.4, or 3.5 or r, and the composition does not comprise certain sequences. In one embodiment of the foregoing, the invention es CFXTEN in Which the Ratio XTEN Radii is at least 20-35 or greater With the proviso that the fusion protein does not comprise a sequence from any one of Table 50 or Table 51. In another embodiment of the foregoing, the invention es CFXTEN in Which the Ratio XTEN Radii is at least 20-35 or greater With the proviso that the fusion protein does not comprise a sequence haVing an AG family XTEN sequence. In another embodiment of the foregoing, the invention es CFXTEN in Which the Ratio XTEN Radii is at least 20-35 or greater With the proviso that the fusion protein does not comprise a sequence selected from GTPGSGTASSSP (SEQ ID NO: 31), GSSTPSGATGSP (SEQ ID NO: 32), GSSPSASTGTGP (SEQ ID NO: 33), GASPGTSSTGSP (SEQ ID NO: 34). In another embodiment of the foregoing, the invention provides CFXTEN in Which the Ratio XTEN Radii is at least 20-35 or greater With the proviso that the fusion protein does not comprise any one of the sequences selected from TASSSP (SEQ ID NO: 31), GSSTPSGATGSP (SEQ ID NO: 32), STGTGP (SEQ ID NO: 33), GASPGTSSTGSP (SEQ ID NO: 34) and GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSG SETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTST EPSEGSAP (SEQ ID NO: 59). In another embodiment of the foregoing, the ion provides CFXTEN in Which the Ratio XTEN Radii is at least 20-35 or greater With the proviso that the fusion n does not comprise a sequence selected from GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSG SETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTST EPSEGSAP (SEQ ID NO: 59), PGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTS STGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTP GSGTASSS (SEQ ID NO: 71), or TSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSG ATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGA SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTG SPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSG TASSSPGSSTPSGATGS (SEQ ID NO: 80). In another embodiment of the foregoing, the invention provides CFXTEN in which the Ratio XTEN Radii is at least 2.0-3.5 or greater with the proviso that the fusion protein does not comprise an XTEN ce consisting of GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSG SETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTST EPSEGSAP (SEQ ID NO: 59), ASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTS STGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTP GSGTASSS (SEQ ID NO: 71), or PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSG ATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGA SPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTG SPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSG TASSSPGSSTPSGATGS (SEQ ID NO: 80).

In one aspect, the present invention provides methods to create CFXTEN with XTEN inserted to maximize the steric interference of FVIII binding agents that would otherwise bind to FVIII and lize procoagulant activity or result in the clearance or degradation of FVIII. Accordingly, in one embodiment, the invention provides a method comprising the steps of selecting a FVIII sequence with at least 90% sequence ty to a sequence of Table 1, selecting three or more XTEN from Table 4 in which the Ratio XTEN Radii is at least 2.0, or 2.1, or 2.2, or 2.3, or 2.4, or 2.5, or 2.6, or 2.7, or 2.8, or 2.9, or 3.0, or 3.1, or 3.2, or 3.3, or 3.4, or 3.5 or greater, creating expression constructs designed to locate said XTEN at or proximal to locations selected from Table 5, Table 6, Table 7, Table 8, and Table 9, wherein the three or more XTEN are at least 300 to 400 amino acids, expressing and recovering the resulting CFXTEN, and assaying the resulting fusion proteins in an assay described herein in order to m the reduced binding of the CFXTEN fusion protein. By the inventive method, a CFXTEN exhibits at least 5% reduced, or at least 10% reduced, or at least 15% reduced, or at least 20% reduced, or at least 25% reduced, or at least 40% reduced, or at least 50% reduced, or at least 60% reduced, or at least 70% reduced, or at least 80% reduced binding to a FVIII binding agent including, but not limited to the antibodies of Table 10, and exhibits procoagulant ty.

. CFXTEN Fusion Protein Configurations with Spacer and Cleavage Sequences In another aspect, the invention provides CFXTEN configured with one or more spacer ces orated into or adjacent to the XTEN that are designed to incorporate or e a functionality or property to the composition, or as an aid in the assembly or manufacture of the fusion protein compositions. Such properties include, but are not limited to, inclusion of ge ce(s) to permit release of ents, inclusion of amino acids compatible with nucleotide restrictions sites to permit linkage of XTEN-encoding nucleotides to FVIII-encoding nucleotides or that facilitate construction of expression s, and linkers designed to reduce steric hindrance in regions of CFXTEN fusion proteins.

In an embodiment, a spacer sequence can be introduced between an XTEN sequence and a FVIII component to decrease steric hindrance such that the FVIII component may assume its desired tertiary structure and/or interact appropriately with its target substrate or processing enzyme. For spacers and methods of identifying desirable spacers, see, for example, George, et al. (2003) Protein Engineering :871—879, specif1cally incorporated by reference herein. In one embodiment, the spacer comprises one or more peptide sequences that are between 1—50 amino acid residues in length, or about 1—25 residues, or about 1-10 residues in length. Spacer sequences, exclusive of cleavage sites, can comprise any of the l L amino acids, and will ably have XTEN-like properties in that the majority of residues will be hydrophilic amino acids that are sterically unhindered such as, but not limited to, glycine (G), e (A), serine (S), threonine (T), glutamate (E), proline (P) and aspartate (D). The spacer can be a single glycine residue, polyglycines or polyalanines, or is predominately a mixture of combinations of glycine, serine and alanine residues. In one embodiment, a spacer ce, exclusive of ge site amino acids, has about 1 to 10 amino acids that consist of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P) and are substantially devoid of secondary structure; e. g., less than about 10%, or less than about 5% as determined by the Chou-Fasman and/or GOR algorithms. In one embodiment, the spacer ce is GPEGPS (SEQ ID NO: 1612). In another embodiment, the spacer sequence is GPEGPS (SEQ ID NO: 1612) linked to a cleavage ce of Table 12. In addition, spacer ces are designed to avoid the introduction of T-cell es which can, in part, be achieved by avoiding or limiting the number of hydrophobic amino acids utilized in the spacer; the determination of epitopes is described above and in the Examples.

In a particular ment, the CFXTEN fusion protein ses one or more spacer ces linked at the junction(s) between the payload FVIII sequence and the one or more XTEN incorporated into the fusion protein, wherein the spacer sequences comprise amino acids that are compatible with nucleotides encoding restriction sites. In another embodiment, the CFXTEN fusion protein comprises one or more spacer sequences linked at the junction(s) n the payload FVIII ce and the one more XTEN incorporated into the fusion protein wherein the spacer ces comprise amino acids that are compatible with nucleotides encoding restriction sites and the amino acids and the one more spacer sequence amino acids are chosen from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P). In r embodiment, the CFXTEN fusion protein comprises one or more spacer ces linked at the junction(s) between the payload FVIII sequence and one more XTEN incorporated into the fusion protein wherein the spacer sequences comprise amino acids that are compatible with nucleotides encoding restriction sites and the one more spacer sequences are chosen from the sequences of Table 11. The exact sequence of each spacer sequence is chosen to be compatible with cloning sites in expression vectors that are used for a ular CFXTEN construct. In one embodiment, the spacer sequence has properties compatible with XTEN. In one embodiment, the spacer ce is GAGSPGAETA (SEQ ID NO: 178). For XTEN sequences that are incorporated internal to the FVIII sequence, each XTEN would generally be flanked by two spacer sequences comprising amino acids compatible with restriction sites, while XTEN attached to the N— or C-terminus would only require a single spacer sequence at the junction of the two components and r at the opposite end for incorporation into the vector. As would be apparent to one of ordinary skill in the art, the spacer sequences comprising amino acids compatible with ction sites that are internal to FVIII could be d from the construct when an entire CFXTEN gene is tically generated.

Table 11: Spacer Seguences Compatible with ction Sites Spacer Sequence Restriction Enzyme GSPG (SEQ ID NO: 174) BsaI ETET (SEQ ID NO: 175) BsaI PGSSS (SEQ ID NO: 176) BbsI GAP AscI GPA FseI GPSGP (SEQ ID NO: 177) SfiI AAA SacII TG AgeI GT KpnI GAGSPGAETA (SEQ ID NO: 178) SfiI ASS XhoI In another aspect, the present invention provides CFXTEN configurations with cleavage sequences incorporated into the spacer sequences. In some embodiments, spacer sequences in a CFXTEN fusion protein composition comprise one or more cleavage sequences, which are identical or ent, wherein the cleavage sequence may be acted on by a protease, as shown in , to release FVIII, a FVIII component (e. g., the B domain) or XTEN sequence(s) from the fusion n. In one embodiment, the incorporation of the cleavage sequence into the CFXTEN is designed to permit release of the FVIII component that becomes active or more active (with respect to its ability serve as a membrane binding site for factors IXa and X) upon its release from the XTEN. In the foregoing embodiment, the gulant activity of FVIII component of the CFXTEN is increased after cleavage by at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% compared to the intact CFXTEN. The cleavage sequences are located sufficiently close to the FVIII sequences, generally within 18, or within 12, or within 6, or within 2 amino acids of the FVIII sequence, such that any remaining residues attached to the FVIII after cleavage do not appreciably interfere with the activity (e.g., such as binding to a clotting protein) of the FVIII, yet provide sufficient access to the protease to be able to effect cleavage of the cleavage ce. In some cases, the CFXTEN comprising the cleavage sequences will also have one or more spacer ce amino acids between the FVIII and the cleavage ce or the XTEN and the cleavage sequence to facilitate access of the protease; the spacer amino acids comprising any natural amino acid, including e, serine and alanine as preferred amino acids. In one embodiment, the cleavage site is a sequence that can be cleaved by a protease endogenous to the mammalian subject such that the CFXTEN can be cleaved after administration to a subject. In such case, the CFXTEN can serve as a prodrug or a circulating depot for the FVIII. In a particular construct of the foregoing, the CFXTEN would have one or two XTEN linked to the N— and/or the C-terminus of a FVIII-BDD via a cleavage sequence that can be acted upon by an activated coagulation factor, and would have an additional XTEN located n the processing amino acids at position R740 and R1689 such that the XTEN could be released, g a form of FVIII similar to native activated FVIII. In one embodiment of the foregoing construct, the FVIII that is released from the fusion protein by cleavage of the cleavage ce exhibits at least about a two-fold, or at least about a three-fold, or at least about a four-fold, or at least about a five-fold, or at least about a six-fold, or at least about a eight-fold, or at least about a ld, or at least about a 20-fold se in activity compared to the intact CFXTEN fusion protein.

Examples of ge sites contemplated by the ion include, but are not limited to, a polypeptide sequence cleavable by a mammalian endogenous protease selected from FXIa, FXIIa, kallikrein, FVIIIa, FVIIIa, FXa, FIIa (thrombin), Elastase-2, me B, MMP-l2, MMP-l3, MMP-l7 or MMP-20, or by mmalian proteases such as TEV, enterokinase, ssionTM protease (rhinovirus 3C protease), and sortase A. Sequences known to be cleaved by the foregoing proteases and others are known in the art. Exemplary cleavage sequences contemplated by the invention and the respective cut sites within the sequences are presented in Table 12, as well as sequence variants thereof.

For CFXTEN comprising incorporated cleavage sequence(s), it is generally preferred that the one or more cleavage sequences are substrates for activated clotting ns. For example, thrombin ated clotting factor II) acts on the sequence LTPRSLLV (SEQ ID NO: 1618) [Rawlings N.D., et al. (2008) Nucleic Acids Res, 36: D320], which is cut after the arginine at position 4 in the sequence. Active FIIa is produced by cleavage of FII by FXa in the presence of phospholipids and calcium and is down stream from factor VIII in the coagulation pathway. Once activated, its natural role in coagulation is to cleave fibrinogen, which then in turn, begins clot formation. FIIa activity is tightly controlled and only occurs when coagulation is necessary for proper hemostasis. By incorporation of the LTPRSLLV sequence (SEQ ID NO: 1618) into the CFXTEN between and linking the FVIII and the XTEN components, the XTEN is removed from the adjoining FVIII concurrent with activation of either the extrinsic or intrinsic coagulation pathways when coagulation is required physiologically, thereby ively releasing FVIII.

In another embodiment, the invention provides CFXTEN with incorporated FXIa cleavage sequences between the FVIII and XTEN component(s) that are acted upon only by initiation of the intrinsic coagulation system, wherein a gulant form of FVIII is released from XTEN by FXIa to participate in the coagulation cascade. While not intending to be bound by any particular theory, it is believed that the CFXTEN of the foregoing embodiment would sequester the FVIII away from the other ation s except at the site of active clotting, thus allowing for larger doses (and therefore longer dosing als) with minimal safety concerns.

] Thus, cleavage ces, particularly those susceptible to the gulant activated clotting proteins listed in Table 12, would provide for sustained release of FVIII that, in certain embodiments of the CFXTEN, can provide a higher degree of activity for the FVIII component released from the intact form of the CFXTEN, as well as additional safety margin for high doses of CFXTEN administered to a subject. In one embodiment, the invention provides CFXTEN comprising one or more cleavage sequences operably positioned to release the FVIII from the fusion protein upon cleavage, wherein the one or more cleavage sequences has at least about 86%, or at least about 92%, or 100% sequence identity to a sequence selected from Table 12.

In some embodiments, only the two or three amino acids flanking both sides of the cut site (four to six amino acids total) are orated into the cleavage sequence that, in turn, is incorporated into the CFXTEN of the embodiments, providing, e.g., XTEN release sites. In other embodiments, the incorporated cleavage ce of Table 12 can have one or more deletions or insertions or one or two or three amino acid tutions for any one or two or three amino acids in the known sequence, wherein the deletions, insertions or tutions result in reduced or enhanced susceptibility but not an absence of susceptibility to the protease, resulting in an ability to tailor the rate of release of the FVIII from the XTEN. Exemplary substitutions within cleavage sequences that are utilized in the CFXTEN of the invention are shown in Table 12.

Table 12: Protease Cleavage Seguences Eggaggmjg @2312? 8&3; Minimalcmsne SENgD FXIa KLTRtAET 179 KD/FL/T/RlVA/VE/GT/GV FXIa DFTRVVVG 18o KD/FL/T/RlVA/VE/GT/GV FXIIa VGG 181 NA Kallikrein SPFRlSTGG 182 -/-/FL/RY¢SR/RT/—/— FVIIa LQVRVIVGG 183 NA FIXa PLGRVIVGG 184 R¢-/—/—/— FXa IEGRtTVGG 185 IA/E/GFP/RlSTI/VFSHG FIIa (thrombin) LTPRVSLLV 186 A/R¢SAG/—/—/— Elastase-2 LGPVVSGVP 187 —/—/—/v1ATt—/—/—/— me-B VAGDISLEE 188 V/-/-/Dt-/-/-/- MMP-12 GPAGVLGGA 189 G/PA/—/GtL/—/G/— 19o MMP-13 GPAGVLRGA 191 GtL/—/GA/— 192 MMP-17 APLGVLRLR 193 -/PS/-/—¢LQ/—/LT/— MMP-2O PALPtLVAQ 194 NA TEV ENLYFQtG 195 ENLYFQtG/s 196 Enterokinase DDDKMVGG 197 DDDKMVGG 198 (Pizgieizzieoigll) 200 LEVLFQtGP 199 IGP Sortase A LPKTtGSEs 201 L/P/KEAD/TlG/JEKS/S 202 lindicates cleavage site NA: not applicable the listing of multiple amino acids before, between, or after a slash indicate alternative amino acids that can be substituted at the position; - indicates that any amino acid may be substituted for the corresponding amino acid indicated in the middle column 6. Exemplary CFXTEN Fusion Protein Sequences Non-limiting examples of sequences of fusion proteins containing a single FVIII linked to one or more XTEN are presented in Table 21 . The exemplary amino acid sequences of Table 21 (and the DNA sequences that encode them) contain his tags for ation purposes that, as would be apparent to one of skill in the art, can be deleted from the sequence without having an effect on the procoagulant activity of the CFXTEN fusion protein. In one embodiment, the CFXTEN of Table 21 further comprise amino acids on the N—terminus corresponding to that of native human FVIII (namely, the sequence MQIELSTCFFLCLLRFCFS (SEQ ID NO: 1611)) to aid in the expression and secretion of the CFXTEN fusion protein. In one embodiment, a CFXTEN composition comprises a fusion protein haVing at least about 80% sequence identity compared to a CFXTEN from Table 21, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% sequence identity as ed to a CFXTEN from Table 21, when optimally aligned. In another embodiment, a CFXTEN composition comprises a fusion protein from Table 21 in which the C- terminal his-his-his-his-his-his ce (SEQ ID NO: 1700) deleted. However, the invention also contemplates substitution of any of the FVIII sequences of Table l for a FVIII component of the CFXTEN of Table 21, and/or substitution of any ce of any one of Tables 3, 4, and l3-l7 for an XTEN component of the CFXTEN of Table 21. Generally, the resulting CFXTEN of the foregoing examples retain at least a n of the procoagulant actiVity of the corresponding FVIII not linked to the XTEN. In the foregoing fusion proteins hereinabove described in this aph, the CFXTEN fusion protein can further se one or more cleavage ces; e.g., a sequence from Table 12, the cleavage sequence being located between the FVIII and the XTEN sequences or between nt FVIII domains linked by XTEN. In some embodiments comprising cleavage sequence(s), the intact CFXTEN composition has less actiVity but a longer half-life in its intact form compared to a corresponding FVIII not linked to the XTEN, but is designed such that upon administration to a subject, the FVIII ent is gradually released from the fusion n by cleavage at the ge sequence(s) by endogenous proteases, whereupon the FVIII component exhibits procoagulant activity.

The CFXTEN compositions of the embodiments can be evaluated for actiVity using assays or in vivo parameters as described herein (e. g., in vitro coagulation assays, assays of Table 49, or a pharmacodynamic effect in a preclinical hemophilia model or in clinical trials in humans, using methods as described in the Examples or other methods known in the art for assessing FVIII actiVity) to determine the suitability of the configuration or the FVIII sequence variant, and those CFXTEN compositions ding after cleavage of any incorporated XTEN-releasing ge sites) that retain at least about %, or about 40%, or about 50%, or about 55%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95% or more activity compared to native FVIII sequence are considered suitable for use in the treatment of related conditions.

V). PROPERTIES OF THE CFXTEN COMPOSITIONS OF THE INVENTION (a) Pharmacokinetic ties of CFXTEN It is an object of the present invention to provide CFXTEN fusion proteins and pharmaceutical compositions comprising CFXTEN with enhanced cokinetics compared to FVIII not linked to XTEN. The pharmacokinetic properties of a FVIII enhanced by linking a given XTEN to the FVIII include, but are not limited to, terminal ife, area under the curve (AUC), Cmax, volume of distribution, maintaining the biologically active CFXTEN above a minimum effective blood unit concentration for a longer period of time compared to the FVIII not linked to XTEN. The enhanced properties permit less frequent dosing and/or a longer-lived procoagulant effect compared to a comparable dose of FVIII not linked to XTEN. ement of one or more of these properties can resulting benefits in the treatment of factor VIII-related conditions.

Exogenously administered factor VIII has been reported to have a terminal half-life in humans of imately 12-14 hours when complexed with normal von Willebrand factor n, whereas in the absence of von Willebrand factor, the half-life of factor VIII is reduced to 2 hours (Tuddenham EG, et al., Br J Haematol. (1982) 52(2):259-267; Bjorkman, S., et al. Clin Pharmacokinet. (2001) 40:815). As a result of the enhanced properties conferred by XTEN, the CFXTEN, when used at the dose and dose regimen determined to be appropriate for the t and its underlying condition, can achieve a circulating concentration resulting in a desired procoagulant or al effect for an extended period of time compared to a comparable dose of the corresponding FVIII not linked to XTEN. As used herein, a “comparable dose” means a dose with an equivalent moles/kg or International Units/kg (IU/kg) for the composition that is administered to a subject. It will be understood in the art that a rable dose" of FVIII not linked to XTEN would represent a lesser weight of drug but would have essentially the same IUs or mole-equivalents of CFXTEN in the dose.

An international unit (“IU”) of factor VIII is defined in the art as the ant actiVity t in 1 ml of normal human plasma. A normal, non-hemophilic individual human is expected to have about 100 IU/dL factor VIII actiVity. In hemophilia A, the doses required to treat are dependent on the condition. For minor bleeding, doses of native or recombinant factor VIII of 20 to 40 IU/kg are typically administered, as necessary. For moderate bleeding, doses of 30 to 60 IU/kg are administered as necessary, and for major bleeding, doses of 80 to 100 IU/kg may be required, with repeat doses of 20 to IU/kg given every 8 to 12 hours until the bleeding is ed. For prophylaxis against bleeding in patients with severe hemophilia A, the usual doses of native or inant FVIII preparations are 20 to 40 lU/kg body weight at intervals of about 2 to 3 days. A standard equation for estimating an appropriate dose of a composition comprising FVIII is: Required units = body weight (kg) x desired factor VIII rise (IU/dL or % of normal) x 0.5 (IU/kg per IU/dL).

In many cases, the therapeutic levels for FVIII in subjects of ent ages or degree of disease have been established and are available in published literature or are stated on the drug label for approved products containing the FVIII. For example, the Subcommittee on Factor VIII and Factor IX of the Scientific and Standardization Committee of the ational y on Thrombosis and Haemostasis posted, on the ISTH Website 29 November, 2000, that the most widely used measure of hemophilia A is established by determining the circulating concentrations of plasma FVIII procoagulant levels, with persons with <l% (< 0.01 IU/ml) factor VIII defined as severe; 1-5% (0.01 - 0.05 IU/ml) as moderately severe; and >5-40% (0.05 - <0.40 IU/ml) as mild, where normal is 1 IU/ml of factor VIIIC (100%). The eutic levels can be established for new compositions, including those CFXTEN and pharmaceutical compositions comprising CFXTEN of the disclosure, using rd s. In practicing the present invention, it will be understood that any dosage of CFXTEN that is effective may be used for treating bleeding episodes or maintaining hemostasis. The methods for establishing the therapeutic levels and dosing schedules for a given composition are known to those of skill in the art (see, e. g., Goodman & Gilman's The Pharmacological Basis of Therapeutics, 11th Edition, McGraw—Hill (2005)). For example, by using dose-escalation studies in subjects with the target condition to determine efficacy or a desirable pharmacologic effect, appearance of adverse events, and determination of ating blood levels, the therapeutic blood levels for a given subject or population of subjects can be determined for a given drug or biologic. The dose escalation studies would evaluate the activity of a CFXTEN through studies in a subject or group of hemophilia A subjects. The studies would monitor blood levels of procoagulant, as well as physiological or clinical parameters as known in the art or as described herein for one or more parameters associated with the factor VIII-related condition, or clinical ters associated with a beneficial outcome, together with observations and/or measured parameters to determine the no effect dose, adverse events, minimum effective dose and the like, together with measurement of pharmacokinetic parameters that establish the determined or derived ating blood levels. The s can then be ated with the dose administered and the blood concentrations of the therapeutic that are coincident with the foregoing determined parameters or effect levels. By these methods, a range of doses and blood concentrations can be correlated to the m effective dose as well as the maximum dose and blood concentration at which a desired effect occurs or is maintained and the period for which it can be maintained, y establishing the therapeutic blood levels and dosing le for the composition.

Thus, by the foregoing methods, a Cmin blood level is established, below which the CFXTEN fusion protein would not have the desired pharmacologic effect and a Cmax blood level, above which side effects such as thrombosis may occur (Brobrow, RS, JABFP (2005) ]47-149), establishing the therapeutic window for the composition.

One of skill in the art can, by the means disclosed herein or by other methods known in the art, confirm that the administered CFXTEN remains at therapeutic blood levels to maintain hemostasis for the d interval or requires adjustment in dose or length or sequence of XTEN. Further, the determination of the riate dose and dose frequency to keep the CFXTEN within the therapeutic window ishes the therapeutically ive dose regimen; the schedule for administration of multiple consecutive doses using a therapeutically effective dose of the fusion protein to a subject in need thereof resulting in consecutive Cmax peaks and/or Cmin troughs that remain above therapeutically- effective concentrations and result in an improvement in at least one measured parameter relevant for the target condition. In one embodiment, the CFXTEN or a pharmaceutical itions comprising CFXTEN administered at an appropriate dose to a subject results in blood concentrations of the CFXTEN fusion protein that remains above the minimum effective concentration to maintain hemostasis for a period at least about two-fold longer compared to the corresponding FVIII not linked to XTEN and administered at a comparable dose; alternatively at least about three-fold longer; alternatively at least about four-fold longer; alternatively at least about five-fold longer; alternatively at least about siX-fold longer; alternatively at least about seven-fold longer; atively at least about eight-fold longer; alternatively at least about nine-fold longer, alternatively at least about ten-fold longer, or at least about twenty-fold longer or greater compared to the corresponding FVIII not linked to XTEN and administered at a able dose. As used herein, an “appropriate dose” means a dose of a drug or biologic that, when stered to a subject, would result in a desirable therapeutic or cologic effect (e.g., hemostasis) and/or a blood concentration within the therapeutic window.

In practicing the invention, CFXTEN with longer terminal half-life are generally preferred, so as to improve patient convenience, to increase the al between doses and to reduce the amount of drug required to achieve a sustained . The enhanced PK parameters allow for reduced dosing of the subject compositions, ed to FVIII not linked to XTEN, particularly for those hemophilia A subjects receiving routine prophylaxis.

As described more fully in the es pertaining to pharmacokinetic characteristics of fusion proteins comprising XTEN, it was observed that increasing the total length of the XTEN, singly or in combination, confers a disproportionate increase in the terminal ife of a fusion n comprising the XTEN. Accordingly, the invention provides CFXTEN fusion proteins and pharmaceutical compositions comprising CFXTEN n the CFXTEN exhibits an enhanced half-life when administered to a subject. In some embodiments, the invention provides monomeric CFXTEN fusion proteins comprising one or more XTEN wherein the number and location of the XTEN are ed to confer an increase in the terminal half-life for the CFXTEN administered to a subject compared to the corresponding FVIII not linked to the XTEN and administered at a comparable dose, n the increase is at least about two-fold longer, or at least about three-fold, or at least about old, or at least about five-fold, or at least about six-fold, or at least about seven-fold, or at least about eight-fold, or at least about nine-fold, or at least about ten-fold, or at least about d, or at least a 20-fold, or at least a 40-fold or greater increase in terminal half-life compared to the FVIII not linked to the XTEN. In other embodiments, the invention provides CXTEN compositions and pharmaceutical compositions comprising CFXTEN wherein the administration of a composition to a subject in need thereof results in a terminal half-life that is at least 12 h greater, or at least about 24 h greater, or at least about 48 h greater, or at least about 96 h greater, or at least about 144 h greater, or at least about 7 days greater, or at least about 14 days greater, or at least about 21 days greater compared to a able dose of FVIII not linked to XTEN. In another embodiment, administration of a coagulation-effective dose of a CFXTEN fusion protein to a subject in need f can result in a gain in time between consecutive doses ary to maintain blood levels of about 0.1 IU/ml of at least 48 h, or at least 72 h, or at least about 96 h, or at least about 120 h, or at least about 7 days, or at least about 14 days, or at least about 21 days n consecutive doses compared to a FVIII not linked to XTEN and administered at a able dose.

In one ment, the present invention provides CFXTEN fusion ns and ceutical compositions comprising CFXTEN that exhibit, when administered to a t in need thereof, an increase in AUC of at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about a 100%, or at least about 150%, or at least about 200%, or at least about 300%, or at least about 500%, or at least about 1000%, or at least about a 2000% compared to the corresponding FVIII not linked to the XTEN and administered to a subject at a comparable dose. The pharmacokinetic parameters of a CFXTEN can be determined by standard methods involving dosing, the taking of blood samples at timed intervals, and the assaying of the n using ELISA, HPLC, radioassay, clotting assays, the assays of Table 49, or other methods known in the art or as described herein, followed by standard calculations of the data to derive the half-life and other PK parameters.

In one embodiment, a smaller IU amount of about two-fold less, or about three-fold less, or about four-fold less, or about five-fold less, or about siX-fold less, or about eight-fold less, or about 10- fold less or greater of the fusion protein is administered in comparison to the corresponding FVIII not linked to the XTEN under a dose regimen needed to maintain hemostasis and the fusion protein achieves a comparable area under the curve as the corresponding IU amount of the FVIII not linked to the XTEN needed to maintain hemostasis. In another embodiment, the CFXTEN fusion protein or a pharmaceutical compositions comprising CFXTEN requires less frequent administration for routine laxis of a hemophilia A subject, wherein the dose of fusion protein is administered about every four days, about every seven days, about every 10 days, about every 14 days, about every 21 days, or about monthly to the subject, and the fusion protein achieves a comparable area under the curve as the corresponding FVIII not linked to the XTEN and administered to the subject. In yet other embodiments, an accumulative smaller IU amount of about 5%, or about 10%, or about 20%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90% less of the fusion protein is administered to a subject in comparison to the corresponding IU amount of the FVIII not linked to the XTEN under a dose regimen needed to maintain a blood concentration of 0.1 IU/ml, yet the fusion protein achieves at least a able area under the curve as the corresponding FVIII not linked to the XTEN. The accumulative smaller IU amount is measure for a period of at least about one week, or about 14 days, or about 21 days, or about one month.

In one aspect, the invention provides CFXTEN compositions designed to reduce g by FVIII g agents, thereby increasing the al half-life of CFXTEN administered to a subject, while still retaining procoagulant activity. It is believed that the CFXTEN of the present ion have comparatively higher and/or sustained activity achieved by reduced active clearance of the molecule by the addition of unstructured XTEN to the FVIII coagulation factor. The nce mechanisms to remove FVIII from the circulation have yet to be fully elucidated. Uptake, elimination, and inactivation of coagulation proteins can occur in the atory system as well as in the extravascular space.

Coagulation factors are complex ns that interact with a large number of other ns, lipids, and receptors, and many of these interactions can contribute to the elimination of CFs from the circulation.

The n von Willebrand factor is an example of a FVIII binding agent that binds to FVIII. Factor VIII and von Willebrand factor (VWF) circulate in the blood as a tight, non-covalently linked complex in which VWF serves as a carrier that likely butes to the protection of FVIII from active ge mechanisms, yet nevertheless results in a limitation on the terminal half-life of FVIII. For example: (i) VWF stabilizes the heterodimeric ure of FVIII; (ii) VWF protects FVIII from proteolytic degradation by phospholipid-binding proteases like activated protein C and activated FX (FXa); (iii) VWF interferes with binding of FVIII to negatively d phospholipid surfaces exposed within activated platelets; (iV) VWF inhibits binding of FVIII to activated FIX (FIXa), thereby denying FVIII access to the ivating complex; and (V) VWF prevents the cellular uptake of FVIII (Lenting, P.J., et al., J Thrombosis and tasis (2007) 5(7):1353-1360). In addition, LDL receptor-related protein (LRPl, also known as 0t2-macrogobulin receptor or CD91) has been identified as a candidate clearance receptor for FVIII, with LRPl binding sites identified on both chains of the heterodimer form of FVIII (Lenting PJ, et al.,. J Biol Chem (1999) 274: 23734—23739; Saenko EL, et al., J Biol Chem (1999) 274: 37692). LRPs are involved in the clearance of a diversity of ligands including proteases, inhibitors of the Kunitz type, protease serpin complexes, lipases and oteins (Narita, et al., Blood (1998) 2:555-560). It has been shown that the light chain, but not the heavy chain, of factor VIII binds to surface-exposed LRPl receptor protein (Lentig et al. (J Biol Chem (1999) 274(34):23734-23739; and US. Pat. No. 6,919,311), which suggests that LRPl may play an essential role in the active clearance of proteins like FVIII. While the VWF—FVIII interaction is of high affinity (<1 nM), the complex is nevertheless in a dynamic equilibrium, such that a small but cant portion of the FVIII molecules (5—8%) circulate as a free protein (Leyte A, et al., Biochem J (1989) 257: 679—683; Noe DA.

Haemostasis (1996) 26: 289—3 03). As such, a portion of native FVIII is unprotected by VWF, allowing active clearance mechanisms to remove the unprotected FVIII from the circulation.

In one embodiment, the invention provides CFXTEN that associate with VWF but have enhanced protection from active clearance receptors conferred by the incorporation of two more XTEN at one or more locations within the FVIII molecule (e. g., locations selected from Table 5, Table 6, Table 7, Table 8, and Table 9 or FIGS. 8-9), wherein the XTEN interfere with the interaction of the resulting CFXTEN with those clearance receptors with the result that the cokinetic properties of the CFXTEN is enhanced compared to the corresponding FVIII not linked to XTEN. In another embodiment, the invention provides CFXTEN that have reduced binding affinity with VWF of at least % less, or about 10%, or about 20%, or about 40%, or about 50%, or about 60%, or about 70% less, but are heless configured to have enhanced protection from active clearance ors conferred by the incorporation ofXTEN at one or more locations within the FVIII molecule, wherein the XTEN interfere with the interaction of factor VIII with those receptors. In the foregoing ments, the CFXTEN have an increased terminal half-life of at least about 12 h, or 24 h, or 48 h, or 72 h, or 96 h, or 120 h, or 144 h, or 7 days, or 10 days, or 14 days, or 21 days compared to the FVIII not linked to XTEN. The invention provides a method to create CFXTEN with d clearance wherein the CFXTEN fusion proteins created with the multiple insertions are evaluated for inhibition of binding to clearance receptors, compared to FVIII not linked to XTEN, using in vitro binding assays or in vivo pharmacokinetic models described herein or other assays known in the art, and selecting those that demonstrate reduced binding yet retain procoagulant FVIII actiVity. In addition, the ing fusion proteins can be optimized to have increased Ratio XTEN Radii of at least 2.0-3.5 in order to achieve pharmacokinetic properties that are further enhanced. Table 5, Table 6, Table 7, Table 8, and Table 9 and FIGS. 8-9 provide non-limiting examples ofXTEN insertion points within the factor VIII sequence. Using such insertion points, the invention plates CFXTEN compositions that have configurations with multiple XTEN ed with about 100, or about 200, or about 300, or about 400, or about 500 amino acids separating at least three XTEN to further increase the protection against active clearance mechanisms and, hence, increase the terminal half-life of the CFXTEN. Not to be bound by a particular theory, the XTEN of the CFXTEN compositions with high net charge (e.g., CFXTEN comprising AE family XTEN) are expected, as described above, to have less non-specific interactions with various vely-charged surfaces such as blood vessels, tissues, or various receptors, which would further contribute to reduced active clearance.

Conversely, the XTEN of the CFXTEN compositions with a low (or no) net charge (e. g., CFXTEN comprising AG family XTEN) are expected to have a higher degree of interaction with surfaces that, while contributing to active clearance, can potentiate the actiVity of the associated coagulation factor, given the known contribution of cell (e.g., platelets) and vascular surfaces to the coagulation process and the intensity of activation of coagulation factors (Zhou, R., et al., Biomaterials (2005) 26(16):2965-2973; London, F., et al. Biochemistry (2000) 39(32):9850—985 8). The invention, in part, takes advantage of the fact that certain ligands wherein d binding to a clearance or, either as a result of a decreased on—rate or an increased te, may be effected by the obstruction of a receptor site by an inserted XTEN forming random coil, resulting in the reduced binding. The choice of the particular configuration of the CFXTEN fusion protein can be tested by methods disclosed herein to confirm those configurations that reduce the degree of binding to a clearance receptor such that a reduced rate of active clearance is achieved. In one embodiment, the CFXTEN comprises a XTEN sequence that has one or more XTEN inserted at locations selected from Table 5, Table 6, Table 7, Table 8, and Table 9 or FIGS. 8-9 n the terminal half-life of the CFXTEN is increased at least about two-fold, or at least about three- fold, or at least about four-fold, or at least about five-fold, or at least about six-fold, or at least about eight-fold, or at least about ten-fold, or at least about -fold ed to a FVIII not linked to an XTEN. In another ment, the CFXTEN ses a FVIII-XTEN sequence that has a first and at least a second XTEN inserted at a first and second location selected from Table 5, Table 6, Table 7, Table 8, and Table 9 or FIGS. 8-9 n the terminal half-life of the CFXTEN is increased at least about two-fold, or at least about three-fold, or at least about old, or at least about five-fold, or at least about six-fold, or at least about eight-fold, or at least about ten-fold, or at least about twenty-fold compared to a FVIII not linked to an XTEN. In yet another embodiment, the CFXTEN comprises a FVIII-XTEN sequence that incorporates multiple XTEN sequences using three of more XTEN ion locations selected from Table 5, Table 6, Table 7, Table 8, and Table 9 or FIGS. 8-9 separated by about 100, or about 200, or about 300, or about 400, or about 500 amino acids, wherein the terminal half-life of the CFXTEN is increased at least about two-fold, or at least about fold, or at least about four-fold, or at least about ld, or at least about six-fold, or at least about eight-fold, or at least about ten-fold, or at least about twenty-fold compared to a FVIII not linked to an XTEN. In the foregoing embodiments hereinabove described in this paragraph, the XTEN incorporated into the CFXTEN configurations can be identical or they can be different, and can have at least about 80%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99%, sequence identity to a sequence from any one of Tables 3, 4, and 13-17, and can optionally include one or more cleavage sequences from Table 12, facilitating release of one or more of the XTEN from the CFXTEN fusion protein.

In one embodiment, the invention provides CFXTEN that e the pharmacokinetics of the fusion protein by linking one or more XTEN to the FVIII component of the fusion n wherein the fusion protein has an increase in apparent molecular weight factor of at least about two-fold, or at least about three-fold, or at least about four-fold, or at least about five-fold, or at least about six-fold, or at least about seven-fold, or at least about eight-fold, or at least about ld, or at least about twelve-fold, or at least about fifteen-fold, and wherein the terminal half-life of the CFXTEN when administered to a subject is increased at least about two-fold, or at least about four-fold, or at least about eight-fold, or at least about 10-fold or more ed to the corresponding FVIII not linked to XTEN. In the foregoing ment, wherein at least two XTEN molecules are incorporated into the , the XTEN can be identical or they can be of a ent sequence composition, net charge, or length. The XTEN can have at least about 80%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99%, sequence identity to a sequence from any one of Tables 3, 4, and 13-17, and can optionally e one or more cleavage sequences from Table 12, facilitating release of one or more of the XTEN from the CFXTEN fusion protein.

Thus, the invention provides CFXTEN compositions in which the degree of activity, bioavailability, half-life or physicochemical teristic of the fusion n can be tailored by the selection and placement of the type and length of the XTEN in the CFXTEN compositions. Accordingly, the ion contemplates itions in which a FVIII from Table 1 and XTEN or XTEN fragment from any one of Tables 3, 4, or 13-17 are produced, for example, in a configuration selected from any one of formulae I-VIII or the XTEN are inserted at locations selected from Table 5, Table 6, Table 7, Table 8, and Table 9 or FIGS. 8-9 such that the construct has the desired property.

The invention provides methods to produce the CFXTEN compositions that can maintain the FVIII ent at therapeutic levels in a t in need thereof for at least a two-fold, or at least a three-fold, or at least a four-fold, or at least a five-fold greater period of time compared to comparable dosages of the corresponding FVIII not linked to XTEN. In one embodiment of the method, the subject is receiving routine prophylaxis to prevent bleeding episodes. In another embodiment of the method, the subject is receiving treatment for a bleeding episode. In another embodiment of the method, the subject is receiving treatment to raise the circulating blood concentration of gulant FVIII above 1%, or above 1-5%, or above 5-40% relative to FVIII concentrations in normal plasma. “Procoagulant” as used herein has its general g in the art and generally refers to an activity that promotes clot formation, either in an in vitro assay or in vivo. The method to produce the compositions that can maintain the FVIII component at therapeutic levels includes the steps of selecting one or more XTEN appropriate for conjugation to a FVIII to provide the desired pharmacokinetic ties in View of a given dose and dose n, creating a gene uct that encodes the CFXTEN in one of the configurations disclosed herein, transforming an appropriate host cell with an expression vector comprising the encoding gene, expressing the fusion protein under suitable culture conditions, recovering the , administration of the CFXTEN to a mammal followed by assays to verify the pharmacokinetic properties and the actiVity of the CFXTEN fusion protein (e.g., the ability to maintain hemostasis or serve as a procoagulant) and the safety of the administered composition. Those compositions exhibiting the desired properties are selected for further use. CFXTEN created by the methods provided herein can result in increased efficacy of the administered composition by, amongst other properties, ining the ating concentrations of the procoagulant FVIII component at therapeutic levels for an enhanced period of time.

The invention provides methods to assay the CFXTEN fusion proteins of differing composition or configuration in order to provide CFXTEN with the desired degree of procoagulant and therapeutic ty and pharmacokinetic properties, as well as a sufficient safety profile. c in vitro and in vivo assays or animal models are used to assess the activity and functional characteristics of each configured CFXTEN and/or FVHI component to be incorporated into CFXTEN, including but not d to the assays of the Examples, those assays of Table 49, as well as the following assays or other such assays known in the art for assaying the properties and effects of FVHI. onal assays can be conducted that allow determination of coagulation activity, such as one-stage clotting assay and two- stage clotting assay (Barrowcliffe TW, Semin Thromb Hemost. (2002) 28(3):247-256), activated l prothrombin (aPTT) assays (Belaaouaj AA et al., J. Biol. Chem. (2000) 275:27123-8; DiaZ-Collier JA.

Haemost (1994) 71 :339-46), chromogenic FVIH assays (Lethagen, S., et al., Scandinavian J Haematology (1986) 37:448—453), or animal model pharmacodynamic assays including bleeding time or thrombelastography (TEG or ROTEM), among others. Other assays include determining the g affinity of a CFXTEN for the target substrate using binding or itive binding assays, such as Biacore assays with chip-bound receptors or binding proteins or ELISA assays, as described in US Patent ,534,617, assays described in the Examples herein, radio-receptor assays, or other assays known in the art. Other assays to determine the binding of FVIH tors to CFXTEN include the Bethesda assay or the Nijmegen modification of the Bethesda assay. The foregoing assays can also be used to assess FVHI sequence variants (assayed as single components or as CFXTEN fusion proteins) and can be compared to the native FVIII to ine whether they have the same degree of procoagulant actiVity as the native CF, or some fraction thereof such that they are suitable for ion in CFXTEN; e. g., at least about %, or at least about 20$, or about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% of the activity compared to the native FVIH.

Dose optimization is important for all drugs. A therapeutically effective dose or amount of the CFXTEN varies according to s such as the disease state, age, seX, and weight of the dual, and the ability of the administered fusion protein to elicit a desired response in the individual. For example, a standardized single dose of FVIII for all patients presenting with diverse bleeding ions or abnormal clinical parameters (e.g., neutralizing antibodies) may not always be ive. Hemophilia A patients with trauma, who have undergone surgery, or that have high titers of FVIII inhibitory antibodies generally will require higher and more frequent dosing. Generally, dosage level is adjusted in frequency, duration, and units in keeping with the severity and duration of each patient's bleeding episode.

Accordingly, the CFXTEN is included in the pharmaceutically acceptable carrier, delivery vehicle, or stabilizer in an amount sufficient to deliver to a patient a therapeutically effective amount of the fusion protein to stop bleeding, as ed by standard clotting assays. A consideration of these factors is well within the purview of the ordinarily skilled clinician for the purpose of determining the therapeutically or pharmacologically effective amount of the CFXTEN and the appropriated dosing schedule, versus that amount that would result in insufficient potency such that clinical improvement or the arrest of bleeding is not achieved.

The invention provides methods to establish a dose regimen for the CFXTEN pharmaceutical compositions of the invention. The methods include administration of consecutive doses of a therapeutically ive amount of the CFXTEN pharmaceutical composition using variable periods of time between doses to determine that interval of dosing sufficient to achieve and/or maintain the desired parameter, blood level or clinical effect; such consecutive doses of a eutically effective amount at the effective interval establishes the therapeutically effective dose regimen for the CFXTEN for a factor elated disease state or condition. A prophylactically effective amount refers to an amount of CFXTEN required for the period of time necessary to prevent a physiologic or al result or event; e.g., d onset of a bleeding episode or ining blood concentrations of procoagulant FVIH or equivalent above a threshold level (e. g., 1-5% to 5-40% of normal). In the methods of treatment, the dosage amount of the CFXTEN that is administered to a subject ranges from about 5 to 300 lU/kg/dose, or from about 10 to 100 dose, or from about 20 to about 65 lU/kg/dose, or from about 20 to about 40 lU/kg/dose for a subject. A suitable dosage may also depend on other factors that may influence the response to the drug; e. g., bleeding episodes generally requiring higher doses at more frequent intervals compared to prophylaxis.

In some embodiments, the method comprises administering a therapeutically-effective amount of a pharmaceutical composition comprising a CFXTEN fusion protein composition and at least one pharmaceutically acceptable r to a subject in need f, wherein the administration results in a greater improvement in at least one ter or physiologic condition associated with a FVIII deficiency or coagulopathy, or s in a more favorable clinical e mediated by the FVIH component of the CFXTEN ed to the effect on the ter, condition or clinical outcome mediated by administration of a pharmaceutical composition comprising a FVIII not linked to XTEN and administered at a comparable dose. Non-limiting examples of parameters that are improved include blood concentration of procoagulant FVIII, a reduced ted partial prothrombin (aPTT) assay time, a reduced one-stage or two-stage clotting assay time, delayed onset of a bleeding episode, a reduced chromogenic FVIII assay time, a d ng time, resolution of a bleeding event, or a reduced Bethesda titer to the CFXTEN relative to native FVIII. In one embodiment of the foregoing, the improvement is achieved by administration of the CFXTEN pharmaceutical composition at a dose that achieves a circulating concentration of procoagulant FVIII (or equivalent) above a threshold level (e.g., 1-5% to 5-40% of normal FVIII levels), thereby establishing the therapeutically effective dose. In another embodiment of the foregoing, the improvement is achieved by stration of multiple consecutive doses of the CFXTEN pharmaceutical composition using a therapeutically effective dose regimen that maintains a ating concentration of gulant FVIII (or equivalent) above a threshold level (e.g., 1-5% to 5-40% of normal FVIII ) for the length of the dosing period. In another embodiment of the , the administration of at least two consecutive doses of the CFXTEN pharmaceutical composition using a therapeutically effective dose regimen maintains a circulating tration of procoagulant FVIII (or equivalent) above about 1%,, 2%, 3%, 4%, 5%, 10%, 15%, 20%, %, or 40% of normal FVIII levels for a period that is at least about three-fold longer; alternatively at least about old longer; alternatively at least about five-fold longer; alternatively at least about six- fold longer; alternatively at least about seven-fold ; alternatively at least about eight-fold longer; alternatively at least about old longer or at least about ten-fold longer ed to a FVIII not linked to XTEN and administered using a eutically effective dose regimen ] In one embodiment, the CFXTEN or a pharmaceutical compositions comprising CFXTEN administered at a therapeutically effective dose regimen results in a gain in time of at least about three- fold ; alternatively at least about four-fold longer; alternatively at least about five-fold longer; alternatively at least about siX-fold longer; alternatively at least about seven-fold longer; alternatively at least about eight-fold longer; alternatively at least about nine-fold longer or at least about ten-fold longer between at least two consecutive Cmax peaks and/or Cmin troughs for blood levels of the fusion protein compared to the corresponding biologically active n of the fusion protein not linked to the XTEN and administered at a comparable dose regimen to a t. In another embodiment, the CFXTEN administered at a therapeutically effective dose regimen results in a comparable improvement in one, or two, or three or more measured parameters using less frequent dosing or a lower total dosage in IUs of the fusion protein of the pharmaceutical composition compared to the corresponding biologically active protein component(s) not linked to the XTEN and administered to a subject using a therapeutically effective dose regimen for the FVIII. The measured parameters include any of the clinical, biochemical, or physiological parameters disclosed herein, or others known in the art for assessing subjects with factor VIII-related conditions. (b) Pharmacology and Pharmaceutical Properties of CFXTEN The present invention es CFXTEN compositions comprising FVIII covalently linked to XTEN that have enhanced pharmaceutical and pharmacology properties compared to FVIII not linked to XTEN, as well as s to enhance the therapeutic and/or procoagulant effect of the FVIII components of the compositions. In addition, the invention provides CFXTEN compositions with enhanced properties compared to those art-known fusion proteins of factor VIII containing n, immunoglobulin polypeptide partners, polypeptides of shorter length and/or polypeptide partners with repetitive sequences. In addition, CFXTEN fusion proteins provide significant advantages over chemical conjugates, such as pegylated constructs of FVIII, notably the fact that recombinant CFXTEN fusion proteins can be made in host cell expression systems, which can reduce time and cost at both the research and development and manufacturing stages of a product, as well as result in a more homogeneous, defined product with less toxicity from both the product and metabolites of the CFXTEN compared to pegylated conjugates.

As therapeutic agents, the CFXTEN ses a number of advantages over therapeutics not comprising XTEN, including one or more of the following non-limiting properties: increased solubility, increased thermal stability, reduced genicity, increased nt molecular , reduced renal clearance, reduced proteolysis, d metabolism, ed eutic efficiency, less frequent dosage regimen with increased time between doses capable of maintaining hemostasis in a subject with ilia A, the ability to administer the CFXTEN composition aneously or uscularly, a “tailored” rate of tion when administered subcutaneously or intramuscularly, ed lyophilization ity, ed serum/plasma stability, increased terminal half-life, increased solubility in blood stream, decreased binding by neutralizing antibodies, decreased active clearance, tailored substrate binding affinity, stability to ation, stability to freeze-thaw, stability to proteases, stability to tination, ease of administration, compatibility with other pharmaceutical excipients or carriers, persistence in the subject, increased stability in storage (e. g., increased shelf-life), and the like. The net effect of the enhanced properties is that the use of a CFXTEN composition can result in an overall enhanced therapeutic effect compared to a FVIII not linked to XTEN, result in economic benefits associated with less nt dosing, and/or result in improved patient compliance when administered to a subject with a factor VIII-related condition.

The invention provides CFXTEN compositions and pharmaceutical compositions comprising CFXTEN wherein the stration of the composition results in an improvement in at least one of the clinical or biochemical parameters disclosed herein as being useful for assessing the subject diseases, conditions or disorders. Non-limiting examples of parameters that are improved include blood concentrations of procoagulant FVIII, a reduced activated partial prothrombin (aPTT) assay time, a reduced one-stage or two-stage clotting assay time, delayed onset of a ng episode, a reduced chromogenic FVIII assay time, a reduced bleeding time, resolution of a bleeding event, or a reduced Bethesda titer to the CFXTEN relative to native FVIII. The enhanced cokinetic properties of the subject CFXTEN permits using an accumulatively lower IU dose of fusion protein to maintain the parameter compared to the corresponding FVIII component not linked to the XTEN. In one embodiment, the total dose in IUs of an CFXTEN of the embodiments needed to achieve and maintain the improvement in the at least one parameter for about 2-7 days is at least about three-fold lower, or at least about four-fold, or at least about ld, or at least about six-fold, or at least about eight-fold, or at least about 10-fold lower compared to the corresponding FVIII component not linked to the XTEN. In another embodiment, the total dose in IUs of a subject CFXTEN needed to achieve and maintain the improvement in the at least one parameter over two, three or four consecutive doses is at least about three-fold lower, or at least about old, or at least about five-fold, or at least about six-fold, or at least about eight-fold, or at least about 10-fold lower compared to the corresponding FVIII component not linked to the XTEN. Alternatively, the ion provides certain embodiments of CFXTEN wherein the period between consecutive administrations that results in achieving and ining the improvement in at least one parameter is at least about three-fold, or at least about four-fold, or at least about five-fold, or at least about six-fold, or at least about eight-fold, or at least about d longer ed to the corresponding FVIII component not linked to the XTEN and administered at a comparable IU dose. Alternatively, the invention provides certain embodiments of CFXTEN wherein administration of 25 IU/kg results in a 30% improvement in a aPTT assay (or similar coagulation assay) time in a hemophilia A subject compared to 25 IU/kg of the corresponding FVIII not linked to XTEN when assayed at about 2-7 days after administration. In yet another embodiment, the invention provides CFXTEN n stration of 25 IU/kg results in a 30% improvement in a ng time assay time in a hemophilia A subject compared to 25 IU/kg of the corresponding FVIII not linked to XTEN when assayed at about 2-7 days after administration.

In one embodiment, XTEN as a fusion partner ses the solubility of the FVIII payload. ingly, where enhancement of the pharmaceutical or physicochemical properties of the FVIII is desirable, such as the degree of aqueous solubility or stability, the length and/or the motif family composition of the XTEN sequences incorporated into the fusion protein may each be selected to confer a different degree of solubility and/or stability on the respective fusion proteins such that the overall pharmaceutical properties of the CFXTEN composition are enhanced. The CFXTEN fusion proteins can be constructed and assayed, using methods described herein, to confirm the physicochemical properties and the choice of the XTEN length sequence or location adjusted, as needed, to result in the desired properties. In one embodiment, the CFXTEN has an aqueous solubility that is at least about 25% greater compared to a FVIII not linked to the XTEN, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 75%, or at least about 100%, or at least about 200%, or at least about 300%, or at least about 400%, or at least about 500%, or at least about 1000% greater than the corresponding FVIII not linked to XTEN.

The invention provides s to produce and recover expressed CFXTEN from a host cell with enhanced solubility and ease of recovery ed to FVIII not linked to XTEN. In one embodiment, the method includes the steps of transforming a eukaryotic host cell with a polynucleotide encoding a CFXTEN with one or more XTEN ents of cumulative sequence length r than about 100, or greater than about 200, or greater than about 400, or greater than about 600, or greater than about 800, or greater than about 1000, or r than about 2000, or greater than about 3000 amino acid residues, expressing the CFXTEN fusion protein in the host cell under suitable culture and induction conditions, and recovering the sed fusion protein in soluble form. In one embodiment, the one or more XTEN of the CFXTEN fusion proteins each have at least about 80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% sequence identity compared to one or more XTEN selected from any one of Tables 4, and 13-17, or fragments thereof, and the FVIII have at least about 80% ce identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or 100% sequence identity compared to a FVIII selected from Table 1, and the CFXTEN components are in an N— to C-terminus configuration selected from any one of the configuration embodiments sed herein.

VI). USES OF THE CFXTEN COMPOSITIONS ] The invention provides methods and regimens for ing a beneficial effect in a factor VIII- related condition by the administration of compositions comprising CFXTEN. As used herein, “factor VIII-related condition” is intended to include, but is not limited to factor VIII def1ciencies, bleeding disorders related to factor VIII deficiency, hemophilia A, neutralization of factor VIII by VIII antibodies or other factor VIII inhibitors, and bleeding es resulting from trauma or surgery or vascular injury and other such conditions that can be ameliorated or corrected by administration of FVIII to a subject. The inventive methods achieve a cial effect while addressing disadvantages and/or limitations of other methods of treatment using factor VIII preparations that have a vely short terminal half-life, require frequent administrations, are neutralized by inhibitors or have unfavorable pharmacoeconomics.

Hemostasis is regulated by multiple protein factors, and such proteins, as well as analogues thereof, have found utility in the ent of factor VIII-related conditions. However, the use of commercially-available FVIII has met with less than optimal s in the management of subjects afflicted with such ions. In particular, dose optimization and frequency of dosing is important for FVIII used in maintaining ating FVIII concentrations above threshold levels needed for hemostasis, as well as the treatment or prevention of bleeding episodes in hemophilia A subjects. The fact that commercially-available FVIII products have a short half-life necessitates frequent dosing in order to achieve clinical benefit, which results in difficulties in the management of such patients.

As ished by the Subcommittee on Factor VIII and Factor IX of the Scientific and Standardization Committee of the International Society on Thrombosis and tasis (posted on the ISTH Website 29 November, 2000), the most widely used measure of the severity of hemophilia A is ished by determining the circulating concentrations of plasma FVIII procoagulant levels, with persons with <1% (< 0.01 IU/ml) factor VIII defined as severe; 1-5% (0.01 - 0.05 IU/ml) as moderately severe; and >5-40% (0.05 - <0.40 IU/ml) as mild, where normal is 1 IU/ml of factor VIIIC (100%).

The invention es methods of treating a subject ing from or at risk of developing a factor VIII-related condition. More particularly, the invention provides s for treating or preventing controlling bleeding in subject. The subject can be any animal but preferably is a human. In one embodiment, the method comprises administering a coagulation-effective amount of a CFXTEN composition to the subject in need thereof In another embodiment, the method comprises the step of administering to the subject with a bleed a coagulation-effective amount of a pharmaceutical ition that includes a CFXTEN, wherein the stration results in an arrest or attenuation of the bleeding.

As used herein, “coagulation-effective amount” is an amount of a FVIII composition that, when administered to a subject, is sufficient to effect hemostasis or other beneficial or desired therapeutic (including preventative) result. In practicing the present invention, it will be understood that a ation-effective amount can be administered in one or more administrations. Precise ationeffective s of the pharmaceutical composition to be administered will be guided by the judgment of the practitioner, however, the unit dose will generally depend on the severity or cause of the bleeding and the amount of pre-eXisting FVIII in the subject. In a particular embodiment of the method of treating a bleed, a coagulation-effective amount of a pharmaceutical compositions comprising CFXTEN is administered to a subject suffering from a bleeding episode, wherein the administration s in the resolution of the bleeding for a duration at least two-fold, or at least three-fold, or at least four-fold longer ed to a FVIII not linked to XTEN and administered to a comparable subject with a comparable bleed at a comparable dose.

] In another embodiment, the administration of a coagulation-effective amount of a CFXTEN composition to a subject with a factor VIII-related condition results in a 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70% or greater improvement of one or more biochemical, logical or clinical parameters associated with the FVIII condition, compared to the FVIII not linked to XTEN, when measured at between 2 and 7 days after administration. In another embodiment, the administration of a coagulation-effective amount of a CFXTEN composition to the subject in need thereof results in an improvement of one or more biochemical, physiological or al parameters associated with the FVIII condition for a period at least two-fold , or at least four-fold longer, or at least five-fold longer, or at least siX-fold longer compared to period achieved by a FVIII not linked to XTEN and stered at a comparable dose. Non-limiting examples of ters that are improved for a longer duration e blood concentrations of procoagulant FVIII, a reduced activated partial prothrombin (aPTT) assay time, a reduced one-stage or two-stage clotting assay time, delayed onset of a bleeding episode, a reduced chromogenic FVIII assay time, a reduced bleeding time, among other FVIII-related parameters known in the art. In the foregoing embodiments of the paragraph, the administered CFXTEN comprises a FVIII with at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% sequence identity to a factor VIII of Table l and one or more XTEN sequences with at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% sequence identity to an XTEN of Table 4 inserted into the FVIII at one or more locations ed from Table 5, Table 6, Table 7, Table 8, and Table 9, or as depicted in FIGS. 8-9. In certain embodiments, at least one XTEN ion site of the CFXTEN is selected from amino acids 32, 220, 224, 336, 339, 390, 399, 416, 603, 1656, 1711, 1725, 1905 and 1910 (numbered relative to mature native human FVHI).

In a particular embodiment of the method of treatment, a coagulation-effective amount of CFXTEN fusion protein stered to a subject suffering from hemophilia A is sufficient to increase the ating FVIII gulant concentration to greater than 0.05 IU/ml and to maintain hemostasis for at least about 24 h, or at least about 48 h, or at least about 72 h, or at least about 96 h, or at least about 120 h, or at least about 144 h, or at least about 168 h, or greater. In another embodiment, the administration of a coagulation-effective amount of a pharmaceutical composition comprising CFXTEN to a t in need thereof results in a greater reduction in a one-stage clotting assay time of at least about 5%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or more in a blood sample from the subject at 2-7 days after the administration compared to the assay time in a subject after administration of a comparable amount of the ponding FVIII not linked to XTEN. In another embodiment, the administration of a therapeutically effective amount of a CFXTEN or a pharmaceutical compositions comprising CFXTEN to a subject in need thereof s in a r reduction in the activated partial prothrombin time of at least about 5%, or about 10%, or about %, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or more in a blood sample from the subject 2-7 days after administration compared to the activated partial prothrombin time in a subject after administration of a comparable amount of the corresponding FVIII not linked to XTEN. In another embodiment, the administration of a CFXTEN or a pharmaceutical compositions comprising CFXTEN to a subject in need thereof using a therapeutically effective amount results in maintenance of activated partial prothrombin times Within 30% of normal in a blood sample from the subject for a period of time that is at least ld, or at least about three-fold, or at least about old longer compared to that of a FVIII not linked to XTEN and administered to a subject using a comparable dose.

In one embodiment of the method of treatment, the CFXTEN fusion protein is formulated and administered as a pharmaceutical composition comprising the CFXTEN in ure With a pharmaceutically able excipient. s for making pharmaceutical formulations are well known in the art. Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co., Easton, Pa. 1990 (See, also, Wang and Hanson, Parenteral Formulations of Proteins and es: Stability and Stabilizers, Journal of Parenteral Science and Technology Technical Report No. , 10, Supp. 42-2S (1988)).

In another aspect, the invention provides a regimen for treating a hemophilia A patient, said regimen comprising a composition sing a CFXTEN fusion protein. In one embodiment of the regimen for treating a hemophilia A patient, the regimen further ses the step of determining the amount of pharmaceutical composition comprising the CFXTEN needed to achieve hemostasis in the patient. In some embodiments of the regimen, (i) a smaller 1U amount of about two-fold less, or about three-fold less, or about four-fold less, or about five-fold less, or about six-fold less, or about eight-fold less, or about d less of the pharmaceutical composition comprising CFXTEN is administered to a subject in need thereof in comparison to the corresponding coagulation factor not linked to the XTEN under an otherwise same dose regimen, and the fusion protein achieves a comparable area under the curve (based on IU/ml) and/or a comparable therapeutic effect as the corresponding FVIII not linked to the XTEN; (ii) the pharmaceutical composition is administered less frequently (e. g., every three days, about every seven days, about every 10 days, about every 14 days, about every 21 days, or about monthly) in comparison to the corresponding FVIII not linked to the XTEN under an otherwise same dose amount, and the fusion protein es a comparable area under the curve and/or a comparable therapeutic effect as the corresponding coagulation factor not linked to the XTEN; or (iii) an lative smaller IU amount of at least about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90% less of the pharmaceutical composition is stered in comparison to the corresponding FVIII not linked to the XTEN under an otherwise same dose schedule and the CFXTEN fusion protein achieves a comparable therapeutic effect as the corresponding FVIII not linked to the XTEN. The accumulative smaller IU amount is measured for a period of at least about one week, or about 14 days, or about 21 days, or about one month. In the foregoing embodiments, the eutic effect can be determined by any of the measured parameters described herein, including but not limited to blood concentration of procoagulant FVIII, a reduced activated partial prothrombin (aPTT) assay time, a reduced age or two-stage ng assay time, delayed onset of a bleeding episode, a reduced chromogenic FVIII assay time, a reduced bleeding time, resolution of a bleeding event, or a reduced Bethesda titer to the CFXTEN relative to native FVIII, fibrinogen levels, or other assays known in the art for assessing opathies of FVIII. In r embodiment, the invention provides CFXTEN for use in a regimen for a treating a hemophilia A subject sing administering an CFXTEN composition in two or more successive doses to the subject at an effective amount, wherein the adminstration results in at least a 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 90% greater improvement of at least one, two, or three parameters associated with the disease compared to a FVIII not linked to XTEN and stered using a comparable dose.

] In one aspect, the present invention relates to a method of ting or ng the bleeding in a patient, optionally a haemophilia A patient, having pre-eXisting inhibitor(s) against FVIII. Inhibitory antibodies against FVIII commonly develop in hemophiliacs, where the overall incidence of developing an inhibitor is 15 -3 0%, particularly in haemophiliacs who are heavily exposed to FVIII concentrates (Algiman et al. Natural antibodies to factor VIII (anti-hemophilic ) in healthy individuals. PNAS USA (1992) 89: 3795-3799). However, inhibitory antibodies also occur in ts in auto-immune disorders, malignancies (such as lymphoproliferative disorders, lymphomas and solid tumors), during pregnancy and in the post-partum state. Inhibition can also occur when antibodies interfere with the binding of FVIII to FIX and FX. Simultaneously or alternatively, anti-FVIII antibodies can interfere with the binding of von Willebrand factor and/or phospholipids to FVIII, ing coagulation and/or half-life of FVIII. The presence of inhibitory antibodies is often first detected with ms such as easy ng and uncontrolled bleeding, and is y referred to as acquired hemophilia. Anti-FVIII antibodies can be determined by different methods including quantitation of anti-FVIII activity in coagulation assays, ELISA for FVIII inhibitors and purification using chromatography and immunoadsorption (Algiman et al., 1992). Accordingly, the inventive s are used in the treatment or prevention of any condition associated with or characterized by the presence of inhibitory antibodies to FVIII. In one embodiment, the invention provides a method of treating a patient haVing a pre-eXisting inhibitor t FVIII, the method comprising the step of stering to the patient a coagulation- effective amount of a CFXTEN fusion n that must be administered to achieve hemostasis, wherein the coagulation-effective amount of fusion protein stered is reduced in comparison to the amount of FVIII not linked to XTEN (or native FVIII) that must be administered to achieve hemostasis. In the method, the reduced amount of CFXTEN is about two-fold, or three-fold, or four-fold, or five-fold less in IU/kg compared to the corresponding FVIII not linked to XTEN. In another embodiment of the method, the amount of CFXTEN that is administered as a dose to achieve hemostasis is at least 20 to 40 IU/kg less, or 30 to 60 IU/kg less, or 40 to 80 IU/kg less, or 60 to 100 IU/kg less, or 100 to 140 IU/kg less, or 120 to 180 IU/kg less, or 140 to 200 IU/kg less compared to the corresponding FVIII not linked to XTEN or to native FVIII required to achieve hemostasis. In another embodiment, the invention provides a method of treating a bleeding episode in a hemophilia A subject haVing a titer of at least 10, or 20, or 30, or 40, or 50, or 75, or 100, or 150, or 200 or more Bethesda units against a FVIII not linked to XTEN, wherein the dose of CFXTEN fusion protein required to arrest the bleeding epidose is at least two-fold, or three-fold, or four-fold, or ld, or six-fold, or seven-fold, or eight-fold, or nine-fold, or 10-fold less in comparison to the amount of FVIII not linked to XTEN (or native FVIII) that must be administered to achieve hemostasis in a comparable subject. It will be understood by one of skill in the art that the amount of procoagulant administered to maintain hemostasis will depend on the severity of FVIII deficiency and/or the frequency or duration of bleeding.

] A particular object of the present ion relates to use of CFXTEN with d g by FVIII inhibitors that bind the A2 and/or C2 domains of Factor VIII as a drug. Such a drug is advantageously used for maintaining hemostasis in a patient suffering from haemophilia, wherein such patient has circulating FVIII inhibitors ed against the A2 domain and/or C2 domain of Factor VIII.

In one embodiment, the ion es a method of treatment, the method comprising the step of administering to the patient with a A2 domain-binding inhibitor a coagulation-effective amount of a CFXTEN fusion protein, wherein the CFXTEN exhibits at least 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80% or less binding to an inhibitor that binds the A2 domain of FVIII, compared to the FVIII not linked to XTEN or to native FVIII, and wherein the administration results in asis.

In another embodiment, the invention provides a method of treatment, the method comprising the step of administering to the patient with a C2 domain-binding inhibitor a coagulation-effective amount of a CFXTEN fusion protein, wherein the CFXTEN exhibits at least 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80% or less binding to an inhibitor that binds the C2 domain of FVIII, compared to the FVIII not linked to XTEN or to native FVIII, and wherein the stration results in hemostasis.

The reduced binding of the subject CFXTEN can be assayed directly by ELISA that detects FVIII inhibitors, or measured indirectly by demonstration of reduced inhibition of FVIII actiVity of the CFXTEN compared to native FVIII in the presence of an inhibitor as measured by a factor VIII genic test or one-step assay as described herein, or other suitable coagulation methods known in the art. Alternatively, the subject CFXTEN can be measured for reduced (or absence of) inhibition in the presence of known tors by use of a modified Bethesda assay. According to a particular aspect of the present ion, a CFXTEN useful in the methods has reduced reactivity to one or more antibodies from Table 10, as well as naturally-occurring antibodies found in hemophilia patients. For testing purposes, such and other inhibitory antibodies can be obtained from humans (i.e. from the serum of patients which have inhibitory dies) or can be obtained from mice, guinea pigs, horses, goats, non- human primates and other mammals by immunization with FVIII, or fragments thereof, more particularly with a fragment comprising the all or part of the A2 or C2 domain, whether in polyclonal or onal form.

The invention further contemplates that the CFXTEN used in accordance with the methods ed herein can be administered in conjunction with other treatment methods and compositions (e. g., other coagulation proteins) useful for treating factor VIII-related conditions, or conditions for which coagulation factor is adjunctive therapy; e. g., bleeding episodes due to injury or surgery.

In another aspect, the invention provides s of preparing a drug for a factor VIII-related condition, comprising combining a factor VIII ce selected from Table 1 with one or more XTEN selected from Table 4 inserted in one or more ion sites selected from Table 5, Table 6, Table 7, Table 8, and Table 9 to result in a drug that retains at least a portion of the activity of the native FVIII.

The invention provides a method of preparing a pharmaceutical ition, comprising the step of combining the drug of the foregoing embodiment with at least one pharmaceutically acceptable carrier.

In one embodiment of the method of preparing a drug for a factor VIII-related condition, the factor VIII has a sequence with at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% sequence identity compared to a sequence selected from Table 1 and the one or more XTEN has a ce with at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% sequence identity compared to a sequence selected from any one of Tables 3, 4, and 13-17, or a fragment thereof, wherein the one or more XTEN are inserted in one or more locations selected from Table 5, Table 6, Table 7, Table 8, and Table 9. In a particular embodiment of the foregoing, at least one XTEN insertion site is selected from amino acids 32, 220, 224, 336, 339, 390, 399, 416, 603, 1656, 1711, 1725, 1905 and 1910 (numbered ve to mature native human FVIII).In another embodiment of the method, the CFXTEN ses a sequence with at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% sequence identity compared to a sequence selected from any one of Table 21.

In another aspect, the invention provides a method of making the CFXTEN itions to achieve desired pharmacokinetic, pharmacologic or pharmaceutical properties. In general, the steps in the design and production of the inventive fusion protein compositions, as illustrated in FIGS. 11-13, include: (1) the selection of a FVIII (e. g., native proteins, sequences of Table 1, analogs or derivatives with activity) to treat the particular condition; (2) selecting one or more XTEN (e. g., sequences with at least 80% identity to sequences set forth in Table 4) that will confer the desired pharmacokinetic and physicochemical characteristics on the resulting CFXTEN (e.g., the administration of the CFXTEN composition to a subject results in the fusion protein being maintained above 0.05-0.4 IU/ml for a greater period compared to FVIII not linked to XTEN); (3) establishing a desired N— to C-terminus configuration of the CFXTEN to achieve the desired y or PK parameters (e.g., selecting one or more insertion sites from Table 5, Table 6, Table 7, Table 8, and Table 9); (4) establishing the design of the expression vector encoding the configured CFXTEN; (5) transforming a suitable host with the expression vector; and (6) sing and recovering the resultant isolated CFXTEN fusion protein. In one embodiment of the method of making , the XTEN for insertion are evaluated by the application of Equation IV to maximize the Ratio XTEN Radii for the fusion protein construct, with the XTEN resulting in values r than 2.0, or 2.1, or 2.2, or 2.3, or 2.4, or 2.5, or 2.6, or 2.7, or 2.8, or 2.9. or 3.0 being preferred.

For those CFXTEN for which an increase in half-life or an increased period of time spent above the minimum coagulation-effective concentration is desired, the XTEN chosen for incorporation lly have at least about 144, or about 288, or about 432, or about 576, or about 864, or about 875, or about 912, or about 923 amino acid es where a single XTEN is to be incorporated into the CFXTEN. In another embodiment, the CFXTEN comprises a first XTEN of the foregoing lengths, and at least a second XTEN of about 36, or about 42, or about 72, or about 144, or about 288, or about 576, or about 864, or about 875, or about 912, or about 923, or about 1000 or more amino acid residues. The location of the XTEN within the fusion protein can include one, two, three, four, five or more locations selected from Table 5, Table 6, Table 7, Table 8, and Table 9 or FIGS. 8-9. In one ment, the method of design includes an insertion ofXTEN into the FVIII of at least one site selected from amino acids 32, 220, 224, 336, 339, 390, 399, 416, 603, 1656, 1711, 1725, 1905 and 1910 (numbered relative to mature native human FVIII).

In another aspect, the invention provides methods of making CFXTEN compositions to improve ease of manufacture, result in increased stability, increased water solubility, and/or ease of formulation, as compared to the native FVIII. In one embodiment, the invention includes a method of increasing the water solubility of a FVIII comprising the step of linking the FVIII with at least about 80%, or about 90%, or about 95% identity to a sequence from Table 1 to one or more XTEN at one, two, three, four, five or more locations ed from Table 5, Table 6, Table 7, Table 8, and Table 9 or FIGS. 8-9 wherein the XTEN is a sequence with at least about 80%, or about 90%, or about 95% sequence identity compared to a sequence from any one of Tables 3, 4, and 13-17 such that a higher concentration in soluble form of the resulting CFXTEN can be achieved, under physiologic conditions, compared to the FVIII in an un-fused state. In a particular embodiment, the CFXTEN comprises a FVIII linked to two, three, four, or five XTEN having at least about 24, or about 36, or about 48, or about 60, or about 72, or about 84, or about 96, or about 144, or about 288 amino acid residues inserted at sites selected from Table 5, Table 6, Table 7, Table 8, and Table 9 or FIGS. 8-9, in which the solubility of the fusion protein under physiologic conditions is at least fold r than the corresponding FVIII not linked to XTEN, or atively, at least four-fold, or ld, or six-fold, or seven-fold, or eight-fold, or nine- fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 50-fold, or at least 60-fold or greater than FVIII not linked to XTEN. Factors that contribute to the ty ofXTEN to confer increased water solubility of CFs when incorporated into a fusion n include the high solubility of the XTEN fusion partner and the low degree of self-aggregation between molecules ofXTEN in solution, as well as expanding the hydrophilicity of FVIII external loops into which the XTEN is ed. In some embodiments, the method results in a CFXTEN fiJsion protein wherein the water solubility is at least about 20%, or at least about 30% greater, or at least about 50% greater, or at least about 75% greater, or at least about 90% greater, or at least about 100% greater, or at least about 150% greater, or at least about 200% greater, or at least about 400% r, or at least about 600% greater, or at least about 800% greater, or at least about 1000% greater, or at least about 2000% greater under physiologic conditions, compared to the ed FVIII. In one embodiment, the XTEN of the CFXTEN fusion n is a sequence with at least about 80%, or about 90%, or about 95% sequence identity compared to a sequence from any one of Tables 3, 4, and 13-17. In another embodiment, the invention includes a method of increasing the shelf-life of a FVIII comprising the step of linking the FVIII with one or more XTEN at one or more sites selected from Table 5, Table 6, Table 7, Table 8, and Table 9, wherein the shelf-life of the resulting CFXTEN is extended compared to the FVIII in an un-fused state. As used , life refers to the period of time over which the procoagulant activity of a FVIII or CFXTEN that is in solution, lyophilized or in some other storage formulation remains stable without undue loss of activity or that remains within release specifications established for the pharmaceutical composition. A FVIII that degrades or aggregates lly has reduced functional activity or reduced ilability compared to one that remains in solution. Factors that contribute to the ability of the method to extend the shelf life of FVIII when incorporated into a fusion protein include increased water solubility, reduced self-aggregation in solution, and increased heat stability of the XTEN fusion partner. In particular, the low tendency ofXTEN to aggregate facilitates methods of formulating pharmaceutical preparations containing higher drug concentrations of CFs, and the heat-stability ofXTEN contributes to the property of CFXTEN fusion proteins to remain soluble and functionally active for extended periods. The method results in CFXTEN fusion proteins with prolonged or ed shelf-life that exhibit greater activity ve to a FVIII standard that has been ted to the same storage and handling conditions. The standard may be the un—fused full-length FVIII or a commercially-available FVIII pharmaceutical composition. In one embodiment, the method includes the step of formulating the isolated CFXTEN with one or more pharmaceutically acceptable excipients that e the ability of the XTEN to retain its unstructured conformation and for the CFXTEN to remain soluble in the formulation for a time that is greater than that of the corresponding ed FVIII. In one embodiment, the method comprises linking a FVIII selected from Table 1 to one or more XTEN ed from any one of Tables 3, 4, and 13-17 inserted at one or more sites selected from Table 5, Table 6, Table 7, Table 8, and Table 9 and admixing with at least one pharmaceutically acceptable excipient to create a pharmaceutical composition that s greater than about 100% of the procoagulant activity, or greater than about 105%, 110%, 120%, 130%, 150% or 200% of the procoagulant activity of a FVIII standard subjected to the same storage and handling conditions when compared at a time point of at least 90 days, or at least 6 months, or at least 12 months. Shelf-life may also be assessed in terms of onal activity remaining after storage, normalized to functional activity when storage began. In some embodiments, CFXTEN ceutical compositions of the invention retain about 50% more procoagulant activity, or about 60%, 70%, 80%, or 90% more of the gulant ty of a FVIII standard when ted to the same conditions for the same period of up to 2 weeks, or 4 weeks, or 6 weeks or longer under various temperature conditions. In one embodiment, the CFXTEN pharmaceutical composition retains at least about 50%, or about 60%, or at least about 70%, or at least about 80%, and most preferably at least about 90% or more of its original activity in solution when heated at 80°C for 10 min. In another embodiment, the CFXTEN pharmaceutical composition retains at least about 50%, preferably at least about 60%, or at least about 70%, or at least about 80%, or alternatively at least about 90% or more of its original activity in solution when heated or maintained at 37°C for about 7 days. In r embodiment, CFXTEN pharmaceutical composition retains at least about 80% or more of its functional activity after exposure to a temperature of about 30°C to about 70°C over a period of time of about one hour to about 18 hours. In the foregoing embodiments hereinabove described in this paragraph, the retained activity of the CFXTEN ceutical compositions is at least about two-fold, or at least about three-fold, or at least about four- fold, or at least about five-fold, or at least about six-fold greater at a given time point than that of a corresponding pharmaceutical composition comprising FVIII not linked to the XTEN.

VII). THE NUCLEIC ACIDS SEQUENCES OF THE INVENTION The present invention provides ed polynucleic acids encoding CFXTEN ic fusion proteins and ces complementary to polynucleic acid molecules encoding CFXTEN chimeric fusion proteins, including homologous variants thereof. In another aspect, the invention asses methods to produce polynucleic acids encoding CFXTEN chimeric fusion proteins and sequences complementary to polynucleic acid molecules encoding CFXTEN chimeric fusion protein, ing homologous variants thereof. In general, and as illustrated in FIGS. 11-13, the methods of producing a cleotide sequence coding for a CFXTEN fusion protein and expressing the ing gene product include assembling nucleotides encoding FVIII and XTEN, ligating the ents in frame, incorporating the encoding gene into an expression vector appropriate for a host cell, transforming the appropriate host cell with the expression vector, and culturing the host cell under conditions causing or permitting the fusion n to be expressed in the transformed host cell, thereby producing the biologically-active CFXTEN polypeptide, which is recovered as an isolated fusion protein by standard protein purification methods known in the art. Standard recombinant techniques in molecular biology is used to make the polynucleotides and expression vectors of the present invention.

In accordance with the invention, c acid sequences that encode CFXTEN (or its complement) are used to generate recombinant DNA molecules that direct the expression of CFXTEN fusion ns in appropriate host cells. For the purposes of the invention, nucleic acid encoding a signal peptide corresponding to that of native human FVIII (encoding MQIELSTCFFLCLLRFCFS (SEQ ID NO: 1611)) can be added to any of the encoding constructs described herein to aid in the expression and secretion of the CFXTEN fusion protein. In one embodiment, the nucleic acid add is ATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGT (SEQ ID NO: 1613), or the complement f.

] Several cloning strategies are le for performing the present invention, many of which is used to generate a construct that comprises a gene coding for a fusion protein of the CFXTEN composition of the present invention, or its complement. In some embodiments, the cloning strategy is used to create a gene that encodes a monomeric CFXTEN that comprises at least a first FVIII and at least a first XTEN polypeptide, or their complement. In one embodiment of the ing, the gene comprises a sequence encoding a FVIII or sequence variant. In other embodiments, the cloning strategy is used to create a gene that encodes a monomeric CFXTEN that comprises nucleotides encoding at least a first molecule of FVIII or its complement and a first and at least a second XTEN or their complement that is used to transform a host cell for expression of the fusion protein of the CFXTEN composition. In the foregoing ments hereinabove described in this paragraph, the genes can further comprise nucleotides encoding spacer sequences that also encode cleavage sequence(s).

In designing a desired XTEN sequences, it was discovered that the non-repetitive nature of the XTEN of the inventive compositions is ed despite use of a ”building block" molecular approach in the on of the XTEN-encoding sequences. This was ed by the use of a library of polynucleotides encoding peptide sequence motifs, described above, that are then ligated and/or multimerized to create the genes encoding the XTEN sequences (see FIGS. 11 and 12 and Examples).

Thus, while the XTEN(s) of the expressed fusion protein may consist of le units of as few as four ent sequence , because the motifs themselves consist of non-repetitive amino acid sequences, the overall XTEN sequence is rendered non-repetitive. Accordingly, in one embodiment, the XTEN- encoding polynucleotides comprise multiple polynucleotides that encode non-repetitive sequences, or motifs, operably linked in frame and in which the resulting expressed XTEN amino acid sequences are non-repetitive.

In one approach, a uct is first prepared containing the DNA sequence corresponding to CFXTEN fusion protein. DNA encoding the FVIII of the compositions is obtained synthetically, from a commercial , or from a cDNA library prepared using standard methods from tissue or isolated cells believed to possess FVIII mRNA and to express it at a able level. If necessary, the coding sequence can be obtained using conventional primer extension procedures as described in Sambrook, et al. to detect precursors and processing intermediates of mRNA that may not have been reverse- , supra, transcribed into cDNA. One can then use polymerase chain reaction (PCR) methodology to amplify the target DNA or RNA coding sequence to obtain sufficient material for the ation of the CFXTEN ucts containing the FVIII gene. Assays can then be conducted to confirm that the hybridizing full- length genes are the desired FVIII gene(s). By these conventional methods, DNA can be conveniently obtained from a cDNA y prepared from such sources. The FVIII encoding gene(s) can also created by standard synthetic procedures known in the art (e. g., automated nucleic acid synthesis using, for example one of the methods bed in Engels et al. (Agnew. Chem. Int. Ed. Engl., 28:716-734 1989)), using DNA ces obtained from publicly available databases, patents, or literature references. Such procedures are well known in the art and well described in the scientific and patent literature. For example, sequences can be obtained from Chemical Abstracts Services (CAS) Registry Numbers (published by the American Chemical Society) and/or GenBank Accession Numbers (e.g., Locus ID, NP_XXXXX, and XP_XXXXX) Model Protein identifiers available through the National Center for Biotechnology Information (NCBI) webpage, ble on the world wide web at ncbi.nlm.nih.gov that correspond to entries in the CAS Registry or GenBank database that contain an amino acid sequence of the protein of interest or of a fragment or variant of the protein. In one embodiment, the FVIII ng gene s a protein ce from Table l, or a fragment or variant thereof A gene or polynucleotide encoding the FVIII portion of the subject CFXTEN protein, in the case of an expressed fusion n that comprises a single FVIII, is then cloned into a construct, which is a plasmid or other vector under control of appropriate transcription and translation ces for high level protein expression in a biological system. In a later step, a second gene or polynucleotide coding for the XTEN is genetically fused to the nucleotides encoding the N- and/or C-terminus of the FVIII gene by cloning it into the uct adjacent and in frame with the ) coding for the FVIII. This second step occurs through a ligation or multimerization step. In the foregoing embodiments hereinabove described in this paragraph, it is to be understood that the gene constructs that are d can alternatively be the ment of the respective genes that encode the respective fusion proteins.

The gene encoding for the XTEN can be made in one or more steps, either fully synthetically or by synthesis combined with enzymatic processes, such as ction enzyme-mediated cloning, PCR and overlap extension, including methods more fully described in the Examples. The methods disclosed herein can be used, for example, to ligate short sequences of polynucleotides encoding XTEN into longer XTEN genes of a desired length and sequence. In one embodiment, the method ligates two or more codon—optimized oligonucleotides encoding XTEN motif or segment sequences of about 9 to 14 amino acids, or about 12 to 20 amino acids, or about 18 to 42 amino acids, or about 42 to about 144 amino acids, or about 144 to about 288 amino acids, or 288 to about 864 amino acids or longer, or any combination of the ing ranges of motif or segment lengths.

Alternatively, the disclosed method is used to multimerize XTEN-encoding sequences into longer sequences of a desired length; e. g., a gene encoding 36 amino acids ofXTEN can be dimerized into a gene encoding 72 amino acids, then 144, then 288, etc. Even with multimerization, XTEN polypeptides can be constructed such that the XTEN-encoding gene has low or virtually no repetitiveness through design of the codons selected for the motifs of the shortest unit being used, which can reduce recombination and increase stability of the encoding gene in the ormed host.

Genes encoding XTEN with petitive sequences are assembled from oligonucleotides using standard techniques of gene sis. The gene design can be performed using algorithms that optimize codon usage and amino acid composition. In one method of the invention, a library of relatively short XTEN-encoding cleotide constructs is created and then assembled, as described above. The resulting genes are then led with genes encoding FVIII or regions of FVIII, as illustrated in FIGS. 11 and 12, and the resulting genes used to transform a host cell and produce and r the CFXTEN for evaluation of its properties, as described herein.

In another aspect, the invention provides isolated c acids comprising a polynucleotide sequence encoding the CFXTEN fusion protein ments described herein. In one embodiment, the isolated nucleic acid comprises a polynucleotide sequence selected from (a) a sequence having at least about 80% sequence identity, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, to about 100% sequence identity compared to a ce of comparable length selected from Table 21, when optimally aligned, or (b) the ment of the polynucleotide of (a). In another embodiment, the isolated nucleic acid comprises the sequence ATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGT (SEQ ID NO: 1613) linked to the 5’ end of the nucleic acid of (a) or the complement of the ce linked to the 3’ end of (b).

Polynucleotide libraries In another , the invention provides libraries of cleotides that encode XTEN sequences that are used to assemble genes that encode XTEN of a desired length and ce.

In n ments, the XTEN-encoding library constructs comprise polynucleotides that encode polypeptide segments of a fixed length. As an initial step, a library of oligonucleotides that encode motifs of 9-14 amino acid residues can be assembled. In a preferred embodiment, libraries of oligonucleotides that encode motifs of 12 amino acids are assembled.

The XTEN-encoding sequence ts can be dimerized or multimerized into longer encoding sequences, as depicted schematically in . Dimerization or multimerization can be performed by ligation, overlap extension, PCR assembly or similar cloning techniques known in the art.

This process of can be repeated multiple times until the resulting XTEN-encoding sequences have reached the organization of sequence and desired , providing the XTEN-encoding genes. As will be iated, a library of polynucleotides that encodes, e. g., 12 amino acid motifs can be dimerized and/or ligated into a library of polynucleotides that encode 36 amino acids. Libraries ng motifs of different lengths; e. g., 9-14 amino acid motifs leading to libraries encoding 27 to 42 amino acids are plated by the invention. In turn, the library of polynucleotides that encode 27 to 42 amino acids, and preferably 36 amino acids (as described in the Examples) can be serially dimerized into a library ning successively longer lengths of polynucleotides that encode XTEN sequences of a desired length for incorporation into the gene encoding the CFXTEN fusion protein, as disclosed herein.

] A more efficient way to optimize the DNA sequence encoding XTEN is based on combinatorial libraries. The gene encoding XTEN can be designed and synthesized in segment such that multiple codon versions are obtained for each segment. These segments can be randomly assembled into a library of genes such that each library member encodes the same amino acid sequences but library members comprise a large number of codon versions. Such libraries can be screened for genes that result in high-level expression and/or a low abundance of truncation ts. The process of combinatorial gene assembly is illustrated in . The genes in are led from 6 base fragments and each fragment is available in 4 ent codon versions. This allows for a theoretical diversity of 4096.

In some embodiments, libraries are assembled of cleotides that encode amino acids that are limited to specific sequence XTEN families; e. g., the AD, AE, AF, AG, AM, or AQ sequences of Table 4. In other embodiments, libraries comprise sequences that encode two or more of the motif family sequences from Table 3. The names and sequences of representative, non-limiting cleotide sequences of libraries that encode 36mers are presented in Tables 13-17, and the methods used to create them are described more fully in the respective Examples. In other embodiments, ies that encode XTEN are constructed from segments of polynucleotide codons linked in a randomized sequence that encode amino acids wherein at least about 80%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% of the codons are selected from the group consisting of condons for glycine (G), e (A), serine (S), threonine (T), glutamate (E) and proline (P) amino acids. The libraries can be used, in turn, for serial dimerization or ligation to achieve polynucleotide sequence libraries that encode XTEN sequences, for example, of 42, 48, 72, 144, 288, 576, 864, 875, 912, 923, 1318 amino acids, or up to a total length of about 3000 amino acids, as well as intermediate lengths, in which the encoded XTEN can have one or more of the properties disclosed herein, when expressed as a component of a CFXTEN filSiOIl protein. In some cases, the polynucleotide y ces may also include additional bases used as ”sequencing islands,” described more fully below. is a schematic rt of entative, non-limiting steps in the assembly of a XTEN cleotide construct and a CFXTEN polynucleotide construct in the ments of the invention. Individual oligonucleotides 501 are annealed into sequence motifs 502 such as a 12 amino acid motif (“12-mer”), which is ligated to additional sequence motifs from a library to create a pool that encompasses the desired length of the XTEN 504, as well as ligated to a smaller concentration of an oligo containing BbsI, and KpnI restriction sites 503. The resulting pool of ligation products is gelpurified and the band with the desired length ofXTEN is cut, resulting in an isolated XTEN gene with a stopper sequence 505. The XTEN gene is cloned into a stuffer vector. In this case, the vector encodes an optional CBD sequence 506 and a GFP gene 508. Digestion is than performed with BbsI/HindIII to remove 507 and 508 and place the stop codon. The resulting product is then cloned into a BsaI/HindIII digested vector containing a gene encoding the FVIII, resulting in the gene 500 encoding an FVIII- XTEN fusion protein.

One may clone the library of XTEN-encoding genes into one or more expression vectors known in the art. To facilitate the fication of well-expressing library members, one can construct the library as fusion to a reporter protein. miting examples of suitable reporter genes are green cent protein, luciferace, alkaline phosphatase, and beta-galactosidase. By screening, one can identify short XTEN sequences that can be expressed in high concentration in the host organism of choice. Subsequently, one can generate a library of random XTEN dimers and repeat the screen for high level of sion. Subsequently, one can screen the resulting constructs for a number of properties such as level of expression, se stability, or binding to antiserum.

One aspect of the invention is to provide polynucleotide sequences encoding the components of the fusion protein wherein the creation of the sequence has undergone codon optimization. Of ular interest is codon optimization with the goal of improving expression of the polypeptide compositions and to improve the genetic stability of the encoding gene in the production hosts. For example, codon optimization is of particular importance for XTEN sequences that are rich in glycine or that have very repetitive amino acid sequences. Codon optimization is performed using er programs (Gustafsson, C., et al. (2004) Trends Biotechnol, 22: 346-53), some of which ze ribosomal pausing (Coda Genomics Inc.). In one embodiment, one can perform codon optimization by constructing codon libraries where all members of the library encode the same amino acid sequence but where codon usage is varied. Such libraries can be screened for highly expressing and genetically stable members that are particularly suitable for the large-scale production of XTEN-containing products. When designing XTEN sequences one can consider a number of properties. One can minimize the repetitiveness in the ng DNA sequences. In addition, one can avoid or minimize the use of codons that are rarely used by the production host (e.g. the AGG and AGA arginine codons and one leucine codon in E. coli). In the case of E. coli, two glycine codons, GGA and GGG, are rarely used in highly expressed proteins. Thus codon optimization of the gene encoding XTEN sequences can be very desirable. DNA sequences that have a high level of glycine tend to have a high GC content that can lead to ility or low expression levels. Thus, when possible, it is preferred to choose codons such that the GC-content of XTEN- encoding sequence is suitable for the tion organism that will be used to cture the XTEN.

In one embodiment, polynucleotide libraries are constructed using the disclosed methods n all members of the library encode the same amino acid sequence but where codon usage for the respective amino acids in the sequence is varied or optimized for the intended host cell. Such libraries can be screened for highly sing and genetically stable members that are particularly suitable for the large-scale production of XTEN-containing products. In one ment, the libraries are optimized for expression in a eukaryotic host cell.

Optionally, one can sequence clones in the library to eliminate isolates that contain undesirable sequences. The initial library of short XTEN ces allows some variation in amino acid sequence.

For instance one can randomize some codons such that a number of hilic amino acids can occur in a particular position. During the s of iterative multimerization one can screen the ing library members for other characteristics like solubility or protease resistance in addition to a screen for high- level expression.

Once the gene that encodes the XTEN of desired length and ties is selected, it is genetically fused at the desired location to the nucleotides encoding the FVIII gene(s) by cloning it into the construct adjacent and in frame with the gene coding for FVIII, or alternatively between nucleotides encoding adjacent domains of the FVIII, or alternatively within a ce encoding a given FVIII domain, or atively in frame with nucleotides ng a spacer/cleavage sequence linked to a terminal XTEN. The invention provides various permutations of the foregoing, depending on the CFXTEN to be encoded. For example, a gene encoding a CFXTEN fusion protein sing a FVIII and two XTEN, such as embodied by formula VI, as depicted above, the gene would have cleotides encoding FVIII, encoding two XTEN, which can be identical or different in composition and sequence length. In one non-limiting embodiment of the foregoing, the FVIII polynucleotides would encode factor VIII and the cleotides encoding the C-terminus XTEN would encode an XTEN of 288 amino acids and the polynucleotides encoding an internal XTEN adjacent to the C-terminus of the A2 domain would encode an XTEN of 144 amino acids. The step of cloning the FVIII genes into the XTEN construct can occur through a ligation or erization step, as shown in . The constructs encoding CFXTEN fusion ns can be ed in different configurations of the components XTEN, CF, and spacer sequences, such as the configurations of formulae I-VIII. In one embodiment, the construct comprises polynucleotide sequences complementary to, or those that encode a monomeric polypeptide of components in the following order (5’ to 3’) FVIII, an XTEN internal to the B domain, and a inal XTEN. In another embodiment, the construct comprises polynucleotide sequences complementary to, or those that encode a monomeric polypeptide of components in the following order (5’ to 3’) FVIIII, spacer sequence linked to the C-terminus, and XTEN. The spacer polynucleotides can optionally comprise sequences ng cleavage sequences. As will be apparent to those of skill in the art, multiple permutations of FVIII domains and inserted XTEN are possible.

] Homology, sequence similarity or sequence identity of nucleotide or amino acid sequences may also be determined conventionally by using known software or er ms such as the BestFit or Gap pairwise comparison programs (GCG Wisconsin Package, Genetics er Group, 575 Science Drive, Madison, Wis. 53711). BestFit uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics. 1981. 2: 482-489), to find the best segment of identity or similarity between two sequences. Gap performs global alignments: all of one sequence with all of another similar sequence using the method ofNeedleman and Wunsch, (Journal of Molecular Biology. 1970. 48:443- 453). When using a sequence alignment program such as BestFit, to ine the degree of sequence homology, similarity or identity, the default setting may be used, or an appropriate scoring matrix may be ed to optimize identity, similarity or homology scores.

Nucleic acid sequences that are “complementary” are those that are capable of base-pairing according to the rd Watson-Crick mentarity rules. As used herein, the term ementary ces” means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as d as being capable of hybridizing to the polynucleotides that encode the CFXTEN sequences under stringent conditions, such as those described herein.

The resulting polynucleotides encoding the CFXTEN ic fusion proteins can then be dually cloned into an expression vector. The nucleic acid sequence is inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan.

Such techniques are well known in the art and well described in the scientific and patent literature.

Various vectors are publicly available. The vector may, for example, be in the form of a plasmid, cosmid, Viral particle, or phage that may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be uced.

Thus, the vector may be an autonomously replicating vector, i.e., a vector, which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e. g., a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the some(s) into which it has been integrated.

Representative plasmids are illustrated in , with encoding regions for different configurations of FVIII and XTEN ents portrayed.

The invention provides for the use of d vectors containing replication and control sequences that are compatible with and recognized by the host cell, and are operably linked to the CFXTEN gene for lled expression of the CFXTEN fusion ns. The vector ordinarily carries a replication site, as well as sequences that encode proteins that are capable of providing phenotypic ion in transformed cells. Such vector sequences are well known for a variety of bacteria, yeast, and Viruses. Useful expression vectors that can be used include, for example, segments of chromosomal, non-chromosomal and synthetic DNA sequences. ”Expression vector” refers to a DNA construct containing a DNA sequence that is operably linked to a suitable control sequence capable of effecting the expression of the DNA encoding the fusion protein in a suitable host. The requirements are that the vectors are replicable and Viable in the host cell of choice. Low— or high-copy number s may be used as desired.

Other suitable vectors include, but are not limited to, derivatives of SV40 and pcDNA and known bacterial plasmids such as col El, pCRl, , pMal-C2, pET, pGEX as described by Smith, et al., Gene 57:31-40 (1988), pMB9 and derivatives thereof, plasmids such as RP4, phage DNAs such as the numerous derivatives of phage I such as NM98 9, as well as other phage DNA such as M13 and tous single stranded phage DNA; yeast plasmids such as the 2 micron plasmid or tives of the 2m d, as well as centomeric and integrative yeast shuttle vectors; vectors useful in eukaryotic cells such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as ds that have been modified to employ phage DNA or the expression control sequences; and the like. Yeast expression systems that can also be used in the present invention include, but are not limited to, the non-fusion pYES2 vector (Invitrogen), the fusion pYESHisA, B, C (Invitrogen), pRS vectors and the like.

The control sequences of the vector include a er to effect transcription, an optional operator sequence to control such ription, a sequence encoding suitable mRNA ribosome binding sites, and sequences that control ation of transcription and ation. The promoter may be any DNA sequence, Which shows transcriptional activity in the host cell of choice and may be derived from genes ng proteins either homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of the DNA encoding the FVIH polypeptide variant in mammalian cells are the SV40 promoter (Subramani et al., Mol. Cell. Biol. 1 (1981), 854-864), the MT-l (metallothionein gene) er (Palmiter et al., e 222 (1983), 809- 814), the CMV promoter (Boshart et al., Cell 41 :521-530, 1985) or the adenovirus 2 major late promoter (Kaufman and Sharp, Mol. Cell. Biol, 2:1304-1319, 1982). The vector may also carry sequences such as UCOE (ubiquitous chromatin g ts).

Examples of suitable promoters for use in ntous fungus host cells are, for instance, the ADH3 promoter or the tpiA promoter. Examples of other useful promoters are those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei ic proteinase, A. niger neutral (X- amylase, A. niger acid stable (x-amylase, A. niger or A. awamoriglucoamylase (gluA), ucor miehei lipase, A. oryzae alkaline se, A. oryzae triose phosphate isomerase or A. nidulans acetamidase.

Preferred are the TAKA—amylase and gluA promoters.

] Promoters suitable for use in expression vectors with prokaryotic hosts include the B-lactamase and lactose promoter s [Chang et al., , 275:615 (1978); Goeddel et al., Nature, 281 :544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci.

USA, 80:21-25 (1983)], all is operably linked to the DNA encoding CFXTEN ptides. Promoters for use in bacterial systems can also contain a Shine-Dalgarno (S.D.) sequence, operably linked to the DNA encoding CFXTEN polypeptides.

The invention contemplates use of other expression systems including, for example, a baculovirus sion system With both non-fusion transfer vectors, such as, but not limited to pVL941 Summers, et al., Virology 84:390-402 (1978)), pVL1393 rogen), pVL1392 (Summers, et al., Virology 84:390- 402 (1978) and anitrogen) and pBlueBaclll (anitrogen), and fusion transfer vectors such as, but not d to, pAc7 00 (Summers, et al., Virology 84:390-402 ), pAc701 and pAc70- 2 (same as pAc700, With different reading frames), pAc36O anitrogen) and pBlueBacHisA, B, C (; ogen) can be used.

Examples of suitable promoters for directing the transcription of the DNA encoding the FVIH polypeptide variant in mammalian cells are the CMV promoter (Boshart et al., Cell 41 :521-530, 1985), the SV40 promoter (Subramani et al., Mol. Cell. Biol. 1 (1981), 854-864), the MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222 (1983), 809-814), the adenovirus 2 major late promoter (Kaufman and Sharp, Mol. Cell. Biol, 2:1304-1319, 1982). The vector may also carry sequences such as UCOE (ubiquitous chromatin opening elements).

] The DNA sequences encoding the CFXTEN may also, if necessary, be operably connected to a suitable terminator, such as the hGH terminator (Palmiter et al., Science 222, 1983, pp. 809-814) or the TP11 terminators (Alber and Kawasaki, J. Mol. Appl. Gen. 1, 1982, pp. 419-434) or ADH3 (McKnight et al., The EMBO J. 4, 1985, pp. 2093-2099). sion vectors may also contain a set of RNA splice sites located downstream from the promoter and upstream from the insertion site for the CFXTEN sequence itself, including splice sites obtained from adenovirus. Also contained in the expression vectors is a polyadenylation signal located ream of the insertion site. Particularly preferred polyadenylation signals include the early or late polyadenylation signal from SV40 (Kaufman and Sharp, ibid.), the enylation signal from the adenovirus 5 Elb region, the hGH terminator (DeNoto et al. Nucl. Acids Res. 3730, 1981). The expression vectors may also include a noncoding viral leader sequence, such as the adenovirus 2 tripartite leader, located between the promoter and the RNA splice sites; and er sequences, such as the SV40 enhancer.

To direct the CFXTEN of the present invention into the secretory pathway of the host cells, a secretory signal sequence (aka, a leader sequence, a prepro sequence, or a pre sequence) may be ed in the recombinant . The ory signal sequence is operably linked to the DNA sequences encoding the CFXTEN, usually positioned 5’ to the DNA sequence encoding the CFXTEN fusion n. The secretory signal sequence may be that, normally associated with the native FVIII protein or may be from a gene encoding another secreted protein. miting examples include OmpA, PhoA, and DsbA for E. coli expression, ppL-alpha, DEX4, invertase signal peptide, acid phosphatase signal peptide, CPY, or INUl for yeast expression, and 1L2L, SV40, IgG kappa and IgG lambda for mammalian expression. Signal sequences are typically proteolytically removed from the protein during the ocation and secretion process, generating a defined N-terminus. Methods are sed in Arnau, er al., Protein Expression and Purification 48: 1-13 (2006).

The procedures used to ligate the DNA sequences coding for the CFXTEN, the er and optionally the terminator and/or secretory signal sequence, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook, J. et al., “Molecular Cloning: A Laboratory Manual,” 3ml edition, Cold Spring Harbor Laboratory Press, 2001). In this manner, a chimeric DNA molecule coding for a monomeric CFXTEN fusion protein is generated within the construct. Optionally, this chimeric DNA molecule may be transferred or cloned into another construct that is a more appropriate expression vector. At this point, a host cell capable of expressing the chimeric DNA molecule can be transformed with the chimeric DNA molecule.

Non-limiting examples of ian cell lines for use in the present invention are the COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), BHK-21 (ATCC CCL 10)) and BHK-293 (ATCC CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977), 0 cells (ATCC CRL 10314), CHO-Kl (ATCC CCL 61), CHO-S rogen 11619-012), and 293-F (Invitrogen ), and the parental and derivative cell lines known in the art useful for expression of FVIII. A tk-ts13 BHK cell line is also available from the ATCC under accession number CRL 1632. In addition, a number of other cell lines may be used within the present invention, including Rat Hep I (Rat hepatoma; ATCC CRL 1600), Rat Hep 11 (Rat hepatoma; ATCC CRL 1548), TCMK (ATCC CCL 139), Human lung (ATCC HB 8065), NCTC 1469 (ATCC CCL 9.1), CHO (ATCC CCL 61) and DUKX cells (Urlaub and Chasin, Proc. Natl.

Acad. Sci. USA 77:4216-4220, 1980).

] Examples of suitable yeasts cells e cells of Saccharomyces spp. or Schizosaccharomyces spp., in particular strains of Saccharomyces cerevisiae or Saccharomyces kluyveri . Methods for transforming yeast cells With heterologous DNA and producing heterologous polypeptides there from are described, e.g. in US. Pat. No. 4,599,311, US. Pat. No. 373, US. Pat. No. 4,870,008, 5,037,743, and US. Pat. No. 4,845,075, all of Which are hereby incorporated by reference. Transformed cells are selected by a phenotype determined by a selectable marker, commonly drug resistance or the y to grow in the absence of a particular nutrient, e. g. leucine. A preferred vector for use in yeast is the PCT] vector disclosed in US. Pat. No. 4,931,373. The DNA ces encoding the CFXTEN may be preceded by a signal sequence and optionally a leader sequence, e.g. as described above. Further examples of suitable yeast cells are s of Kluyveromyces, such as K. lactis, Hansenula e. g. H. polymorpha or Pichia P. pastoris (cf. Gleeson et al., J. Gen. Microbiol. , , e.g. 132, 1986, pp. 3459-3465; US. Pat. No. 4,882,279). Examples of other fungal cells are cells of filamentous fungi, e. g. Aspergillus spp., Neurospora spp., Fusarium spp. or derma spp., in particular strains ofA. oryzae, A. nidulans or A. niger. The use ofAspergillus spp. for the expression of proteins is described in, e. g., EP 272 277, EP 238 023, EP 184 438 The transformation of F. oxysporum may, for instance, be carried out as described by Malardier et al., 1989 Gene 78: 147-156. The transformation of Trichoderma spp. may be performed for instance as described in EP 244 234.

Other suitable cells that can be used in the present invention include, but are not d to, yotic host cells strains such as Escherichia coli, (e. g., strain DH5-oc), Bacillus subtilis, Salmonella typhimurium, or strains of the genera of Pseudomonas, Streptomyces and Staphylococcus. Non-limiting examples of suitable prokaryotes include those from the genera: planes; Archaeoglobus; Bdellovibrio; Borrelia; Chloroflexus; Enterococcus; Escherichia; Lactobacillus; Listeria; Oceanobacillus; ccus; Pseudomonas; Staphylococcus; Streptococcus; Streptomyces; plasma; and Vibrio.

Methods of transfecting mammalian cells and sing DNA sequences introduced in the cells are described in e. g., Kaufman and Sharp, J. Mol. Biol. 159 (1982), 601-621; Southern and Berg, J.

Mol. Appl. Genet. 1 (1982), 327-341; Loyter et al., Proc. Natl. Acad. Sci. USA 79 (1982), 422-426; Wigler et al., Cell 14 (1978), 725; Corsaro and Pearson, Somatic Cell Genetics 7 (1981), 603, Graham and van der Eb, Virology 52 (1973), 456; and Neumann et al., EMBO J. 1 (1982), 841-845.

Cloned DNA sequences are uced into cultured mammalian cells by, for example, calcium phosphate-mediated transfection (Wigler et al., Cell 14:725-732, 1978; Corsaro and Pearson, Somatic Cell cs 616, 1981; Graham and Van der Eb, Virology 52d:456-467, 1973), transfection With many commercially available reagents such as FuGENEG Roche Diagnostics, Mannheim, y) or ctamine (anitrogen) or by electroporation (Neumann et al., EMBO J. 1:841-845, 1982). To identify and select cells that express the exogenous DNA, a gene that confers a able phenotype (a selectable marker) is generally introduced into cells along With the gene or cDNA of interest. Preferred selectable markers include genes that confer resistance to drugs such as neomycin, hygromycin, puromycin, zeocin, and methotrexate. The selectable marker may be an amplifiable selectable marker. A preferred amplif1able selectable marker is a dihydrofolate reductase (DHFR) ce. r examples of selectable markers are well known to one of skill in the art and include reporters such as enhanced green fluorescent protein , beta-galactosidase (B-gal) or mphenicol transferase (CAT). Selectable markers are reviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers, am, Mass., incorporated herein by reference). The person skilled in the art will easily be able to choose suitable selectable s. Any known selectable marker may be employed so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product.

Selectable markers may be introduced into the cell on a separate d at the same time as the gene of interest, or they may be introduced on the same plasmid. If, on the same plasmid, the selectable marker and the gene of interest may be under the control of different promoters or the same promoter, the latter arrangement producing a dicistronic message. ucts of this type are known in the art (for example, Levinson and Simonsen, US. Pat. No. 4,713,339). It may also be ageous to add additional DNA, known as “carrier DNA,” to the mixture that is introduced into the cells.

After the cells have taken up the DNA, they are grown in an appropriate grth medium, typically 1-2 days, to begin expressing the gene of interest. As used herein the term “appropriate grth medium” means a medium containing nts and other components required for the grth of cells and the expression of the CFXTEN of interest. Media generally include a carbon source, a nitrogen source, essential amino acids, essential sugars, Vitamins, salts, phospholipids, protein and grth factors.

For production of gamma-carboxylated proteins, the medium will contain Vitamin K, preferably at a concentration of about 0.1 [Lg/ml to about 5 [Lg/ml. Drug selection is then d to select for the grth of cells that are expressing the selectable marker in a stable fashion. For cells that have been ected with an amplif1able selectable marker the drug concentration may be increased to select for an increased copy number of the cloned ces, thereby increasing expression levels. Clones of stably transfected cells are then screened for expression of the FVIH polypeptide variant of interest.

The transformed or transfected host cell is then cultured in a suitable nutrient medium under conditions permitting expression of the CFXTEN polypeptide after which the resulting peptide may be recovered from the culture as an isolated fusion protein. The medium used to culture the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be ed according to published recipes (e. g. in catalogues of the American Type Culture Collection). The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

] Gene expression may be measured in a sample ly, for example, by conventional rn blotting, Northern blotting to quantitate the transcription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies may be employed that can recognize specif1c duplexes, including DNA duplexes, RNA es, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The dies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be ed.

Gene expression, alternatively, may be ed by immunological of fluorescent methods, such as immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids or the detection of selectable markers, to tate directly the expression of gene product. Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies may be prepared against a native sequence FVIII ptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to FVIII and ng a specific antibody e.

Examples of selectable markers are well known to one of skill in the art and include reporters such as enhanced green fluorescent protein , beta-galactosidase (5-gal) or chloramphenicol acetyltransferase (CAT). ] sed CFXTEN polypeptide product(s) may be purified via methods known in the art or by methods disclosed herein. Procedures such as gel filtration, affinity purification (e. g., using an anti- FVIII antibody column), salt fractionation, ion exchange chromatography, size ion chromatography, hydroxyapatite adsorption chromatography, hydrophobic ction chromatography and gel electrophoresis may be used; each tailored to recover and purify the fusion protein produced by the respective host cells. Additional purification may be achieved by conventional chemical purification means, such as high performance liquid chromatography. Some expressed CFXTEN may require refolding during isolation and purification. Methods of purification are described in Robert K. Scopes, Protein Purification: Principles and Practice, Charles R. Castor (ed.), Springer-Verlag 1994, and Sambrook, et al., supra. Multi-step purification separations are also described in Baron, et al., Crit. Rev.

Biotechnol. 10:179-90 (1990) and Below, et al., J. Chromatogr. A. -83 . For therapeutic purposes it is preferred that the CFXTEN fusion proteins of the invention are substantially pure. Thus, in a preferred embodiment of the invention the CFXTEN of the ion is purified to at least about 90 to 95% neity, preferably to at least about 98% homogeneity. Purity may be assessed by, e. g., gel electrophoresis, HPLC, and amino-terminal amino acid sequencing..

VIII). PHARMACEUTICAL COMPOSITIONS The present ion provides pharmaceutical compositions sing CFXTEN. In one embodiment, the pharmaceutical composition comprises a CFXTEN fusion protein disclosed herein admixed with at least one pharmaceutically acceptable carrier. CFXTEN polypeptides of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the polypeptide is combined in admixture with a ceutically acceptable carrier vehicle, such as aqueous solutions, buffers, solvents and/or pharmaceutically acceptable suspensions, emulsions, stabilizers or ents. Examples of non-aqueous solvents include propyl ethylene , polyethylene glycol and vegetable oils. Formulations of the pharmaceutical compositions are prepared for storage by mixing the active CFXTEN ingredient having the desired degree of purity with optional logically able carriers, excipients (e. g., sodium chloride, a calcium salt, sucrose, or polysorbate) or stabilizers (e. g., sucrose, trehalose, raffinose, arginine, a calcium salt, glycine or histidine), as described in Remington's Pharmaceutical Sciences 16th edition, Osol, A.

Ed. (1980), in the form of lyophilized formulations or aqueous solutions.

] The pharmaceutical composition may be supplied as a lyophilized powder to be reconstituted prior to administration. In r embodiment, the ceutical composition may be supplied in a liquid form in a Vial, the contents of which can be administered directly to a patient. Alternatively, the composition is supplied as a liquid in a pre-filled syringe for administration of the ition. In another embodiment, the composition is ed as a liquid in a pre-fllled Vial that can be incorporated into a pump.

The pharmaceutical compositions can be administered by any suitable means or route, including subcutaneously, subcutaneously by infusion pump, intramuscularly, and intravenously. It will be appreciated that the preferred route will vary with the disease and age of the ent, and the severity of the condition being treated.

In one embodiment, the CFXTEN pharmaceutical composition in liquid form or after reconstitution (when supplied as a lyophilized powder) comprises coagulation factor VIII with an actiVity of at least 50 IU/ml, or at least 100 IU/ml, or at least 200 IU/ml, or at least 300 IU/ml, or at least 400 IU/ml, or an actiVity of at least 500 IU/ml, or an actiVity of at least 600 IU/ml, which composition is capable of increasing factor VIII actiVity to at least 1.5% of the normal plasma level in the blood for at least about 12 hours, or at least about 24 hours, or at least about 48 hours, or at least about 72 hours, or at least about 96 hours, or at least about 120 hours after administration of the factor VIII ceutical composition to a subject in need of routine prophylaxis. In another embodiment, the CFXTEN pharmaceutical composition in liquid form or after reconstitution (when supplied as a lyophilized powder) comprises coagulation factor VIII with an actiVity of at least 50 IU/ml, or at least 100 IU/ml, or at least 200 IU/ml, or at least 300 IU/ml, or at least 400 IU/ml, or at least 500 IU/ml, or an actiVity of at least 600 IU/ml, which composition is capable of increasing factor VIII actiVity to at least 2.5% of the normal plasma level in the blood for at least about 12 hours, or at least about 24 hours, or at least about 48 hours, or at least about 72 hours, or at least about 96 hours, or at least about 120 hours after administration to a subject in need of e prophylaxis. It is specifically contemplated that the pharmaceutical compositions of the foregoing can be formulated to include one or more ents, s or other ients known in the art to be compatible with administration by the intravenous route or the subcutaneous route or the intramuscular route. Thus, in the embodiments hereinabove described in this paragraph, the pharmaceutical composition is stered subcutaneously, intramuscularly or intravenously.

The compositions of the invention may be formulated using a variety of excipients. Suitable ents include rystalline cellulose (e. g. Avicel PH102, Avicel PH101), polymethacrylate, poly(ethyl acrylate, methyl methacrylate, trimethylammonioethyl methacrylate chloride) (such as Eudragit RS-3OD), hydroxypropyl methylcellulose (Methocel K1 00M, Premium CR Methocel K1 00M, Methocel E5, Opadry®), magnesium stearate, talc, triethyl citrate, aqueous ethylcellulose dispersion (Surelease®), and protamine sulfate. The slow release agent may also comprise a carrier, which can comprise, for example, solvents, dispersion media, coatings, antibacterial and antifungal , isotonic and absorption delaying . Pharmaceutically acceptable salts can also be used in these slow release , for example, mineral salts such as hydrochlorides, hydrobromides, phosphates, or sulfates, as well as the salts of organic acids such as acetates, proprionates, malonates, or benzoates. The composition may also contain liquids, such as water, saline, glycerol, and ethanol, as well as substances such as wetting agents, emulsifying agents, or pH buffering agents. mes may also be used as a carrier.

In another embodiment, the compositions of the present invention are encapsulated in liposomes, which have demonstrated utility in delivering beneficial active agents in a controlled manner over prolonged periods of time. Liposomes are closed bilayer membranes containing an entrapped aqueous volume. Liposomes may also be unilamellar vesicles possessing a single membrane bilayer or multilamellar vesicles with le membrane bilayers, each separated from the next by an aqueous layer. The structure of the resulting ne bilayer is such that the hydrophobic (non-polar) tails of the lipid are oriented toward the center of the bilayer while the hydrophilic (polar) heads orient towards the aqueous phase. In one embodiment, the liposome may be coated with a flexible water soluble polymer that avoids uptake by the organs of the mononuclear phagocyte system, primarily the liver and spleen. Suitable hydrophilic polymers for surrounding the liposomes include, without tion, PEG, nylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropylmethacrylate, polyhydroxethylacrylate, hydroxymethylcellulose hydroxyethylcellulose, polyethyleneglycol, polyaspartamide and hydrophilic peptide ces as described in US. Pat. Nos. 6,316,024; 966; 6,056,973; 6,043,094, the contents of which are incorporated by reference in their entirety. Additional liposomal technologies are described in US. Pat. Nos. 6,759,057; 713; 6,352,716; 6,316,024; 191; 6,126,966; 6,056,973; 6,043,094; 5,965,156; 5,916,588; 5,874,104; 5,215,680; and 479, the contents ofwhich are incorporated herein by reference. These describe liposomes and lipid-coated microbubbles, and methods for their manufacture. Thus, one d in the art, considering both the disclosure of this invention and the disclosures of these other patents could produce a liposome for the ed release of the polypeptides of the present ion.

For liquid formulations, a d property is that the ation be ed in a form that can pass through a 25, 28, 30, 31, 32 gauge needle for intravenous, intramuscular, intraarticular, or subcutaneous administration.

Syringe pumps may also be used as slow e agents. Such devices are described in US.

Pat. Nos. 4,976,696; 4,933,185; 5,017,378; 6,309,370; 6,254,573; 4,435,173; 4,398,908; 6,572,585; ,298,022; 5,176,502; 5,492,534; 5,318,540; and 4,988,337, the contents of which are orated herein by reference. One skilled in the art, considering both the disclosure of this invention and the disclosures of these other patents could produce a syringe pump for the ed release of the compositions of the present invention.

IX). PHARMACEUTICAL KITS In another aspect, the invention provides a kit to tate the use of the CFXTEN polypeptides. The kit comprises the pharmaceutical composition provided herein, a ner and a label or package insert on or associated with the container. Suitable ccrttaitiers include, for example, bottles, Vials, syringes, etc, formed frem a variety 0f als such as glass or plastic. The container holds a pharmaceutical ition as a fermulatien that is effective for treating the EVEN—related condition and may have a sterile access pert (for example the container maybe an intravenous selution bag or a vial having a stepper pierceable by a hypedermie injection needle). The e insert can list the approved indications for the drug, instructions for the reconstitution and/or stration of the drug for the use for the approved indication, appropriate dosage and safety information, and information identifying the lot and expiration of the drug. In another embodiment of the foregoing, the kit can comprise a second container that can carry a suitable t for the pharmaceutical composition, the use of which will provide the user with the appropriate tration to be delivered to the subject.

EXAMPLES Example 1: Construction ofXTEN_AD36 motif segments The following example describes the construction of a collection of codon—optimized genes encoding motif sequences of 36 amino acids. As a first step, a stuffer vector pCWO359 was constructed based on a pET vector and that includes a T7 promoter. pCWO359 s a cellulose binding domain (CBD) and a TEV protease recognition site followed by a stuffer sequence that is flanked by BsaI, BbsI, and KpnI sites. The BsaI and BbsI sites were inserted such that they generate compatible overhangs after digestion. The stuffer sequence is followed by a truncated version of the GFP gene and a His tag. The stuffer sequence ns stop codons and thus E. coli cells carrying the stuffer plasmid pCWO359 form non-fluorescent colonies. The stuffer vector pCWO359 was ed with BsaI and KpnI to remove the stuffer segment and the resulting vector fragment was isolated by agarose gel purification. The ces were designated XTEN_AD3 6, ing the AD family of motifs. Its segments have the amino acid sequence [X]3 where X is a 12mer peptide with the sequences: GESPGGSSGSES (SEQ ID NO: 19), GSEGSSGPGESS (SEQ ID NO: 20), GSSESGSSEGGP (SEQ ID NO: 21), or GSGGEPSESGSS (SEQ ID NO: 22). The insert was obtained by ing the following pairs of phosphorylated synthetic oligonucleotide pairs: Aleor: AGGTGAATCTCCDGGTGGYTCYAGCGGTTCYGARTC (SEQ ID NO: 1619) ADlrev: ACCTGAYTCRGAACCGCTRGARCCACCHGGAGATTC (SEQ ID NO: 1620) AD2for: AGGTAGCGAAGGTTCTTCYGGTCCDGGYGARTCYTC (SEQ ID NO: 1621) AD2rev: ACCTGARGAYTCRCCHGGACCRGAAGAACCTTCGCT (SEQ ID NO: 1622) AD3for: AGGTTCYTCYGAAAGCGGTTCTTCYGARGGYGGTCC (SEQ ID NO: 1623) AD3reV: ACCRCCYTCRGAAGAACCGCTTTCRGARGA (SEQ ID NO: 1624) AD4for: AGGTTCYGGTGGYGAACCDTCYGARTCTGGTAGCTC (SEQ ID NO: 1625) We also annealed the orylated oligonucleotide 3KpnIstopperFor: AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 1626) and the non-phosphorylated oligonucleotide pr_3KpnIstopperReV: CCTCGAGTGAAGACGA (SEQ ID NO: 1627). The ed oligonucleotide pairs were ligated, which resulted in a mixture of ts with varying length that represents the varying number of 12mer repeats ligated to one BbsI/KpnI segment. The products corresponding to the length of 36 amino acids were isolated from the mixture by preparative agarose gel electrophoresis and ligated into the BsaI/KpnI digested stuffer vector pCWO35 9. Most of the clones in the resulting library ated LCWO401 showed green fluorescence after induction, which shows that the sequence ofXTEN_AD36 had been ligated in frame with the GFP gene and that most sequences of XTEN_AD36 had good sion levels.

We screened 96 isolates from library LCWO401 for high level of fluorescence by stamping them onto agar plate containing IPTG. The same isolates were evaluated by PCR and 48 isolates were identified that contained segments with 36 amino acids as well as strong fluorescence. These isolates were sequenced and 39 clones were identified that contained t XTEN_AD36 segments. The file names of the tide and amino acid constructs and the sequences for these segments are listed in Table 13.

Table 13: DNA and Amino Acid Seguences for AD 36-mer motifs (SEQ ID NOS 203-278, res ectivel in order of a earance File name Amino acid sequence Nucleotide sequence LCWO401_001_ GSGGEPSESGSSGESPGG GGTTCTGGTGGCGAACCGTCCGAGTCTGGTAGCTCA GFP-\_A0 1 .ab1 SSGSESGESPGGSSGSES GGTGAATCTCCGGGTGGCTCTAGCGGTTCCGAGTCA GGTGAATCTCCTGGTGGTTCCAGCGGTTCCGAGTCA LCWO401_002_ GSEGSSGPGESSGESPGG GGTAGCGAAGGTTCTTCTGGTCCTGGCGAGTCTTCA GFP-\_B01 .ab1 SSGSESGSSESGSSEGGP GGTGAATCTCCTGGTGGTTCCAGCGGTTCTGAATCA GGTTCCTCCGAAAGCGGTTCTTCCGAGGGCGGTCCA LCWO401_003_ GSSESGSSEGGPGSSESG GGTTCCTCTGAAAGCGGTTCTTCCGAAGGTGGTCCA GFP-\_C0 1 .ab1 SSEGGPGESPGGSSGSES GGTTCCTCTGAAAGCGGTTCTTCTGAGGGTGGTCCA TCTCCGGGTGGCTCCAGCGGTTCCGAGTCA LCWO401_004_ SESGSSGSSESG GGTTCCGGTGGCGAACCGTCTGAATCTGGTAGCTCA GFP-\_D0 1 .abl SSEGGPGSGGEPSESGSS GGTTCTTCTGAAAGCGGTTCTTCCGAGGGTGGTCCA GGTGGTGAACCTTCCGAGTCTGGTAGCTCA LCWO401_007_ GSSESGSSEGGPGSEGSS TCCGAAAGCGGTTCTTCTGAGGGTGGTCCA GFP-\_F0 1 .ab1 GPGESSGSEGSSGPGESS GAAGGTTCTTCCGGTCCAGGTGAGTCTTCA GGTAGCGAAGGTTCTTCTGGTCCTGGTGAATCTTCA LCWO401_008_ GSSESGSSEGGPGESPGG GGTTCCTCTGAAAGCGGTTCTTCCGAGGGTGGTCCA GFP-\_G0 1 .ab1 SSGSESGSEGSSGPGESS GGTGAATCTCCAGGTGGTTCCAGCGGTTCTGAGTCA GGTAGCGAAGGTTCTTCTGGTCCAGGTGAATCCTCA LCWO401_012_ GSGGEPSESGSSGSGGEP GGTTCTGGTGGTGAACCGTCTGAGTCTGGTAGCTCA GFP-\_H0 1 .ab1 SESGSSGSEGSSGPGESS GGTTCCGGTGGCGAACCATCCGAATCTGGTAGCTCA GGTAGCGAAGGTTCTTCCGGTCCAGGTGAGTCTTCA LCWO401_015_ GSSESGSSEGGPGSEGSS GGTTCTTCCGAAAGCGGTTCTTCCGAAGGCGGTCCA GFP-\_A02.ab1 GPGESSGESPGGSSGSES GGTAGCGAAGGTTCTTCTGGTCCAGGCGAATCTTCA GGTGAATCTCCTGGTGGCTCCAGCGGTTCTGAGTCA LCWO401_016_ GSSESGSSEGGPGSSESG TCCGAAAGCGGTTCTTCTGAGGGCGGTCCA GFP-\_B02.ab1 SSEGGPGSSESGSSEGGP GGTTCCTCCGAAAGCGGTTCTTCCGAGGGCGGTCCA GGTTCTTCTGAAAGCGGTTCTTCCGAGGGCGGTCCA File name Amino acid sequence Nucleotide sequence LCW0401_020_ GSGGEPSESGSSGSEGSS GGTTCCGGTGGCGAACCGTCCGAATCTGGTAGCTCA GFP-\_E02.ab1 GPGESSGSSESGSSEGGP GGTAGCGAAGGTTCTTCTGGTCCAGGCGAATCTTCA GGTTCCTCTGAAAGCGGTTCTTCTGAGGGCGGTCCA LCW0401_022_ GSGGEPSESGSSGSSESG GGTTCTGGTGGTGAACCGTCCGAATCTGGTAGCTCA GFP-\_F02.ab1 SSEGGPGSGGEPSESGSS GGTTCTTCCGAAAGCGGTTCTTCTGAAGGTGGTCCA GGTTCCGGTGGCGAACCTTCTGAATCTGGTAGCTCA LCW0401_024_ GSGGEPSESGSSGSSESG GGTTCTGGTGGCGAACCGTCCGAATCTGGTAGCTCA GFP-\_G02.ab1 SSEGGPGESPGGSSGSES GGTTCCTCCGAAAGCGGTTCTTCTGAAGGTGGTCCA GGTGAATCTCCAGGTGGTTCTAGCGGTTCTGAATCA LCW0401_026_ SESGSSGESPGG GGTTCTGGTGGCGAACCGTCTGAGTCTGGTAGCTCA GFP-\_H02.ab1 SSGSESGSEGSSGPGESS GGTGAATCTCCTGGTGGCTCCAGCGGTTCTGAATCA GAAGGTTCTTCTGGTCCTGGTGAATCTTCA LCW0401_027_ SESGSSGESPGG GGTTCCGGTGGCGAACCTTCCGAATCTGGTAGCTCA GFP-\_A03.ab1 SSGSESGSGGEPSESGSS GGTGAATCTCCGGGTGGTTCTAGCGGTTCTGAGTCA GGTTCTGGTGGTGAACCTTCCGAGTCTGGTAGCTCA LCW0401_028_ GSSESGSSEGGPGSSESG GGTTCCTCTGAAAGCGGTTCTTCTGAGGGCGGTCCA GFP-\_B03.ab1 SSEGGPGSSESGSSEGGP GGTTCTTCCGAAAGCGGTTCTTCCGAGGGCGGTCCA GGTTCTTCCGAAAGCGGTTCTTCTGAAGGCGGTCCA LCW0401_030_ GESPGGSSGSESGSEGSS GGTGAATCTCCGGGTGGCTCCAGCGGTTCTGAGTCA GFP-\_C03.ab1 GPGESSGSEGSSGPGESS GGTAGCGAAGGTTCTTCCGGTCCGGGTGAGTCCTCA GAAGGTTCTTCCGGTCCTGGTGAGTCTTCA LCW0401_031_ GSGGEPSESGSSGSGGEP GGTTCTGGTGGCGAACCTTCCGAATCTGGTAGCTCA GFP-\_D03.ab1 SESGSSGSSESGSSEGGP GGTTCCGGTGGTGAACCTTCTGAATCTGGTAGCTCA GGTTCTTCTGAAAGCGGTTCTTCCGAGGGCGGTCCA LCW0401_033_ GSGGEPSESGSSGSGGEP GGTTCCGGTGGTGAACCTTCTGAATCTGGTAGCTCA E03.ab1 SESGSSGSGGEPSESGSS GGTTCCGGTGGCGAACCATCCGAGTCTGGTAGCTCA GGTTCCGGTGGTGAACCATCCGAGTCTGGTAGCTCA LCW0401_037_ GSGGEPSESGSSGSSESG GGTTCCGGTGGCGAACCTTCTGAATCTGGTAGCTCA GFP-\_F03.ab1 SSEGGPGSEGSSGPGESS GGTTCCTCCGAAAGCGGTTCTTCTGAGGGCGGTCCA GGTAGCGAAGGTTCTTCTGGTCCGGGCGAGTCTTCA LCW0401_038_ GSGGEPSESGSSGSEGSS GGTGGTGAACCGTCCGAGTCTGGTAGCTCA GFP-\_G03.ab1 GPGESSGSGGEPSESGSS GAAGGTTCTTCTGGTCCGGGTGAGTCTTCA GGTTCTGGTGGCGAACCGTCCGAATCTGGTAGCTCA LCW0401_039_ GSGGEPSESGSSGESPGG GGTTCTGGTGGCGAACCGTCCGAATCTGGTAGCTCA GFP-\_H03.ab1 SSGSESGSGGEPSESGSS GGTGAATCTCCTGGTGGTTCCAGCGGTTCCGAGTCA GGTTCTGGTGGCGAACCTTCCGAATCTGGTAGCTCA 1_040_ GSSESGSSEGGPGSGGEP GGTTCTTCCGAAAGCGGTTCTTCCGAGGGCGGTCCA GFP-\_A04.ab1 SESGSSGSSESGSSEGGP GGTTCCGGTGGTGAACCATCTGAATCTGGTAGCTCA GGTTCTTCTGAAAGCGGTTCTTCTGAAGGTGGTCCA LCW0401_042_ GSEGSSGPGESSGESPGG GGTAGCGAAGGTTCTTCCGGTCCTGGTGAGTCTTCA GFP-\_C04.ab1 SSGSESGSEGSSGPGESS GGTGAATCTCCAGGTGGCTCTAGCGGTTCCGAGTCA GAAGGTTCTTCTGGTCCTGGCGAGTCCTCA LCW0401_046_ GSSESGSSEGGPGSSESG GGTTCCTCTGAAAGCGGTTCTTCCGAAGGCGGTCCA GFP-\_D04.ab1 SSEGGPGSSESGSSEGGP GGTTCTTCCGAAAGCGGTTCTTCTGAGGGCGGTCCA GGTTCCTCCGAAAGCGGTTCTTCTGAGGGTGGTCCA LCW0401_047_ GSGGEPSESGSSGESPGG GGTTCTGGTGGCGAACCTTCCGAGTCTGGTAGCTCA E04.ab1 SSGSESGESPGGSSGSES GGTGAATCTCCGGGTGGTTCTAGCGGTTCCGAGTCA GGTGAATCTCCGGGTGGTTCCAGCGGTTCTGAGTCA LCW0401_051_ GSGGEPSESGSSGSEGSS GGTTCTGGTGGCGAACCATCTGAGTCTGGTAGCTCA GFP-\_F04.ab1 GPGESSGESPGGSSGSES GGTAGCGAAGGTTCTTCCGGTCCAGGCGAGTCTTCA GGTGAATCTCCTGGTGGCTCCAGCGGTTCTGAGTCA LCW0401_053_ GESPGGSSGSESGESPGG GGTGAATCTCCTGGTGGTTCCAGCGGTTCCGAGTCA GFP-\_H04.ab1 SSGSESGESPGGSSGSES GGTGAATCTCCAGGTGGCTCTAGCGGTTCCGAGTCA GGTGAATCTCCTGGTGGTTCTAGCGGTTCTGAATCA LCW0401_054_ GSEGSSGPGESSGSEGSS GGTAGCGAAGGTTCTTCCGGTCCAGGTGAATCTTCA GFP-\_A05.ab1 GSGGEPSESGSS GAAGGTTCTTCTGGTCCTGGTGAATCCTCA GGTTCCGGTGGCGAACCATCTGAATCTGGTAGCTCA LCW0401_059_ SESGSSGSEGSS GGTTCTGGTGGCGAACCATCCGAATCTGGTAGCTCA GFP-\_D05.ab1 GPGESSGESPGGSSGSES GAAGGTTCTTCTGGTCCTGGCGAATCTTCA GGTGAATCTCCAGGTGGCTCTAGCGGTTCCGAATCA File name Amino acid ce Nucleotide sequence LCW0401_060_ GSGGEPSESGSSGSSESG GGTTCCGGTGGTGAACCGTCCGAATCTGGTAGCTCA E05.ab1 SSEGGPGSGGEPSESGSS GGTTCCTCTGAAAGCGGTTCTTCCGAGGGTGGTCCA GGTTCCGGTGGTGAACCTTCTGAGTCTGGTAGCTCA LCW0401_061_ GSSESGSSEGGPGSGGEP GGTTCCTCTGAAAGCGGTTCTTCTGAGGGCGGTCCA GFP-\_F05.ab1 SESGSSGSEGSSGPGESS GGTTCTGGTGGCGAACCATCTGAATCTGGTAGCTCA GGTAGCGAAGGTTCTTCCGGTCCGGGTGAATCTTCA LCW0401_063_ SESGSSGSEGSS GGTGGTGAACCGTCCGAATCTGGTAGCTCA GFP-\_H05.ab1 GPGESSGSEGSSGPGESS GGTAGCGAAGGTTCTTCTGGTCCTGGCGAGTCTTCA GAAGGTTCTTCTGGTCCTGGTGAATCTTCA LCW0401_066_ GSGGEPSESGSSGSSESG GGTTCTGGTGGCGAACCATCCGAGTCTGGTAGCTCA GFP-\_B06.ab1 SSEGGPGSGGEPSESGSS GGTTCTTCCGAAAGCGGTTCTTCCGAAGGCGGTCCA GGTTCTGGTGGTGAACCGTCCGAATCTGGTAGCTCA LCW0401_067_ GSGGEPSESGSSGESPGG GGTTCCGGTGGCGAACCTTCCGAATCTGGTAGCTCA GFP-\_C06.ab1 GESPGGSSGSES GGTGAATCTCCGGGTGGTTCTAGCGGTTCCGAATCA GGTGAATCTCCAGGTGGTTCTAGCGGTTCCGAATCA LCW0401_069_ GSGGEPSESGSSGSGGEP GGTTCCGGTGGTGAACCATCTGAGTCTGGTAGCTCA GFP-\_D06.ab1 SESGSSGESPGGSSGSES GGTTCCGGTGGCGAACCGTCCGAGTCTGGTAGCTCA GGTGAATCTCCGGGTGGTTCCAGCGGTTCCGAATCA LCW0401_070_ GSEGSSGPGESSGSSESG GGTAGCGAAGGTTCTTCTGGTCCGGGCGAATCCTCA GFP-\_E06.ab1 SSEGGPGSEGSSGPGESS GGTTCCTCCGAAAGCGGTTCTTCCGAAGGTGGTCCA GGTAGCGAAGGTTCTTCCGGTCCTGGTGAATCTTCA LCW0401_078_ GSSESGSSEGGPGESPGG GGTTCCTCTGAAAGCGGTTCTTCTGAAGGCGGTCCA F06.ab1 SSGSESGESPGGSSGSES GGTGAATCTCCGGGTGGCTCCAGCGGTTCTGAATCA GGTGAATCTCCTGGTGGCTCCAGCGGTTCCGAGTCA LCW0401_079_ GSEGSSGPGESSGSEGSS GGTAGCGAAGGTTCTTCTGGTCCAGGCGAGTCTTCA GFP-\_G06.ab1 GPGESSGSGGEPSESGSS GGTAGCGAAGGTTCTTCCGGTCCTGGCGAGTCTTCA GGTTCCGGTGGCGAACCGTCCGAATCTGGTAGCTCA Example 2: Construction ofXTEN_AE36 segments A codon library encoding XTEN sequences of 36 amino acid length was constructed. The XTEN sequence was designated XTEN_AE36. Its segments have the amino acid sequence [X]3 where X is a 12mer peptide with the sequence: GSPAGSPTSTEE (SEQ ID NO: 23), GSEPATSGSETP (SEQ ID NO: 24), TPESGP (SEQ ID NO: 25), or GTSTEPSEGSAP (SEQ ID NO: 26). The insert was obtained by annealing the following pairs of phosphorylated synthetic ucleotide pairs: AElfor: AGGTAGCCCDGCWGGYTCTCCDACYTCYACYGARGA (SEQ ID NO: 1628) AElreV: ACCTTCYTCRGTRGARGTHGGAGARCCWGCHGGGCT (SEQ ID NO: 1629) AE2for: AGGTAGCGAACCKGCWACYTCYGGYTCTGARACYCC (SEQ ID NO: 1630) : ACCTGGRGTYTCAGARCCRGARGTWGCMGGTTCGCT (SEQ ID NO: 1631) AE3for: AGGTACYTCTGAAAGCGCWACYCCKGARTCYGGYCC (SEQ ID NO: 1632) AE3reV: ACCTGGRCCRGAYTCMGGRGTWGCGCTTTCAGARGT (SEQ ID NO: 1633) AE4for: AGGTACYTCTACYGAACCKTCYGARGGYAGCGCWCC (SEQ ID NO: 1634) AE4reV: ACCTGGWGCGCTRCCYTCRGAMGGTTCRGTAGARGT (SEQ ID NO: 1635) We also annealed the phosphorylated oligonucleotide 3KpnIstopperFor: AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 1626) and the non-phosphorylated oligonucleotide pr_3KpnIstopperReV: CCTCGAGTGAAGACGA (SEQ ID NO: 1627). The annealed oligonucleotide pairs were ligated, which ed in a mixture of products with varying length that represents the varying number of 12mer repeats ligated to one BbsI/KpnI segment. The products corresponding to the length of 36 amino acids were isolated from the e by preparative agarose gel electrophoresis and ligated into the BsaI/Kpnl digested stuffer vector pCWO35 9. Most of the clones in the resulting library designated LCWO402 showed green fluorescence after induction which shows that the sequence of E36 had been ligated in frame with the GFP gene and most sequences of XTEN_AE36 show good expression.

We screened 96 es from library LCWO402 for high level of fluorescence by stamping them onto agar plate containing IPTG. The same isolates were ted by PCR and 48 es were identified that contained segments with 36 amino acids as well as strong fluorescence. These isolates were sequenced and 37 clones were identified that contained correct E36 segments. The file names of the nucleotide and amino acid ucts and the sequences for these segments are listed in Table l 4.

Table 14: DNA and Amino Acid Seguences for AE 36-mer motifs (SEQ ID NOS 279-352, res l in order of a earance File name Amino acid seguence Nucleotide sequence LCWO402_002_ GSPAGSPTSTEEGTSE GGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAA GFP-\_A07.ab1 SATPESGPGTSTEPSE GGTACTTCTGAAAGCGCAACCCCGGAGTCCGGCCCA GSAP TCTACCGAACCGTCTGAGGGCAGCGCACCA LCWO402_003_ GTSTEPSEGSAPGTST GGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCA GFP-\_B07.ab1 EPSEGSAPGTSTEPSE GGTACCTCTACTGAACCTTCCGAGGGCAGCGCTCCA GSAP GGTACCTCTACCGAACCTTCTGAAGGTAGCGCACCA LCWO402_004_ GTSTEPSEGSAPGTSE TCTACCGAACCGTCTGAAGGTAGCGCACCA GFP-\_C07.ab1 SATPESGPGTSESATP GGTACCTCTGAAAGCGCAACTCCTGAGTCCGGTCCA ESGP GGTACTTCTGAAAGCGCAACCCCGGAGTCTGGCCCA LCWO402_005_ SEGSAPGTSE GGTACTTCTACTGAACCGTCTGAAGGTAGCGCACCA GFP-\_D07.ab1 SATPESGPGTSESATP GGTACTTCTGAAAGCGCAACCCCGGAATCCGGCCCA ESGP GGTACCTCTGAAAGCGCAACCCCGGAGTCCGGCCCA LCWO402_006_ GSEPATSGSETPGTSE GGTAGCGAACCGGCAACCTCCGGCTCTGAAACCCCA GFP-\_E07.ab1 SATPESGPGSPAGSPT GGTACCTCTGAAAGCGCTACTCCTGAATCCGGCCCA STEE GGTAGCCCGGCAGGTTCTCCGACTTCCACTGAGGAA LCWO402_008_ GTSESATPESGPGSEP GGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCA GFP-\_F07.ab1 ATSGSETPGTSTEPSE GGTAGCGAACCGGCTACTTCTGGCTCTGAGACTCCA GSAP GGTACTTCTACCGAACCGTCCGAAGGTAGCGCACCA LCWO402_009_ GSPAGSPTSTEEGSPA GGTAGCCCGGCTGGCTCTCCAACCTCCACTGAGGAA GFP-\_G07.ab1 GSPTSTEEGSEPATSG GGTAGCCCGGCTGGCTCTCCAACCTCCACTGAAGAA SETP GGTAGCGAACCGGCTACCTCCGGCTCTGAAACTCCA LCWO402_01 1_ GSPAGSPTSTEEGTSE GGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAA GFP-\_A08.ab1 SATPESGPGTSTEPSE GGTACTTCTGAAAGCGCTACTCCTGAGTCTGGTCCA GSAP GGTACCTCTACTGAACCGTCCGAAGGTAGCGCTCCA LCWO402_012_ GSPAGSPTSTEEGSPA GGTAGCCCTGCTGGCTCTCCGACTTCTACTGAGGAA GFP-\_B08.ab1 GSPTSTEEGTSTEPSE GGTAGCCCGGCTGGTTCTCCGACTTCTACTGAGGAA GSAP TCTACCGAACCTTCCGAAGGTAGCGCTCCA LCWO402_013_ GTSESATPESGPGTST GGTACTTCTGAAAGCGCTACTCCGGAGTCCGGTCCA GFP-\_C08.ab1 EPSEGSAPGTSTEPSE GGTACCTCTACCGAACCGTCCGAAGGCAGCGCTCCA GSAP GGTACTTCTACTGAACCTTCTGAGGGTAGCGCTCCA LCWO402_014_ GTSTEPSEGSAPGSPA GGTACCTCTACCGAACCTTCCGAAGGTAGCGCTCCA GFP-\_D08.ab1 GSPTSTEEGTSTEPSE GGTAGCCCGGCAGGTTCTCCTACTTCCACTGAGGAA GSAP GGTACTTCTACCGAACCTTCTGAGGGTAGCGCACCA 2_015_ GSEPATSGSETPGSPA GGTAGCGAACCGGCTACTTCCGGCTCTGAGACTCCA GFP-\_E08.ab1 GSPTSTEEGTSESATP CCTGCTGGCTCTCCGACCTCTACCGAAGAA ESGP GGTACCTCTGAAAGCGCTACCCCTGAGTCTGGCCCA LCWO402_016_ GTSTEPSEGSAPGTSE GGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCA F08.ab1 SATPESGPGTSESATP GGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCA File name Amino acid sequence Nucleotide sequence ESGP GGTACTTCTGAAAGCGCTACTCCTGAATCCGGTCCA LCW0402_020_ GTSTEPSEGSAPGSEP GGTACTTCTACTGAACCGTCTGAAGGCAGCGCACCA GFP-\_G08.ab1 ATSGSETPGSPAGSPT GGTAGCGAACCGGCTACTTCCGGTTCTGAAACCCCA STEE GGTAGCCCAGCAGGTTCTCCAACTTCTACTGAAGAA LCW0402_023_ GSPAGSPTSTEEGTSE GGTAGCCCTGCTGGCTCTCCAACCTCCACCGAAGAA GFP-\_A09.ab1 SATPESGPGSEPATSG GGTACCTCTGAAAGCGCAACCCCTGAATCCGGCCCA SETP GGTAGCGAACCGGCAACCTCCGGTTCTGAAACCCCA 2_024_ TPESGPGSPA GGTACTTCTGAAAGCGCTACTCCTGAGTCCGGCCCA GFP-\_B09.ab1 GSPTSTEEGSPAGSPT GGTAGCCCGGCTGGCTCTCCGACTTCCACCGAGGAA STEE GGTAGCCCGGCTGGCTCTCCAACTTCTACTGAAGAA LCW0402_025_ GTSTEPSEGSAPGTSE GGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCA GFP-\_C09.ab1 SATPESGPGTSTEPSE GGTACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCA GSAP GGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCA LCW0402_026_ GSPAGSPTSTEEGTST CCGGCAGGCTCTCCGACTTCCACCGAGGAA GFP-\_D09.ab1 EPSEGSAPGSEPATSG GGTACCTCTACTGAACCTTCTGAGGGTAGCGCTCCA SETP GGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCA LCW0402_027_ GSPAGSPTSTEEGTST GGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAA GFP-\_E09.ab1 EPSEGSAPGTSTEPSE GGTACTTCTACTGAACCTTCCGAAGGCAGCGCACCA GSAP GGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCA LCW0402_032_ GSEPATSGSETPGTSE GGTAGCGAACCTGCTACCTCCGGTTCTGAAACCCCA GFP-\_H09.ab1 GPGSPAGSPT GGTACCTCTGAAAGCGCAACTCCGGAGTCTGGTCCA STEE GGTAGCCCTGCAGGTTCTCCTACCTCCACTGAGGAA LCW0402_034_ GTSESATPESGPGTST GGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCA GFP-\_A10.ab1 EPSEGSAPGTSTEPSE GGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCA GSAP GGTACTTCTACTGAACCGTCCGAAGGTAGCGCACCA LCW0402_036_ GSPAGSPTSTEEGTST GGTAGCCCGGCTGGTTCTCCGACTTCCACCGAGGAA GFP-\_C10.ab1 EPSEGSAPGTSTEPSE GGTACCTCTACTGAACCTTCTGAGGGTAGCGCTCCA GSAP TCTACTGAACCTTCCGAAGGCAGCGCTCCA LCW0402_039_ GTSTEPSEGSAPGTST GGTACTTCTACCGAACCGTCCGAGGGCAGCGCTCCA GFP-\_E10.ab1 EPSEGSAPGTSTEPSE GGTACTTCTACTGAACCTTCTGAAGGCAGCGCTCCA GSAP GGTACTTCTACTGAACCTTCCGAAGGTAGCGCACCA LCW0402_040_ SGSETPGTSE GGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCA GFP-\_F10.ab1 SATPESGPGTSTEPSE GGTACCTCTGAAAGCGCTACTCCTGAATCTGGCCCA GSAP TCTACTGAACCGTCCGAGGGCAGCGCACCA LCW0402_041_ GTSTEPSEGSAPGSPA GGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCA GFP-\_G10.ab1 GSPTSTEEGTSTEPSE GGTAGCCCAGCAGGTTCTCCTACCTCCACCGAGGAA GSAP GGTACTTCTACCGAACCGTCCGAGGGTAGCGCACCA 2_050_ SGSETPGTSE GGTAGCGAACCGGCAACCTCCGGCTCTGAAACTCCA GFP-\_A11.ab1 SATPESGPGSEPATSG GGTACTTCTGAAAGCGCTACTCCGGAATCCGGCCCA SETP GGTAGCGAACCGGCTACTTCCGGCTCTGAAACCCCA LCW0402_051_ GSEPATSGSETPGTSE GGTAGCGAACCGGCAACTTCCGGCTCTGAAACCCCA B11.ab1 SATPESGPGSEPATSG GGTACTTCTGAAAGCGCTACTCCTGAGTCTGGCCCA SETP GGTAGCGAACCTGCTACCTCTGGCTCTGAAACCCCA LCW0402_059_ GSEPATSGSETPGSEP GGTAGCGAACCGGCAACCTCTGGCTCTGAAACTCCA GFP-\_E11.ab1 ATSGSETPGTSTEPSE GAACCTGCAACCTCCGGCTCTGAAACCCCA GSAP GGTACTTCTACTGAACCTTCTGAGGGCAGCGCACCA LCW0402_060_ GTSESATPESGPGSEP GGTACTTCTGAAAGCGCTACCCCGGAATCTGGCCCA GFP-\_F11.ab1 ATSGSETPGSEPATSG GAACCGGCTACTTCTGGTTCTGAAACCCCA SETP GGTAGCGAACCGGCTACCTCCGGTTCTGAAACTCCA LCW0402_061_ GTSTEPSEGSAPGTST TCTACTGAACCTTCCGAAGGCAGCGCTCCA GFP-\_G11.ab1 EPSEGSAPGTSESATP GGTACCTCTACCGAACCGTCCGAGGGCAGCGCACCA ESGP GGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCA 2_065_ GSEPATSGSETPGTSE GGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCA GFP-\_A12.ab1 SATPESGPGTSESATP GGTACCTCTGAAAGCGCTACTCCGGAATCTGGTCCA ESGP GGTACTTCTGAAAGCGCTACTCCGGAATCCGGTCCA LCW0402_066_ GSEPATSGSETPGSEP GGTAGCGAACCTGCTACCTCCGGCTCTGAAACTCCA GFP-\_B12.ab1 ATSGSETPGTSTEPSE GGTAGCGAACCGGCTACTTCCGGTTCTGAAACTCCA GSAP GGTACCTCTACCGAACCTTCCGAAGGCAGCGCACCA LCW0402_067_ GSEPATSGSETPGTST GGTAGCGAACCTGCTACTTCTGGTTCTGAAACTCCA GFP-\_C12.ab1 EPSEGSAPGSEPATSG GGTACTTCTACCGAACCGTCCGAGGGTAGCGCTCCA SETP GGTAGCGAACCTGCTACTTCTGGTTCTGAAACTCCA File name Amino acid sequence Nucleotide sequence LCW0402_069_ SEGSAPGTST GGTACCTCTACCGAACCGTCCGAGGGTAGCGCACCA GFP-N_D12.ab1 EPSEGSAPGSEPATSG GGTACCTCTACTGAACCGTCTGAGGGTAGCGCTCCA SETP GGTAGCGAACCGGCAACCTCCGGTTCTGAAACTCCA LCW0402_073_ GTSTEPSEGSAPGSEP TCTACTGAACCTTCCGAAGGTAGCGCTCCA GFP-N_F12.ab1 ATSGSETPGSPAGSPT GGTAGCGAACCTGCTACTTCTGGTTCTGAAACCCCA STEE GGTAGCCCGGCTGGCTCTCCGACCTCCACCGAGGAA LCW0402_074_ GSEPATSGSETPGSPA GGTAGCGAACCGGCTACTTCCGGCTCTGAGACTCCA GFP-N_G12.ab1 GSPTSTEEGTSESATP CCAGCTGGTTCTCCAACCTCTACTGAGGAA ESGP GGTACTTCTGAAAGCGCTACCCCTGAATCTGGTCCA LCW0402_075_ GTSESATPESGPGSEP GGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCA GFP-N_H12.ab1 ATSGSETPGTSESATP GGTAGCGAACCTGCTACCTCCGGCTCTGAGACTCCA ESGP GGTACCTCTGAAAGCGCAACCCCGGAATCTGGTCCA ] Example 3: Construction ofXTEN_AF36 segments A codon library encoding sequences of 36 amino acid length was constructed. The sequences were ated F36. Its segments have the amino acid sequence [X]3 where X is a 12mer peptide with the sequence: GSTSESPSGTAP (SEQ ID NO: 27), GTSTPESGSASP (SEQ ID NO: 28), GTSPSGESSTAP (SEQ ID NO: 29), or GSTSSTAESPGP (SEQ ID NO: 30). The insert was obtained by annealing the following pairs of phosphorylated synthetic oligonucleotide pairs: AF 1 for: AGGTTCTACYAGCGAATCYCCKTCTGGYACYGCWCC (SEQ ID NO: 1636) AP 1rev: ACCTGGWGCRGTRCCAGAMGGRGATTCGCTRGTAGA (SEQ ID NO: 1637) AF2for: AGGTACYTCTACYCCKGAAAGCGGYTCYGCWTCTCC (SEQ ID NO: 1638) AF2reV: ACCTGGAGAWGCRGARCCGCTTTCMGGRGTAGARGT (SEQ ID NO: 1639) AF3for: YTCYCCKAGCGGYGAATCTTCTACYGCWCC (SEQ ID NO: 1640) AF3reV: ACCTGGWGCRGTAGAAGATTCRCCGCTMGGRGARGT (SEQ ID NO: 1641) AF4for: AGGTTCYACYAGCTCTACYGCWGAATCTCCKGGYCC (SEQ ID NO: 1642) : RCCMGGAGATTCWGCRGTAGAGCTRGTRGA (SEQ ID NO: 1643) We also annealed the phosphorylated oligonucleotide 3KpnIstopperFor: AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 1626) and the non-phosphorylated oligonucleotide pr_3KpnIstopperRev: CCTCGAGTGAAGACGA (SEQ ID NO: 1627). The annealed oligonucleotide pairs were ligated, which resulted in a e of products with varying length that represents the varying number of 12mer repeats ligated to one BbsI/KpnI segment The products corresponding to the length of 36 amino acids were isolated from the mixture by ative agarose gel electrophoresis and ligated into the BsaI/KpnI ed stuffer vector pCWO35 9. Most of the clones in the resulting library designated LCWO403 showed green fluorescence after induction which shows that the sequence ofXTEN_AF36 had been ligated in frame with the GFP gene and most sequences of XTEN_AF36 show good expression.

We screened 96 isolates from library LCWO403 for high level of fluorescence by stamping them onto agar plate containing IPTG. The same isolates were evaluated by PCR and 48 isolates were identified that ned segments with 36 amino acids as well as strong fluorescence. These isolates were ced and 44 clones were identified that contained correct XTEN_AF36 segments. The file names of the nucleotide and amino acid constructs and the sequences for these segments are listed in Table 15.

Table 15: DNA and Amino Acid Seguences for AF 36-mer motifs (SEQ ID NOS 353-440, res l in order of a earance File name Amino acid sequence tide sequence LCW0403_004_ GTSTPESGSASPGTSP GGTACTTCTACTCCGGAAAGCGGTTCCGCATCTCCA GFP-\_A01.abl SGESSTAPGTSPSGES GGTACTTCTCCTAGCGGTGAATCTTCTACTGCTCCAG STAP GTACCTCTCCTAGCGGCGAATCTTCTACTGCTCCA LCW0403_005_ GTSPSGESSTAPGSTS GGTACTTCTCCGAGCGGTGAATCTTCTACCGCACCA GFP-\_B01.abl STAESPGPGTSPSGES GGTTCTACTAGCTCTACCGCTGAATCTCCGGGCCCAG STAP GTACTTCTCCGAGCGGTGAATCTTCTACTGCTCCA LCW0403_006_ GSTSSTAESPGPGTSP GGTTCCACCAGCTCTACTGCTGAATCTCCTGGTCCAG GFP-\_C01.abl SGESSTAPGTSTPESG GTACCTCTCCTAGCGGTGAATCTTCTACTGCTCCAGG SASP TACTTCTACTCCTGAAAGCGGCTCTGCTTCTCCA LCW0403_007_ GSTSSTAESPGPGSTS GGTTCTACCAGCTCTACTGCAGAATCTCCTGGCCCAG GFP-\_D01.abl STAESPGPGTSPSGES GTTCCACCAGCTCTACCGCAGAATCTCCGGGTCCAG STAP GTACTTCCCCTAGCGGTGAATCTTCTACCGCACCA LCW0403_008_ GSTSSTAESPGPGTSP GGTTCTACTAGCTCTACTGCTGAATCTCCTGGCCCAG GFP-\_E01.abl SGESSTAPGTSTPESG GTACTTCTCCTAGCGGTGAATCTTCTACCGCTCCAGG SASP TACTCCGGAAAGCGGTTCTGCATCTCCA LCW0403_010_ GSTSSTAESPGPGTST GGTTCTACCAGCTCTACCGCAGAATCTCCTGGTCCAG GFP-\_F01.abl PESGSASPGSTSESPS GTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAG GTAP GTTCTACTAGCGAATCTCCTTCTGGCACTGCACCA LCW0403_011_ GSTSSTAESPGPGTST GGTTCTACTAGCTCTACTGCAGAATCTCCTGGCCCAG GFP-\_G01.abl PESGSASPGTSTPESG GTACCTCTACTCCGGAAAGCGGCTCTGCATCTCCAG SASP GTACTTCTACCCCTGAAAGCGGTTCTGCATCTCCA LCW0403_012_ GSTSESPSGTAPGTSP GGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAG H01.abl SGESSTAPGSTSESPS GTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGG GTAP TTCTACTAGCGAATCTCCTTCTGGCACTGCACCA LCW0403_013_ GSTSSTAESPGPGSTS GGTTCCACCAGCTCTACTGCAGAATCTCCGGGCCCA GFP-\_A02.abl STAESPGPGTSPSGES GGTTCTACTAGCTCTACTGCAGAATCTCCGGGTCCAG STAP GTACTTCTCCTAGCGGCGAATCTTCTACCGCTCCA LCW0403_014_ GSTSSTAESPGPGTST GGTTCCACTAGCTCTACTGCAGAATCTCCTGGCCCAG GFP-\_B02.abl PESGSASPGSTSESPS GTACCTCTACCCCTGAAAGCGGCTCTGCATCTCCAG GTAP GTTCTACCAGCGAATCCCCGTCTGGCACCGCACCA LCW0403_015_ GSTSSTAESPGPGSTS GGTTCTACTAGCTCTACTGCTGAATCTCCGGGTCCAG GFP-\_C02.abl STAESPGPGTSPSGES GTTCTACCAGCTCTACTGCTGAATCTCCTGGTCCAGG STAP TACCTCCCCGAGCGGTGAATCTTCTACTGCACCA 3_017_ GSTSSTAESPGPGSTS GGTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAG GFP-\_D02.abl APGSTSSTAE GTTCTACCAGCGAATCCCCGTCTGGCACCGCACCAG SPGP GTTCTACTAGCTCTACCGCTGAATCTCCGGGTCCA LCW0403_018_ GSTSSTAESPGPGSTS GGTTCTACCAGCTCTACCGCAGAATCTCCTGGCCCA GFP-\_E02.abl STAESPGPGSTSSTAE GGTTCCACTAGCTCTACCGCTGAATCTCCTGGTCCAG SPGP CTAGCTCTACCGCTGAATCTCCTGGTCCA LCW0403_019_ PSGTAPGSTS GGTTCTACTAGCGAATCCCCTTCTGGTACTGCTCCAG GFP-\_F02.abl STAESPGPGSTSSTAE GTTCCACTAGCTCTACCGCTGAATCTCCTGGCCCAGG SPGP TTCCACTAGCTCTACTGCAGAATCTCCTGGTCCA LCW0403_023_ GSTSESPSGTAPGSTS GGTTCTACTAGCGAATCTCCTTCTGGTACCGCTCCAG GFP-\_H02.abl APGSTSESPS GTTCTACCAGCGAATCCCCGTCTGGTACTGCTCCAGG GTAP TTCTACCAGCGAATCTCCTTCTGGTACTGCACCA LCW0403_024_ GSTSSTAESPGPGSTS GGTTCCACCAGCTCTACTGCTGAATCTCCTGGCCCAG GFP-\_A03.abl GPGSTSSTAE GTTCTACCAGCTCTACTGCTGAATCTCCGGGCCCAGG SPGP TTCCACCAGCTCTACCGCTGAATCTCCGGGTCCA LCW0403_025_ GSTSSTAESPGPGSTS GGTTCCACTAGCTCTACCGCAGAATCTCCTGGTCCAG GFP-\_B03.abl STAESPGPGTSPSGES GTTCTACTAGCTCTACTGCTGAATCTCCGGGTCCAGG STAP TACCTCCCCTAGCGGCGAATCTTCTACCGCTCCA LCW0403_028_ GSSPSASTGTGPGSST GGTTCTAGCCCTTCTGCTTCCACCGGTACCGGCCCAG D03.abl PSGATGSPGSSTPSGA GTAGCTCTACTCCGTCTGGTGCAACTGGCTCTCCAGG TGSP TAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCA File name Amino acid sequence Nucleotide sequence LCW0403_029_ GTSPSGESSTAPGTST GGTACTTCCCCTAGCGGTGAATCTTCTACTGCTCCAG GFP-\_E03.ab1 PESGSASPGSTSSTAE GTACCTCTACTCCGGAAAGCGGCTCCGCATCTCCAG SPGP GTTCTACTAGCTCTACTGCTGAATCTCCTGGTCCA LCW0403_030_ GSTSSTAESPGPGSTS GGTTCTACTAGCTCTACCGCTGAATCTCCGGGTCCAG GFP-\_F03.ab1 STAESPGPGTSTPESG CCAGCTCTACTGCAGAATCTCCTGGCCCAGG SASP TACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCA 3_031_ GTSPSGESSTAPGSTS GGTACTTCTCCTAGCGGTGAATCTTCTACCGCTCCAG G03.ab1 STAESPGPGTSTPESG GTTCTACCAGCTCTACTGCTGAATCTCCTGGCCCAGG SASP TACTTCTACCCCGGAAAGCGGCTCCGCTTCTCCA LCW0403_033_ GSTSESPSGTAPGSTS GGTTCTACTAGCGAATCCCCTTCTGGTACTGCACCAG GFP-\_H03.ab1 STAESPGPGSTSSTAE CCAGCTCTACTGCTGAATCTCCGGGCCCAGG SPGP TTCCACCAGCTCTACCGCAGAATCTCCTGGTCCA LCW0403_035_ GSTSSTAESPGPGSTS GGTTCCACCAGCTCTACCGCTGAATCTCCGGGCCCA GFP-\_A04.ab1 ESPSGTAPGSTSSTAE GGTTCTACCAGCGAATCCCCTTCTGGCACTGCACCA SPGP GGTTCTACTAGCTCTACCGCAGAATCTCCGGGCCCA LCW0403_036_ AESPGPGTSP GGTTCTACCAGCTCTACTGCTGAATCTCCGGGTCCAG GFP-\_B04.ab1 SGESSTAPGTSTPESG CCCCGAGCGGTGAATCTTCTACTGCACCAG SASP GTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCA LCW0403_039_ PSGTAPGSTS GGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAG GFP-\_C04.ab1 ESPSGTAPGTSPSGES GTTCTACTAGCGAATCCCCGTCTGGTACCGCACCAG STAP GTACTTCTCCTAGCGGCGAATCTTCTACCGCACCA LCW0403_041_ GSTSESPSGTAPGSTS GGTTCTACCAGCGAATCCCCTTCTGGTACTGCTCCAG GFP-\_D04.ab1 ESPSGTAPGTSTPESG GTTCTACCAGCGAATCCCCTTCTGGCACCGCACCAG SASP GTACTTCTACCCCTGAAAGCGGCTCCGCTTCTCCA LCW0403_044_ GTSTPESGSASPGSTS GGTACCTCTACTCCTGAAAGCGGTTCTGCATCTCCAG GFP-\_E04.ab1 STAESPGPGSTSSTAE GTTCCACTAGCTCTACCGCAGAATCTCCGGGCCCAG SPGP GTTCTACTAGCTCTACTGCTGAATCTCCTGGCCCA LCW0403_046_ PSGTAPGSTS GGTTCTACCAGCGAATCCCCTTCTGGCACTGCACCA GFP-\_F04.ab1 ESPSGTAPGTSPSGES GGTTCTACTAGCGAATCCCCTTCTGGTACCGCACCAG STAP GTACTTCTCCGAGCGGCGAATCTTCTACTGCTCCA LCW0403_047_ GSTSSTAESPGPGSTS ACTAGCTCTACCGCTGAATCTCCTGGCCCAG GFP-\_G04.ab1 STAESPGPGSTSESPS CTAGCTCTACCGCAGAATCTCCGGGCCCAG GTAP GTTCTACTAGCGAATCCCCTTCTGGTACCGCTCCA LCW0403_049_ GSTSSTAESPGPGSTS ACCAGCTCTACTGCAGAATCTCCTGGCCCA GFP-\_H04.ab1 STAESPGPGTSTPESG GGTTCTACTAGCTCTACCGCAGAATCTCCTGGTCCAG SASP GTACCTCTACTCCTGAAAGCGGTTCCGCATCTCCA LCW0403_051_ GSTSSTAESPGPGSTS GGTTCTACTAGCTCTACTGCTGAATCTCCGGGCCCAG GFP-\_A05.ab1 STAESPGPGSTSESPS GTTCTACTAGCTCTACCGCTGAATCTCCGGGTCCAGG GTAP TTCTACTAGCGAATCTCCTTCTGGTACCGCTCCA LCW0403_053_ GTSPSGESSTAPGSTS GGTACCTCCCCGAGCGGTGAATCTTCTACTGCACCA GFP-\_B05.ab1 ESPSGTAPGSTSSTAE GGTTCTACTAGCGAATCCCCTTCTGGTACTGCTCCAG SPGP GTTCCACCAGCTCTACTGCAGAATCTCCGGGTCCA LCW0403_054_ GSTSESPSGTAPGTSP GGTTCTACTAGCGAATCCCCGTCTGGTACTGCTCCAG GFP-\_C05.ab1 SGESSTAPGSTSSTAE GTACTTCCCCTAGCGGTGAATCTTCTACTGCTCCAGG SPGP TTCTACCAGCTCTACCGCAGAATCTCCGGGTCCA LCW0403_057_ GSTSSTAESPGPGSTS ACCAGCTCTACCGCTGAATCTCCTGGCCCAG GFP-\_D05.ab1 ESPSGTAPGTSPSGES GTTCTACTAGCGAATCTCCGTCTGGCACCGCACCAG STAP GTACTTCCCCTAGCGGTGAATCTTCTACTGCACCA LCW0403_058_ GSTSESPSGTAPGSTS ACTAGCGAATCTCCTTCTGGCACTGCACCAG GFP-\_E05.ab1 ESPSGTAPGTSTPESG GTTCTACCAGCGAATCTCCGTCTGGCACTGCACCAG SASP GTACCTCTACCCCTGAAAGCGGTTCCGCTTCTCCA LCW0403_060_ GTSTPESGSASPGSTS GGTACCTCTACTCCGGAAAGCGGTTCCGCATCTCCA GFP-\_F05.ab1 ESPSGTAPGSTSSTAE GGTTCTACCAGCGAATCCCCGTCTGGCACCGCACCA SPGP GGTTCTACTAGCTCTACTGCTGAATCTCCGGGCCCA LCW0403_063_ GSTSSTAESPGPGTSP GGTTCTACTAGCTCTACTGCAGAATCTCCGGGCCCA GFP-\_G05.ab1 SGESSTAPGTSPSGES GGTACCTCTCCTAGCGGTGAATCTTCTACCGCTCCAG STAP GTACTTCTCCGAGCGGTGAATCTTCTACCGCTCCA LCW0403_064_ GTSPSGESSTAPGTSP GGTACCTCCCCTAGCGGCGAATCTTCTACTGCTCCAG GFP-\_H05.ab1 SGESSTAPGTSPSGES GTACCTCTCCTAGCGGCGAATCTTCTACCGCTCCAGG STAP TACCTCCCCTAGCGGTGAATCTTCTACCGCACCA LCW0403_065_ GSTSSTAESPGPGTST GGTTCCACTAGCTCTACTGCTGAATCTCCTGGCCCAG File name Amino acid sequence Nucleotide sequence GFP-\_A06.ab1 PESGSASPGSTSESPS GTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCCAGG GTAP TTCTACTAGCGAATCTCCGTCTGGCACCGCACCA LCW0403_066_ GSTSESPSGTAPGTSP GGTTCTACTAGCGAATCTCCGTCTGGCACTGCTCCAG GFP-\_B06.ab1 SGESSTAPGTSPSGES GTACTTCTCCTAGCGGTGAATCTTCTACCGCTCCAGG STAP TACTTCCCCTAGCGGCGAATCTTCTACCGCTCCA LCW0403_067_ GSTSESPSGTAPGTST GGTTCTACTAGCGAATCTCCTTCTGGTACCGCTCCAG GFP-\_C06.ab1 PESGSASPGSTSSTAE GTACTTCTACCCCTGAAAGCGGCTCCGCTTCTCCAGG SPGP TTCCACTAGCTCTACCGCTGAATCTCCGGGTCCA 3_068_ AESPGPGSTS GGTTCCACTAGCTCTACTGCTGAATCTCCTGGCCCAG GFP-\_D06.ab1 STAESPGPGSTSESPS GTTCTACCAGCTCTACCGCTGAATCTCCTGGCCCAGG GTAP TTCTACCAGCGAATCTCCGTCTGGCACCGCACCA LCW0403_069_ GSTSESPSGTAPGTST GGTTCTACTAGCGAATCCCCGTCTGGTACCGCACCA GFP-\_E06.ab1 PESGSASPGTSTPESG GGTACTTCTACCCCGGAAAGCGGCTCTGCTTCTCCAG SASP GTACTTCTACCCCGGAAAGCGGCTCCGCATCTCCA LCW0403_070_ GSTSESPSGTAPGTST GGTTCTACTAGCGAATCCCCGTCTGGTACTGCTCCAG F06.ab1 PESGSASPGTSTPESG GTACTTCTACTCCTGAAAGCGGTTCCGCTTCTCCAGG SASP TACCTCTACTCCGGAAAGCGGTTCTGCATCTCCA e 4: Construction ofXTEN_AG36 segments A codon library encoding ces of 36 amino acid length was constructed. The sequences were designated XTEN_AG36. Its ts have the amino acid sequence [X]3 where X is a 12mer peptide with the sequence: GTPGSGTASSSP (SEQ ID NO: 31), GSSTPSGATGSP (SEQ ID NO: 32), GSSPSASTGTGP (SEQ ID NO: 33), or GASPGTSSTGSP (SEQ ID NO: 34). The insert was obtained by annealing the following pairs of phosphorylated synthetic oligonucleotide pairs: AGlfor: AGGTACYCCKGGYAGCGGTACYGCWTCTTCYTCTCC (SEQ ID l\O: 1644) AGlreV: AGARGAAGAWGCRGTACCGCTRCCMGGRGT (SEQ ID NO: 1645) AG2for: AGGTAGCTCTACYCCKTCTGGTGCWACYGGYTCYCC (SEQ ID l\O: 1646) AG2reV: ACCTGGRGARCCRGTWGCACCAGAMGGRGTAGAGCT (SEQ ID NO: 1647) AG3for: AGGTTCTAGCCCKTCTGCWTCYACYGGTACYGGYCC (SEQ ID l\O: 1648) AG3reV: ACCTGGRCCRGTACCRGTRGAWGCAGAMGGGCTAGA (SEQ ID V0: 1649) AG4for: AGGTGCWTCYCCKGGYACYAGCTCTACYGGTTCTCC (SEQ ID NO: 1650) AG4reV: AGAACCRGTAGAGCTRGTRCCMGGRGAWGC (SEQ ID V0: 1651) We also annealed the phosphorylated oligonucleotide 3KpnIstopperFor: AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 1626) and the osphorylated oligonucleotide pr_3KpnIstopperRev: CCTCGAGTGAAGACGA (SEQ ID NO: 1627). The annealed oligonucleotide pairs were ligated, which resulted in a mixture of ts with g length that represents the varying number of 12mer repeats ligated to one BbsI/KpnI segment. The products corresponding to the length of 36 amino acids were isolated from the mixture by preparative agarose gel electrophoresis and ligated into the BsaI/KpnI digested stuffer vector pCWO35 9. Most of the clones in the resulting library designated LCWO404 showed green fluorescence after induction which shows that the sequence _AG36 had been ligated in frame with the GFP gene and most sequences of XTEN_AG36 show good expression.

] We screened 96 isolates from library LCWO404 for high level of fluorescence by stamping them onto agar plate containing IPTG. The same isolates were evaluated by PCR and 48 isolates were fied that contained segments with 36 amino acids as well as strong fluorescence. These isolates were sequenced and 44 clones were identified that contained correct G36 segments. The file names of the tide and amino acid constructs and the sequences for these segments are listed in Table 16.

Table 16: DNA and Amino Acid Seguences for AG 36-mer motifs (SEQ ID NOS 441-528, res l in order of a earance File name Amino acid sequence Nucleotide sequence LCW0404_001_ GASPGTSSTGSPGTPGS TCCCCGGGCACTAGCTCTACCGGTTCTCCA GFP-\_A07.ab1 GTASSSPGSSTPSGATG GGTACTCCTGGTAGCGGTACTGCTTCTTCTTCTCCAG SP GTAGCTCTACTCCTTCTGGTGCTACTGGTTCTCCA LCW0404_003_ GSSTPSGATGSPGSSPS GGTAGCTCTACCCCTTCTGGTGCTACCGGCTCTCCAG GFP-\_B07.ab1 PGSSTPSGATG GTTCTAGCCCGTCTGCTTCTACCGGTACCGGTCCAGG SP TAGCTCTACCCCTTCTGGTGCTACTGGTTCTCCA LCW0404_006_ GASPGTSSTGSPGSSPS GGTGCATCTCCGGGTACTAGCTCTACCGGTTCTCCAG GFP-\_C07.ab1 ASTGTGPGSSTPSGATG GTTCTAGCCCTTCTGCTTCCACTGGTACCGGCCCAGG SP TAGCTCTACCCCGTCTGGTGCTACTGGTTCCCCA 4_007_ GTPGSGTASSSPGSSTPS GGTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCAG GFP-\_D07.ab1 GATGSPGASPGTSSTGS GTAGCTCTACCCCTTCTGGTGCAACTGGTTCCCCAGG P TGCATCCCCTGGTACTAGCTCTACCGGTTCTCCA LCW0404_009_ GTPGSGTASSSPGASPG GGTACCCCTGGCAGCGGTACTGCTTCTTCTTCTCCAG GFP-\_E07.ab1 TSSTGSPGSRPSASTGT GTGCTTCCCCTGGTACCAGCTCTACCGGTTCTCCAGG GP TTCTAGACCTTCTGCATCCACCGGTACTGGTCCA LCW0404_01 1_ GASPGTSSTGSPGSSTPS GGTGCATCTCCTGGTACCAGCTCTACCGGTTCTCCAG GFP-\_F07.ab1 GATGSPGASPGTSSTGS GTAGCTCTACTCCTTCTGGTGCTACTGGCTCTCCAGG P TGCTTCCCCGGGTACCAGCTCTACCGGTTCTCCA LCW0404_012_ GTPGSGTASSSPGSSTPS GGTACCCCGGGCAGCGGTACCGCATCTTCCTCTCCA GFP-\_G07.ab1 GATGSPGSSTPSGATGS GGTAGCTCTACCCCGTCTGGTGCTACCGGTTCCCCAG P GTAGCTCTACCCCGTCTGGTGCAACCGGCTCCCCA LCW0404_014_ GASPGTSSTGSPGASPG GGTGCATCTCCGGGCACTAGCTCTACTGGTTCTCCAG H07.ab1 TSSTGSPGASPGTSSTGS GTGCATCCCCTGGCACTAGCTCTACTGGTTCTCCAGG P TCCTGGTACCAGCTCTACTGGTTCTCCA LCW0404_015_ GSSTPSGATGSPGSSPS GGTAGCTCTACTCCGTCTGGTGCAACCGGCTCCCCA GFP-\_A08.ab1 ASTGTGPGASPGTSSTG GGTTCTAGCCCGTCTGCTTCCACTGGTACTGGCCCAG SP GTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCA LCW0404_016_ GSSTPSGATGSPGSSTPS GGTAGCTCTACTCCTTCTGGTGCTACCGGTTCCCCAG GFP-\_B08.ab1 GATGSPGTPGSGTASSS GTAGCTCTACTCCTTCTGGTGCTACTGGTTCCCCAGG P TACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCA LCW0404_017_ GSSTPSGATGSPGSSTPS GGTAGCTCTACTCCGTCTGGTGCAACCGGTTCCCCAG C08.ab1 GATGSPGASPGTSSTGS CTACTCCTTCTGGTGCTACTGGCTCCCCAGG P TGCATCCCCTGGCACCAGCTCTACCGGTTCTCCA LCW0404_0 1 8_ GTPGSGTASSSPGSSPS CCTGGTAGCGGTACCGCATCTTCCTCTCCAG GFP-\_D08.ab1 ASTGTGPGSSTPSGATG GCCCTTCTGCATCTACCGGTACCGGTCCAGG SP TAGCTCTACTCCTTCTGGTGCTACTGGCTCTCCA LCW0404_023_ GASPGTSSTGSPGSSPS GGTGCTTCCCCGGGCACTAGCTCTACCGGTTCTCCAG GFP-\_F08.ab1 ASTGTGPGTPGSGTASS GTTCTAGCCCTTCTGCATCTACTGGTACTGGCCCAGG SP TACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCA LCW0404_025_ GSSTPSGATGSPGSSTPS GGTAGCTCTACTCCGTCTGGTGCTACCGGCTCTCCAG GFP-\_G08.ab1 GATGSPGASPGTSSTGS GTAGCTCTACCCCTTCTGGTGCAACCGGCTCCCCAGG P TGCTTCTCCGGGTACCAGCTCTACTGGTTCTCCA LCW0404_029_ GTPGSGTASSSPGSSTPS GGTACCCCTGGCAGCGGTACCGCTTCTTCCTCTCCAG GFP-\_A09.ab1 GATGSPGSSPSASTGTG GTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAGG P TTCTAGCCCGTCTGCATCTACCGGTACCGGCCCA LCW0404_030_ GSSTPSGATGSPGTPGS GGTAGCTCTACTCCTTCTGGTGCAACCGGCTCCCCAG GFP-\_B09.ab1 GTASSSPGTPGSGTASS GTACCCCGGGCAGCGGTACCGCATCTTCCTCTCCAG SP GTACTCCGGGTAGCGGTACTGCTTCTTCTTCTCCA LCW0404_03 1_ GTPGSGTASSSPGSSTPS GGTACCCCGGGTAGCGGTACTGCTTCTTCCTCTCCAG GFP-\_C09.ab1 GATGSPGASPGTSSTGS GTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAGG File name Amino acid sequence Nucleotide sequence P TGCTTCTCCGGGCACCAGCTCTACCGGTTCTCCA LCW0404_034_ GSSTPSGATGSPGSSTPS GGTAGCTCTACCCCGTCTGGTGCTACCGGCTCTCCAG GFP-\_D09.ab1 GATGSPGASPGTSSTGS GTAGCTCTACCCCGTCTGGTGCAACCGGCTCCCCAG P GTGCATCCCCGGGTACTAGCTCTACCGGTTCTCCA LCW0404_035_ GASPGTSSTGSPGTPGS GGTGCTTCTCCGGGCACCAGCTCTACTGGTTCTCCAG GFP-\_E09.ab1 GTASSSPGSSTPSGATG GTACCCCGGGCAGCGGTACCGCATCTTCTTCTCCAG SP GTAGCTCTACTCCTTCTGGTGCAACTGGTTCTCCA LCW0404_036_ GSSPSASTGTGPGSSTPS AGCCCGTCTGCTTCCACCGGTACTGGCCCAG GFP-\_F09.ab1 GATGSPGTPGSGTASSS GTAGCTCTACCCCGTCTGGTGCAACTGGTTCCCCAGG P TACCCCTGGTAGCGGTACCGCTTCTTCTTCTCCA LCW0404_037_ SSTGSPGSSPS GGTGCTTCTCCGGGCACCAGCTCTACTGGTTCTCCAG GFP-\_G09.ab1 PGSSTPSGATG GTTCTAGCCCTTCTGCATCCACCGGTACCGGTCCAGG SP TAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCA LCW0404_040_ GASPGTSSTGSPGSSTPS TCCCCGGGCACCAGCTCTACCGGTTCTCCA GFP-\_H09.ab1 GATGSPGSSTPSGATGS GGTAGCTCTACCCCGTCTGGTGCTACCGGCTCTCCAG P GTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCA LCW0404_041_ GTPGSGTASSSPGSSTPS GGTACCCCTGGTAGCGGTACTGCTTCTTCCTCTCCAG A10.ab1 GATGSPGTPGSGTASSS GTAGCTCTACTCCGTCTGGTGCTACCGGTTCTCCAGG P TACCCCGGGTAGCGGTACCGCATCTTCTTCTCCA LCW0404_043_ GSSPSASTGTGPGSSTPS GGTTCTAGCCCTTCTGCTTCCACCGGTACTGGCCCAG GFP-\_C10.ab1 GATGSPGSSTPSGATGS GTAGCTCTACCCCTTCTGGTGCTACCGGCTCCCCAGG P TACTCCTTCTGGTGCAACTGGCTCTCCA LCW0404_045_ GASPGTSSTGSPGSSPS GGTGCTTCTCCTGGCACCAGCTCTACTGGTTCTCCAG GFP-\_D10.ab1 ASTGTGPGSSPSASTGT GTTCTAGCCCTTCTGCTTCTACCGGTACTGGTCCAGG GP TTCTAGCCCTTCTGCATCCACTGGTACTGGTCCA 4_047_ GTPGSGTASSSPGASPG GGTACTCCTGGCAGCGGTACCGCTTCTTCTTCTCCAG GFP-\_F10.ab1 TSSTGSPGASPGTSSTGS GTGCTTCTCCTGGTACTAGCTCTACTGGTTCTCCAGG P TGCTTCTCCGGGCACTAGCTCTACTGGTTCTCCA LCW0404_048_ GSSTPSGATGSPGASPG GGTAGCTCTACCCCGTCTGGTGCTACCGGTTCCCCAG GFP-\_G10.ab1 TSSTGSPGSSTPSGATGS GTGCTTCTCCTGGTACTAGCTCTACCGGTTCTCCAGG P TAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCA LCW0404_049_ GSSTPSGATGSPGTPGS GGTAGCTCTACCCCGTCTGGTGCTACTGGTTCTCCAG GFP-\_H10.ab1 GTASSSPGSSTPSGATG GTACTCCGGGCAGCGGTACTGCTTCTTCCTCTCCAGG SP TAGCTCTACCCCTTCTGGTGCTACTGGCTCTCCA LCW0404_050_ GASPGTSSTGSPGSSPS GGTGCATCTCCTGGTACCAGCTCTACTGGTTCTCCAG GFP-\_A11.ab1 ASTGTGPGSSTPSGATG GCCCTTCTGCTTCTACCGGTACCGGTCCAGG SP TAGCTCTACTCCTTCTGGTGCTACCGGTTCTCCA LCW0404_051_ GSSTPSGATGSPGSSTPS GGTAGCTCTACCCCGTCTGGTGCTACTGGCTCTCCAG GFP-\_B11.ab1 GATGSPGSSTPSGATGS GTAGCTCTACTCCTTCTGGTGCTACTGGTTCCCCAGG P TAGCTCTACCCCGTCTGGTGCAACTGGCTCTCCA LCW0404_052_ GASPGTSSTGSPGTPGS GGTGCATCCCCGGGTACCAGCTCTACCGGTTCTCCA GFP-\_C11.ab1 GTASSSPGASPGTSSTG GGTACTCCTGGCAGCGGTACTGCATCTTCCTCTCCAG SP CTCCGGGCACCAGCTCTACTGGTTCTCCA LCW0404_053_ GSSTPSGATGSPGSSPS GGTAGCTCTACTCCTTCTGGTGCAACTGGTTCTCCAG GFP-\_D11.ab1 ASTGTGPGASPGTSSTG GTTCTAGCCCGTCTGCATCCACTGGTACCGGTCCAGG SP TGCTTCCCCTGGCACCAGCTCTACCGGTTCTCCA LCW0404_057_ GASPGTSSTGSPGSSTPS GGTGCATCTCCTGGTACTAGCTCTACTGGTTCTCCAG GFP-\_E11.ab1 GATGSPGSSPSASTGTG GTAGCTCTACTCCGTCTGGTGCAACCGGCTCTCCAGG P CCCTTCTGCATCTACCGGTACTGGTCCA LCW0404_060_ GTPGSGTASSSPGSSTPS GGTACTCCTGGCAGCGGTACCGCATCTTCCTCTCCAG GFP-\_F11.ab1 GATGSPGASPGTSSTGS CTACTCCGTCTGGTGCAACTGGTTCCCCAGG P TGCTTCTCCGGGTACCAGCTCTACCGGTTCTCCA LCW0404_062_ GSSTPSGATGSPGTPGS GGTAGCTCTACCCCGTCTGGTGCAACCGGCTCCCCA G11.ab1 GTASSSPGSSTPSGATG GGTACTCCTGGTAGCGGTACCGCTTCTTCTTCTCCAG SP CTACTCCGTCTGGTGCTACCGGCTCCCCA LCW0404_066_ GSSPSASTGTGPGSSPS GGTTCTAGCCCTTCTGCATCCACCGGTACCGGCCCAG GFP-\_H11.ab1 ASTGTGPGASPGTSSTG GTTCTAGCCCGTCTGCTTCTACCGGTACTGGTCCAGG SP TGCTTCTCCGGGTACTAGCTCTACTGGTTCTCCA LCW0404_067_ GTPGSGTASSSPGSSTPS GGTACCCCGGGTAGCGGTACCGCTTCTTCTTCTCCAG GFP-\_A12.ab1 GATGSPGSNPSASTGTG GTAGCTCTACTCCGTCTGGTGCTACCGGCTCTCCAGG P TTCTAACCCTTCTGCATCCACCGGTACCGGCCCA File name Amino acid sequence Nucleotide sequence LCWO404_068_ GSSPSASTGTGPGSSTPS GGTTCTAGCCCTTCTGCATCTACTGGTACTGGCCCAG GFP-\_B12.ab1 GATGSPGASPGTSSTGS GTAGCTCTACTCCTTCTGGTGCTACCGGCTCTCCAGG P TGCTTCTCCGGGTACTAGCTCTACCGGTTCTCCA LCWO404_069_ GSSTPSGATGSPGASPG GGTAGCTCTACCCCTTCTGGTGCAACCGGCTCTCCAG C12.ab1 TSSTGSPGTPGSGTASSS GTGCATCCCCGGGTACCAGCTCTACCGGTTCTCCAG P GTACTCCGGGTAGCGGTACCGCTTCTTCCTCTCCA LCWO404_070_ GATGSPGSSTPS GGTAGCTCTACTCCGTCTGGTGCAACCGGTTCCCCAG GFP-\_D12.ab1 GATGSPGSSTPSGATGS GTAGCTCTACCCCTTCTGGTGCAACCGGCTCCCCAGG P TAGCTCTACCCCTTCTGGTGCAACTGGCTCTCCA LCWO404_073_ GASPGTSSTGSPGTPGS GGTGCTTCTCCTGGCACTAGCTCTACCGGTTCTCCAG GFP-\_E12.ab1 GTASSSPGSSTPSGATG GTACCCCTGGTAGCGGTACCGCATCTTCCTCTCCAGG SP TAGCTCTACTCCTTCTGGTGCTACTGGTTCCCCA LCWO404_075_ GSSTPSGATGSPGSSPS GGTAGCTCTACCCCGTCTGGTGCTACTGGCTCCCCAG GFP-\_F12.ab1 ASTGTGPGSSPSASTGT GTTCTAGCCCTTCTGCATCCACCGGTACCGGTCCAGG GP TTCTAGCCCGTCTGCATCTACTGGTACTGGTCCA LCWO404_080_ GASPGTSSTGSPGSSPS GGTGCTTCCCCGGGCACCAGCTCTACTGGTTCTCCAG GFP-\_G12.ab1 ASTGTGPGSSPSASTGT GTTCTAGCCCGTCTGCTTCTACTGGTACTGGTCCAGG GP TTCTAGCCCTTCTGCTTCCACTGGTACTGGTCCA LCWO404_081_ GASPGTSSTGSPGSSPS GGTGCTTCCCCGGGTACCAGCTCTACCGGTTCTCCAG GFP-\_H12.ab1 ASTGTGPGTPGSGTASS GTTCTAGCCCTTCTGCTTCTACCGGTACCGGTCCAGG SP TACCCCTGGCAGCGGTACCGCATCTTCCTCTCCA ] Example 5: Construction ofXTEN_AE864 XTEN_AE864 was constructed from serial dimerization ofXTEN_AE36 to AE72, 144, 288, 576 and 864. A collection ofXTEN_AE72 segments was constructed from 37 different segments of E36. Cultures of E. coli harboring all 37 different 36-amino acid segments were mixed and plasmid was isolated. This plasmid pool was digested with Bsal/Ncol to generate the small fragment as the . The same plasmid pool was digested with Bbsl/Ncol to generate the large fragment as the vector. The insert and vector fragments were ligated resulting in a doubling of the length and the ligation mixture was transformed into BL21Gold(DE3) cells to obtain colonies of E72.

This library of E72 segments was designated LCWO406. All clones from LCWO406 were combined and dimerized again using the same process as described above yielding library LCWO410 ofXTEN_AE144. All clones from LCWO410 were combined and dimerized again using the same process as described above yielding library LCWO414 of E288. Two isolates LCWO414.001 and LCWO414.002 were ly picked from the y and sequenced to verify the identities. All clones from LCWO414 were combined and dimerized again using the same s as bed above yielding y LCWO418 ofXTEN_AES76. We screened 96 isolates from library LCWO418 for high level of GFP fluorescence. 8 es with right sizes of s by PCR and strong fluorescence were sequenced and 2 isolates (LCWO418.018 and LCWO418.052) were chosen for future use based on sequencing and expression data.

The specific clone pCWO432 ofXTEN_AE864 was constructed by combining LCWO418.018 of XTEN_AE576 and LCWO414.002 ofXTEN_AE288 using the same dimerization process as described above.

Example 6: Construction ofXTEN_AM144 A tion ofXTEN_AM144 segments was constructed starting from 37 ent segments of XTEN_AE36, 44 ts ofXTEN_AF36, and 44 segments ofXTEN_AG36.

Cultures of E. coli that harboring all 125 different 36-amino acid ts were mixed and plasmid was isolated. This plasmid pool was digested with BsaI/NcoI to generate the small fragment as the . The same plasmid pool was digested with BbsI/NcoI to generate the large fragment as the vector. The insert and vector fragments were ligated resulting in a doubling of the length and the ligation mixture was ormed into BL21Gold(DE3) cells to obtain colonies of XTEN_AM72.

This library of XTEN_AM72 segments was designated 1. All clones from LCWO461 were combined and zed again using the same process as described above yielding library LCWO462. 1512 Isolates from library LCWO462 were screened for n expression. Individual colonies were transferred into 96 well plates and cultured overnight as starter cultures. These starter cultures were diluted into fresh autoinduction medium and cultured for 20-3 Oh. Expression was measured using a fluorescence plate reader with excitation at 395 nm and emission at 510 nm. 192 isolates showed high level expression and were submitted for DNA sequencing. Most clones in library LCWO462 showed good expression and similar ochemical properties suggesting that most combinations of XTEN_AM36 segments yield useful XTEN sequences. Thirty isolates from LCWO462 were chosen as a preferred collection of XTEN_AM144 segments for the construction of multifunctional proteins that contain multiple XTEN segments. The file names of the nucleotide and amino acid constructs and the sequences for these ts are listed in Table 17.

Table 17: DNA and amino acid seguences for AM144 segments [SEQ ID NOS 529-594, res ectivel in order of a earance Clone Sequence Trimmed Protein Sequence LCW462_r1 GGTACCCCGGGCAGCGGTACCGCATCTTCCTCTCCAGGTAGC GTPGSGTASSSPGS TCTACCCCGTCTGGTGCTACCGGTTCCCCAGGTAGCTCTACCC STPSGATGSPGSSTP CGTCTGGTGCAACCGGCTCCCCAGGTAGCCCGGCTGGCTCTC SGATGSPGSPAGSP CTACCTCTACTGAGGAAGGTACTTCTGAAAGCGCTACTCCTG TSTEEGTSESATPES AGTCTGGTCCAGGTACCTCTACTGAACCGTCCGAAGGTAGCG GPGTSTEPSEGSAP CTCCAGGTTCTAGCCCTTCTGCATCCACCGGTACCGGCCCAGG GSSPSASTGTGPGS TTCTAGCCCGTCTGCTTCTACCGGTACTGGTCCAGGTGCTTCT GTGPGASP CCGGGTACTAGCTCTACTGGTTCTCCAGGTACCTCTACCGAAC SPGTSTEPS CGTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCGTCTG EGSAPGTSTEPSEG AGGGTAGCGCTCCAGGTAGCGAACCGGCAACCTCCGGTTCTG SAPGSEPATSGSETP LCW462_r5 GGTTCTACCAGCGAATCCCCTTCTGGCACTGCACCAGGTTCTA GSTSESPSGTAPGST CTAGCGAATCCCCTTCTGGTACCGCACCAGGTACTTCTCCGAG SESPSGTAPGTSPSG CGGCGAATCTTCTACTGCTCCAGGTACCTCTACTGAACCTTCC ESSTAPGTSTEPSEG GAAGGCAGCGCTCCAGGTACCTCTACCGAACCGTCCGAGGGC SAPGTSTEPSEGSAP AGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGT GTSESATPESGPGA CCAGGTGCATCTCCTGGTACCAGCTCTACCGGTTCTCCAGGTA SPGTSSTGSPGSSTP GCTCTACTCCTTCTGGTGCTACTGGCTCTCCAGGTGCTTCCCC SGATGSPGASPGTS GGGTACCAGCTCTACCGGTTCTCCAGGTTCTACTAGCGAATCT STGSPGSTSESPSGT GGCACTGCACCAGGTTCTACCAGCGAATCTCCGTCTG ESPSGTAP GCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTTCCGCTT GTSTPESGSASP CTCCA LCW462_r9 TCTACCGAACCTTCCGAGGGCAGCGCACCAGGTACT GTSTEPSEGSAPGT TCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACTTCTGAA SESATPESGPGTSES AGCGCTACTCCTGAATCCGGTCCAGGTACCTCTACTGAACCTT ATPESGPGTSTEPSE Clone ce Trimmed Protein Sequence CTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACCCCGG GSAPGTSESATPES AGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCG GPGTSTEPSEGSAP CACCAGGTACTTCTACTGAACCTTCCGAAGGTAGCGCTCCAG SEGSAPGS GTAGCGAACCTGCTACTTCTGGTTCTGAAACCCCAGGTAGCC EPATSGSETPGSPA CGGCTGGCTCTCCGACCTCCACCGAGGAAGGTGCTTCTCCTG GSPTSTEEGASPGT GCACCAGCTCTACTGGTTCTCCAGGTTCTAGCCCTTCTGCTTC SSTGSPGSSPSASTG TACCGGTACTGGTCCAGGTTCTAGCCCTTCTGCATCCACTGGT TGPGSSPSASTGTG ACTGGTCCA P LCW462_r10 GGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCAGGTACC GSEPATSGSETPGT TCTGAAAGCGCTACTCCGGAATCTGGTCCAGGTACTTCTGAA SESATPESGPGTSES AGCGCTACTCCGGAATCCGGTCCAGGTTCTACCAGCGAATCT ATPESGPGSTSESPS CCTTCTGGCACCGCTCCAGGTTCTACTAGCGAATCCCCGTCTG GTAPGSTSESPSGT GTACCGCACCAGGTACTTCTCCTAGCGGCGAATCTTCTACCGC APGTSPSGESSTAP ACCAGGTGCATCTCCGGGTACTAGCTCTACCGGTTCTCCAGGT GASPGTSSTGSPGS TCTAGCCCTTCTGCTTCCACTGGTACCGGCCCAGGTAGCTCTA SPSASTGTGPGSSTP CCCCGTCTGGTGCTACTGGTTCCCCAGGTAGCTCTACTCCGTC SGATGSPGSSTPSG TGGTGCAACCGGTTCCCCAGGTAGCTCTACTCCTTCTGGTGCT ATGSPGSSTPSGAT TCCCCAGGTGCATCCCCTGGCACCAGCTCTACCGGTT GSPGASPGTSSTGS CTCCA P LCW462_r15 GGTGCTTCTCCGGGCACCAGCTCTACTGGTTCTCCAGGTTCTA GASPGTSSTGSPGS GCCCTTCTGCATCCACCGGTACCGGTCCAGGTAGCTCTACCCC SPSASTGTGPGSSTP TTCTGGTGCAACCGGCTCTCCAGGTACTTCTGAAAGCGCTACC SGATGSPGTSESAT CCGGAATCTGGCCCAGGTAGCGAACCGGCTACTTCTGGTTCT PESGPGSEPATSGSE GAAACCCCAGGTAGCGAACCGGCTACCTCCGGTTCTGAAACT TPGSEPATSGSETP CCAGGTACTTCTGAAAGCGCTACTCCGGAGTCCGGTCCAGGT TPESGPGT ACCTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACTTCT STEPSEGSAPGTSTE ACTGAACCTTCTGAGGGTAGCGCTCCAGGTACCTCTACCGAA PSEGSAPGTSTEPSE CCGTCCGAGGGTAGCGCACCAGGTACCTCTACTGAACCGTCT GSAPGTSTEPSEGS AGCGCTCCAGGTAGCGAACCGGCAACCTCCGGTTCT APGSEPATSGSETP GAAACTCCA LCW462_r16 TCTACCGAACCTTCCGAAGGTAGCGCTCCAGGTAGC GTSTEPSEGSAPGSP CCGGCAGGTTCTCCTACTTCCACTGAGGAAGGTACTTCTACCG AGSPTSTEEGTSTEP AACCTTCTGAGGGTAGCGCACCAGGTACCTCTGAAAGCGCAA SEGSAPGTSESATP CTCCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCCGGCT ESGPGSEPATSGSE CTGAGACTCCAGGTACCTCTGAAAGCGCAACCCCGGAATCTG TPGTSESATPESGP GTCCAGGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAG GSPAGSPTSTEEGT GTACTTCTGAAAGCGCTACTCCTGAGTCTGGTCCAGGTACCTC SESATPESGPGTSTE TACTGAACCGTCCGAAGGTAGCGCTCCAGGTAGCGAACCTGC PSEGSAPGSEPATS TACTTCTGGTTCTGAAACTCCAGGTACTTCTACCGAACCGTCC GSETPGTSTEPSEGS AGCGCTCCAGGTAGCGAACCTGCTACTTCTGGTTCT ATSGSETP GAAACTCCA LCW462_r20 GGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACC GTSTEPSEGSAPGT GAACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACC STEPSEGSAPGTSTE GAACCTTCTGAAGGTAGCGCACCAGGTACTTCTACCGAACCG PSEGSAPGTSTEPSE TCCGAAGGCAGCGCTCCAGGTACCTCTACTGAACCTTCCGAG GSAPGTSTEPSEGS GGCAGCGCTCCAGGTACCTCTACCGAACCTTCTGAAGGTAGC APGTSTEPSEGSAP GCACCAGGTACTTCTACCGAACCTTCCGAGGGCAGCGCACCA GTSTEPSEGSAPGT GGTACTTCTGAAAGCGCTACCCCTGAGTCCGGCCCAGGTACT SESATPESGPGTSES TCTGAAAGCGCTACTCCTGAATCCGGTCCAGGTACTTCTACTG ATPESGPGTSTEPSE AACCTTCCGAAGGTAGCGCTCCAGGTAGCGAACCTGCTACTT GSAPGSEPATSGSE CTGGTTCTGAAACCCCAGGTAGCCCGGCTGGCTCTCCGACCT TPGSPAGSPTSTEE CCACCGAGGAA LCW462_r23 GGTACTTCTACCGAACCGTCCGAGGGCAGCGCTCCAGGTACT GTSTEPSEGSAPGT GAACCTTCTGAAGGCAGCGCTCCAGGTACTTCTACTG STEPSEGSAPGTSTE AACCTTCCGAAGGTAGCGCACCAGGTTCTACCAGCGAATCCC PSEGSAPGSTSESPS CTTCTGGTACTGCTCCAGGTTCTACCAGCGAATCCCCTTCTGG GTAPGSTSESPSGT CACCGCACCAGGTACTTCTACCCCTGAAAGCGGCTCCGCTTCT APGTSTPESGSASP CCAGGTAGCGAACCTGCAACCTCTGGCTCTGAAACCCCAGGT GSEPATSGSETPGT ACCTCTGAAAGCGCTACTCCTGAATCTGGCCCAGGTACTTCTA SESATPESGPGTSTE CTGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTACTGAAC PSEGSAPGTSTEPSE AAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCC GSAPGTSESATPES Clone Sequence Trimmed Protein Sequence CGGAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGT GPGTSESATPESGP CCGGCCCA LCW462_r24 GGTAGCTCTACCCCTTCTGGTGCTACCGGCTCTCCAGGTTCTA GSSTPSGATGSPGS GCCCGTCTGCTTCTACCGGTACCGGTCCAGGTAGCTCTACCCC SPSASTGTGPGSSTP TTCTGGTGCTACTGGTTCTCCAGGTAGCCCTGCTGGCTCTCCG SGATGSPGSPAGSP ACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTCCGACTTCTA TSTEEGSPAGSPTST CTGAGGAAGGTACTTCTACCGAACCTTCCGAAGGTAGCGCTC EEGTSTEPSEGSAP CAGGTGCTTCCCCGGGCACTAGCTCTACCGGTTCTCCAGGTTC GASPGTSSTGSPGS TAGCCCTTCTGCATCTACTGGTACTGGCCCAGGTACTCCGGGC SPSASTGTGPGTPG AGCGGTACTGCTTCTTCCTCTCCAGGTTCTACTAGCTCTACTG SGTASSSPGSTSSTA CTGAATCTCCTGGCCCAGGTACTTCTCCTAGCGGTGAATCTTC ESPGPGTSPSGESST TACCGCTCCAGGTACCTCTACTCCGGAAAGCGGTTCTGCATCT APGTSTPESGSASP LCW462_r27 GGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGGTACT GTSTEPSEGSAPGT TCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCTACT SESATPESGPGTSTE TCCGAAGGTAGCGCACCAGGTACTTCTACTGAACCG PSEGSAPGTSTEPSE TCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCG SESATPES GAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAGTCC GPGTSESATPESGP GGCCCAGGTACTCCTGGCAGCGGTACCGCTTCTTCTTCTCCAG GTPGSGTASSSPGA GTGCTTCTCCTGGTACTAGCTCTACTGGTTCTCCAGGTGCTTC SPGTSSTGSPGASP TCCGGGCACTAGCTCTACTGGTTCTCCAGGTAGCCCTGCTGGC GTSSTGSPGSPAGS TCTCCGACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTCCG PTSTEEGSPAGSPTS ACTTCTACTGAGGAAGGTACTTCTACCGAACCTTCCGAAGGT TEEGTSTEPSEGSAP AGCGCTCCA LCW462_r28 GGTAGCCCAGCAGGCTCTCCGACTTCCACTGAGGAAGGTACT GSPAGSPTSTEEGT TCTACTGAACCTTCCGAAGGCAGCGCACCAGGTACCTCTACT STEPSEGSAPGTSTE TCTGAGGGCAGCGCTCCAGGTACCTCTACCGAACCG PGTSTEPSE TCTGAAGGTAGCGCACCAGGTACCTCTGAAAGCGCAACTCCT GSAPGTSESATPES GAGTCCGGTCCAGGTACTTCTGAAAGCGCAACCCCGGAGTCT GPGTSESATPESGP GGCCCAGGTACCCCGGGTAGCGGTACTGCTTCTTCCTCTCCAG GTPGSGTASSSPGS CTACCCCTTCTGGTGCAACCGGCTCTCCAGGTGCTTC STPSGATGSPGASP TCCGGGCACCAGCTCTACCGGTTCTCCAGGTACCTCTACTGAA GTSSTGSPGTSTEPS CCTTCTGAGGGCAGCGCTCCAGGTACTTCTGAAAGCGCTACC EGSAPGTSESATPE CCGGAGTCCGGTCCAGGTACTTCTACTGAACCGTCCGAAGGT SGPGTSTEPSEGSAP AGCGCACCA LCW462_r3 8 GGTAGCGAACCGGCAACCTCCGGCTCTGAAACTCCAGGTACT GSEPATSGSETPGT TCTGAAAGCGCTACTCCGGAATCCGGCCCAGGTAGCGAACCG SESATPESGPGSEPA GCTACTTCCGGCTCTGAAACCCCAGGTAGCTCTACCCCGTCTG TSGSETPGSSTPSGA GTGCAACCGGCTCCCCAGGTACTCCTGGTAGCGGTACCGCTT TGSPGTPGSGTASS CTTCTTCTCCAGGTAGCTCTACTCCGTCTGGTGCTACCGGCTC SPGSSTPSGATGSP CCCAGGTGCATCTCCTGGTACCAGCTCTACCGGTTCTCCAGGT GASPGTSSTGSPGS AGCTCTACTCCTTCTGGTGCTACTGGCTCTCCAGGTGCTTCCC STPSGATGSPGASP CGGGTACCAGCTCTACCGGTTCTCCAGGTAGCGAACCTGCTA GTSSTGSPGSEPATS CTTCTGGTTCTGAAACTCCAGGTACTTCTACCGAACCGTCCGA GSETPGTSTEPSEGS GGGTAGCGCTCCAGGTAGCGAACCTGCTACTTCTGGTTCTGA APGSEPATSGSETP AACTCCA LCW462_r39 GGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTACC GTSTEPSEGSAPGT TCTACCGAACCGTCCGAGGGCAGCGCACCAGGTACTTCTGAA STEPSEGSAPGTSES AGCGCAACCCCTGAATCCGGTCCAGGTAGCCCTGCTGGCTCT ATPESGPGSPAGSP CCGACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTCCGACT TSTEEGSPAGSPTST TCTACTGAGGAAGGTACTTCTACCGAACCTTCCGAAGGTAGC EEGTSTEPSEGSAP GGTAGCCCGGCTGGTTCTCCGACTTCCACCGAGGAA GSPAGSPTSTEEGT TCTACTGAACCTTCTGAGGGTAGCGCTCCAGGTACC STEPSEGSAPGTSTE TCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTGCTTCCCCG PSEGSAPGASPGTS GGCACCAGCTCTACTGGTTCTCCAGGTTCTAGCCCGTCTGCTT SSPSASTGT CTACTGGTACTGGTCCAGGTTCTAGCCCTTCTGCTTCCACTGG GPGSSPSASTGTGP TACTGGTCCA LCW462_r41 TCTACCCCGTCTGGTGCTACCGGTTCCCCAGGTGCTT GATGSPGA GTACTAGCTCTACCGGTTCTCCAGGTAGCTCTACCCC SPGTSSTGSPGSSTP GTCTGGTGCTACTGGCTCTCCAGGTAGCCCTGCTGGCTCTCCA SGATGSPGSPAGSP ACCTCCACCGAAGAAGGTACCTCTGAAAGCGCAACCCCTGAA TSTEEGTSESATPES Clone Sequence Trimmed Protein Sequence TCCGGCCCAGGTAGCGAACCGGCAACCTCCGGTTCTGAAACC ATSGSETP CCAGGTGCATCTCCTGGTACTAGCTCTACTGGTTCTCCAGGTA GASPGTSSTGSPGS GCTCTACTCCGTCTGGTGCAACCGGCTCTCCAGGTTCTAGCCC STPSGATGSPGSSPS TTCTGCATCTACCGGTACTGGTCCAGGTTCTACCAGCGAATCC PGSTSESPS CCTTCTGGTACTGCTCCAGGTTCTACCAGCGAATCCCCTTCTG GTAPGSTSESPSGT GCACCGCACCAGGTACTTCTACCCCTGAAAGCGGCTCCGCTT APGTSTPESGSASP CTCCA LCW462_r42 GGTTCTACCAGCGAATCTCCTTCTGGCACCGCTCCAGGTTCTA GSTSESPSGTAPGST CTAGCGAATCCCCGTCTGGTACCGCACCAGGTACTTCTCCTAG SESPSGTAPGTSPSG CGGCGAATCTTCTACCGCACCAGGTACCTCTGAAAGCGCTAC ESSTAPGTSESATPE TCCGGAGTCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGG TEPSEGSAP TAGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGC GTSTEPSEGSAPGT ACCAGGTACCTCTACTGAACCTTCTGAGGGCAGCGCTCCAGG STEPSEGSAPGTSES TACTTCTGAAAGCGCTACCCCGGAGTCCGGTCCAGGTACTTCT PGTSTEPSE ACTGAACCGTCCGAAGGTAGCGCACCAGGTAGCTCTACCCCG GSAPGSSTPSGATG TCTGGTGCTACCGGTTCCCCAGGTGCTTCTCCTGGTACTAGCT SPGASPGTSSTGSP CTACCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGG GSSTPSGATGSP CTCTCCA LCW462_r43 GGTTCTACTAGCTCTACTGCAGAATCTCCGGGCCCAGGTACCT GSTSSTAESPGPGTS CTCCTAGCGGTGAATCTTCTACCGCTCCAGGTACTTCTCCGAG PSGESSTAPGTSPSG CGGTGAATCTTCTACCGCTCCAGGTTCTACTAGCTCTACCGCT ESSTAPGSTSSTAES GAATCTCCGGGTCCAGGTTCTACCAGCTCTACTGCAGAATCTC PGPGSTSSTAESPGP CTGGCCCAGGTACTTCTACTCCGGAAAGCGGTTCCGCTTCTCC GTSTPESGSASPGTS AGGTACTTCTCCTAGCGGTGAATCTTCTACCGCTCCAGGTTCT PSGESSTAPGSTSST ACCAGCTCTACTGCTGAATCTCCTGGCCCAGGTACTTCTACCC AESPGPGTSTPESGS CGGAAAGCGGCTCCGCTTCTCCAGGTTCTACCAGCTCTACCG ASPGSTSSTAESPGP CTGAATCTCCTGGCCCAGGTTCTACTAGCGAATCTCCGTCTGG GSTSESPSGTAPGTS CACCGCACCAGGTACTTCCCCTAGCGGTGAATCTTCTACTGCA PSGESSTAP LCW462_r45 TCTACTCCGGAAAGCGGTTCCGCATCTCCAGGTTCTA GTSTPESGSASPGST AATCCCCGTCTGGCACCGCACCAGGTTCTACTAGCT SESPSGTAPGSTSST CTACTGCTGAATCTCCGGGCCCAGGTACCTCTACTGAACCTTC AESPGPGTSTEPSE CGAAGGCAGCGCTCCAGGTACCTCTACCGAACCGTCCGAGGG GSAPGTSTEPSEGS CAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGG APGTSESATPESGP TCCAGGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGG GTSESATPESGPGT TACCTCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCT STEPSEGSAPGTSTE ACTGAACCGTCCGAAGGTAGCGCACCAGGTACTTCTGAAAGC PSEGSAPGTSESATP CCGGAGTCCGGTCCAGGTACCTCTACCGAACCGTCC ESGPGTSTEPSEGS GAAGGCAGCGCTCCAGGTACTTCTACTGAACCTTCTGAGGGT APGTSTEPSEGSAP AGCGCTCCC LCW462_r47 GGTACCTCTACCGAACCGTCCGAGGGTAGCGCACCAGGTACC GTSTEPSEGSAPGT TCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTAGCGAACCG STEPSEGSAPGSEPA TCCGGTTCTGAAACTCCAGGTACTTCTACTGAACCGT TSGSETPGTSTEPSE CTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACCCCGG GSAPGTSESATPES GCCCAGGTACCTCTGAAAGCGCAACCCCGGAGTCCG GPGTSESATPESGP GCCCAGGTGCATCTCCGGGTACTAGCTCTACCGGTTCTCCAG GASPGTSSTGSPGS GTTCTAGCCCTTCTGCTTCCACTGGTACCGGCCCAGGTAGCTC SPSASTGTGPGSSTP TACCCCGTCTGGTGCTACTGGTTCCCCAGGTAGCTCTACTCCG SGATGSPGSSTPSG TCTGGTGCAACCGGTTCCCCAGGTAGCTCTACTCCTTCTGGTG ATGSPGSSTPSGAT CTACTGGCTCCCCAGGTGCATCCCCTGGCACCAGCTCTACCG GSPGASPGTSSTGS CA P LCW462_r54 GGTAGCGAACCGGCAACCTCTGGCTCTGAAACTCCAGGTAGC GSEPATSGSETPGS GAACCTGCAACCTCCGGCTCTGAAACCCCAGGTACTTCTACT EPATSGSETPGTSTE GAACCTTCTGAGGGCAGCGCACCAGGTAGCGAACCTGCAACC PGSEPATS TCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCT GSETPGTSESATPES GAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGC GPGTSTEPSEGSAP GCACCAGGTAGCTCTACTCCGTCTGGTGCTACCGGCTCTCCAG GSSTPSGATGSPGS GTAGCTCTACCCCTTCTGGTGCAACCGGCTCCCCAGGTGCTTC STPSGATGSPGASP TCCGGGTACCAGCTCTACTGGTTCTCCAGGTAGCTCTACCCCG GTSSTGSPGSSTPSG TCTGGTGCTACCGGTTCCCCAGGTGCTTCTCCTGGTACTAGCT ATGSPGASPGTSST CTACCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGG GSPGSSTPSGATGS Clone Sequence d Protein Sequence CTCTCCA P LCW462_r55 GGTACTTCTACCGAACCGTCCGAGGGCAGCGCTCCAGGTACT GTSTEPSEGSAPGT TCTACTGAACCTTCTGAAGGCAGCGCTCCAGGTACTTCTACTG STEPSEGSAPGTSTE AACCTTCCGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCTA PSEGSAPGTSESATP CTCCGGAGTCCGGTCCAGGTACCTCTACCGAACCGTCCGAAG ESGPGTSTEPSEGS CTCCAGGTACTTCTACTGAACCTTCTGAGGGTAGCG APGTSTEPSEGSAP CTCCAGGTTCTACTAGCGAATCTCCGTCTGGCACTGCTCCAGG GSTSESPSGTAPGTS TACTTCTCCTAGCGGTGAATCTTCTACCGCTCCAGGTACTTCC PSGESSTAPGTSPSG CCTAGCGGCGAATCTTCTACCGCTCCAGGTAGCCCGGCTGGC ESSTAPGSPAGSPTS TCTCCTACCTCTACTGAGGAAGGTACTTCTGAAAGCGCTACTC TEEGTSESATPESGP CTGAGTCTGGTCCAGGTACCTCTACTGAACCGTCCGAAGGTA GTSTEPSEGSAP GCGCTCCA LCW462_r57 GGTACTTCTACTGAACCTTCCGAAGGTAGCGCTCCAGGTAGC GTSTEPSEGSAPGS GAACCTGCTACTTCTGGTTCTGAAACCCCAGGTAGCCCGGCT EPATSGSETPGSPA GGCTCTCCGACCTCCACCGAGGAAGGTAGCCCGGCAGGCTCT GSPTSTEEGSPAGSP CCGACCTCTACTGAGGAAGGTACTTCTGAAAGCGCAACCCCG TSTEEGTSESATPES GAGTCCGGCCCAGGTACCTCTACCGAACCGTCTGAGGGCAGC GPGTSTEPSEGSAP GCACCAGGTACCTCTACTGAACCTTCCGAAGGCAGCGCTCCA GTSTEPSEGSAPGT GGTACCTCTACCGAACCGTCCGAGGGCAGCGCACCAGGTACT STEPSEGSAPGTSES TCTGAAAGCGCAACCCCTGAATCCGGTCCAGGTAGCTCTACT ATPESGPGSSTPSG CCGTCTGGTGCAACCGGCTCCCCAGGTTCTAGCCCGTCTGCTT SSPSASTG CCACTGGTACTGGCCCAGGTGCTTCCCCGGGCACCAGCTCTA TGPGASPGTSSTGS CTGGTTCTCCA P _r61 GGTAGCGAACCGGCTACTTCCGGCTCTGAGACTCCAGGTAGC GSEPATSGSETPGSP CCTGCTGGCTCTCCGACCTCTACCGAAGAAGGTACCTCTGAA AGSPTSTEEGTSES AGCGCTACCCCTGAGTCTGGCCCAGGTACCTCTACTGAACCTT ATPESGPGTSTEPSE CCGAAGGCAGCGCTCCAGGTACCTCTACCGAACCGTCCGAGG STEPSEGS GCAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCG APGTSESATPESGP GTCCAGGTACCTCTACTCCGGAAAGCGGTTCCGCATCTCCAG GTSTPESGSASPGST GTTCTACCAGCGAATCCCCGTCTGGCACCGCACCAGGTTCTA SESPSGTAPGSTSST CTAGCTCTACTGCTGAATCTCCGGGCCCAGGTACTTCTGAAA AESPGPGTSESATP GCGCTACTCCGGAGTCCGGTCCAGGTACCTCTACCGAACCGT ESGPGTSTEPSEGS CCGAAGGCAGCGCTCCAGGTACTTCTACTGAACCTTCTGAGG APGTSTEPSEGSAP GTAGCGCTCCA LCW462_r64 GGTACTTCTACCGAACCGTCCGAGGGCAGCGCTCCAGGTACT GTSTEPSEGSAPGT TCTACTGAACCTTCTGAAGGCAGCGCTCCAGGTACTTCTACTG STEPSEGSAPGTSTE AACCTTCCGAAGGTAGCGCACCAGGTACCTCTACCGAACCGT PSEGSAPGTSTEPSE CTGAAGGTAGCGCACCAGGTACCTCTGAAAGCGCAACTCCTG GSAPGTSESATPES AGTCCGGTCCAGGTACTTCTGAAAGCGCAACCCCGGAGTCTG GPGTSESATPESGP GCCCAGGTACTCCTGGCAGCGGTACCGCATCTTCCTCTCCAG GTPGSGTASSSPGS GTAGCTCTACTCCGTCTGGTGCAACTGGTTCCCCAGGTGCTTC STPSGATGSPGASP TCCGGGTACCAGCTCTACCGGTTCTCCAGGTTCCACCAGCTCT GTSSTGSPGSTSSTA ACTGCTGAATCTCCTGGTCCAGGTACCTCTCCTAGCGGTGAAT ESPGPGTSPSGESST CTGCTCCAGGTACTTCTACTCCTGAAAGCGGCTCTGC APGTSTPESGSASP TTCTCCA LCW462_r67 GGTAGCCCGGCAGGCTCTCCGACCTCTACTGAGGAAGGTACT GSPAGSPTSTEEGT AGCGCAACCCCGGAGTCCGGCCCAGGTACCTCTACC SESATPESGPGTSTE GAACCGTCTGAGGGCAGCGCACCAGGTACTTCTGAAAGCGCA PSEGSAPGTSESATP ACCCCTGAATCCGGTCCAGGTAGCGAACCGGCTACTTCTGGC ESGPGSEPATSGSE TCTGAGACTCCAGGTACTTCTACCGAACCGTCCGAAGGTAGC TPGTSTEPSEGSAP GGTAGCCCGGCTGGTTCTCCGACTTCCACCGAGGAA GSPAGSPTSTEEGT GGTACCTCTACTGAACCTTCTGAGGGTAGCGCTCCAGGTACC STEPSEGSAPGTSTE TCTACTGAACCTTCCGAAGGCAGCGCTCCAGGTACTTCTACC PSEGSAPGTSTEPSE GAACCGTCCGAGGGCAGCGCTCCAGGTACTTCTACTGAACCT GSAPGTSTEPSEGS TCTGAAGGCAGCGCTCCAGGTACTTCTACTGAACCTTCCGAA APGTSTEPSEGSAP GGTAGCGCACCA LCW462_r69 GGTACTTCTCCGAGCGGTGAATCTTCTACCGCACCAGGTTCTA ESSTAPGST CTAGCTCTACCGCTGAATCTCCGGGCCCAGGTACTTCTCCGAG SSTAESPGPGTSPSG ATCTTCTACTGCTCCAGGTACCTCTGAAAGCGCTACT ESSTAPGTSESATPE TCTGGCCCAGGTACCTCTACTGAACCGTCTGAGGGT SGPGTSTEPSEGSAP AGCGCTCCAGGTACTTCTACTGAACCGTCCGAAGGTAGCGCA GTSTEPSEGSAPGSS Clone Sequence Trimmed Protein Sequence CCAGGTTCTAGCCCTTCTGCATCTACTGGTACTGGCCCAGGTA TGPGSSTPS CTCCTTCTGGTGCTACCGGCTCTCCAGGTGCTTCTCC GATGSPGASPGTSS GGGTACTAGCTCTACCGGTTCTCCAGGTACTTCTACTCCGGAA TGSPGTSTPESGSAS AGCGGTTCCGCATCTCCAGGTACTTCTCCTAGCGGTGAATCTT PGTSPSGESSTAPGT CTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTCTACTGC SPSGESSTAP TCCA LCW462_r70 GGTACCTCTGAAAGCGCTACTCCGGAGTCTGGCCCAGGTACC GTSESATPESGPGT TCTACTGAACCGTCTGAGGGTAGCGCTCCAGGTACTTCTACTG GSAPGTSTE AACCGTCCGAAGGTAGCGCACCAGGTAGCCCTGCTGGCTCTC PSEGSAPGSPAGSP CGACTTCTACTGAGGAAGGTAGCCCGGCTGGTTCTCCGACTT TSTEEGSPAGSPTST CTACTGAGGAAGGTACTTCTACCGAACCTTCCGAAGGTAGCG EEGTSTEPSEGSAP CTCCAGGTTCTAGCCCTTCTGCTTCCACCGGTACTGGCCCAGG GSSPSASTGTGPGS TAGCTCTACCCCTTCTGGTGCTACCGGCTCCCCAGGTAGCTCT STPSGATGSPGSSTP ACTCCTTCTGGTGCAACTGGCTCTCCAGGTAGCGAACCGGCA SGATGSPGSEPATS ACTTCCGGCTCTGAAACCCCAGGTACTTCTGAAAGCGCTACT GSETPGTSESATPES CCTGAGTCTGGCCCAGGTAGCGAACCTGCTACCTCTGGCTCT GPGSEPATSGSETP GAAACCCCA LCW462_r72 GGTACTTCTACCGAACCGTCCGAAGGCAGCGCTCCAGGTACC GTSTEPSEGSAPGT TCTACTGAACCTTCCGAGGGCAGCGCTCCAGGTACCTCTACC STEPSEGSAPGTSTE GAACCTTCTGAAGGTAGCGCACCAGGTAGCTCTACCCCGTCT PSEGSAPGSSTPSG GGTGCTACCGGTTCCCCAGGTGCTTCTCCTGGTACTAGCTCTA ATGSPGASPGTSST CCGGTTCTCCAGGTAGCTCTACCCCGTCTGGTGCTACTGGCTC GSPGSSTPSGATGS TCCAGGTACTTCTGAAAGCGCAACCCCTGAATCCGGTCCAGG ATPESGPGS TAGCGAACCGGCTACTTCTGGCTCTGAGACTCCAGGTACTTCT EPATSGSETPGTSTE ACCGAACCGTCCGAAGGTAGCGCACCAGGTTCTACTAGCGAA PSEGSAPGSTSESPS TCTCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCGT TSESPSGT CTGGCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTTCCG PESGSASP CTTCTCCA LCW462_r73 GGTACCTCTACTCCTGAAAGCGGTTCTGCATCTCCAGGTTCCA GTSTPESGSASPGST CTACCGCAGAATCTCCGGGCCCAGGTTCTACTAGCTC SSTAESPGPGSTSST TACTGCTGAATCTCCTGGCCCAGGTTCTAGCCCTTCTGCATCT AESPGPGSSPSAST ACTGGTACTGGCCCAGGTAGCTCTACTCCTTCTGGTGCTACCG GTGPGSSTPSGATG GCTCTCCAGGTGCTTCTCCGGGTACTAGCTCTACCGGTTCTCC SPGASPGTSSTGSP AGGTAGCGAACCGGCAACCTCCGGCTCTGAAACCCCAGGTAC GSEPATSGSETPGT CTCTGAAAGCGCTACTCCTGAATCCGGCCCAGGTAGCCCGGC SESATPESGPGSPA TCCGACTTCCACTGAGGAAGGTTCTACTAGCGAATC GSPTSTEEGSTSESP TCCTTCTGGCACTGCACCAGGTTCTACCAGCGAATCTCCGTCT STSESPSGT GGCACTGCACCAGGTACCTCTACCCCTGAAAGCGGTTCCGCT APGTSTPESGSASP TCTCCC LCW462_r78 GGTAGCCCGGCTGGCTCTCCTACCTCTACTGAGGAAGGTACT GSPAGSPTSTEEGT TCTGAAAGCGCTACTCCTGAGTCTGGTCCAGGTACCTCTACTG SESATPESGPGTSTE AACCGTCCGAAGGTAGCGCTCCAGGTTCTACCAGCGAATCTC PSEGSAPGSTSESPS CTTCTGGCACCGCTCCAGGTTCTACTAGCGAATCCCCGTCTGG GTAPGSTSESPSGT TACCGCACCAGGTACTTCTCCTAGCGGCGAATCTTCTACCGCA APGTSPSGESSTAP CCAGGTACCTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGT GTSTEPSEGSAPGSP AGCCCGGCAGGTTCTCCTACTTCCACTGAGGAAGGTACTTCT AGSPTSTEEGTSTEP ACCGAACCTTCTGAGGGTAGCGCACCAGGTAGCGAACCTGCA SEGSAPGSEPATSG ACCTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACT SETPGTSESATPESG CCTGAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGC PGTSTEPSEGSAP AGCGCACCA LCW462_r79 GGTACCTCTACCGAACCTTCCGAAGGTAGCGCTCCAGGTAGC SEGSAPGSP CCGGCAGGTTCTCCTACTTCCACTGAGGAAGGTACTTCTACCG AGSPTSTEEGTSTEP AACCTTCTGAGGGTAGCGCACCAGGTACCTCCCCTAGCGGCG SEGSAPGTSPSGESS AATCTTCTACTGCTCCAGGTACCTCTCCTAGCGGCGAATCTTC TAPGTSPSGESSTAP TACCGCTCCAGGTACCTCCCCTAGCGGTGAATCTTCTACCGCA GTSPSGESSTAPGST CCAGGTTCTACCAGCGAATCCCCTTCTGGTACTGCTCCAGGTT SESPSGTAPGSTSES CTACCAGCGAATCCCCTTCTGGCACCGCACCAGGTACTTCTAC PSGTAPGTSTPESGS CCCTGAAAGCGGCTCCGCTTCTCCAGGTAGCGAACCTGCAAC PATSGSETP CTCTGGCTCTGAAACCCCAGGTACCTCTGAAAGCGCTACTCCT GTSESATPESGPGT GAATCTGGCCCAGGTACTTCTACTGAACCGTCCGAGGGCAGC STEPSEGSAP GCACCA Clone Sequence Trimmed Protein Sequence LCW462_r87 GGTAGCGAACCGGCAACCTCTGGCTCTGAAACCCCAGGTACC SGSETPGT TCTGAAAGCGCTACTCCGGAATCTGGTCCAGGTACTTCTGAA SESATPESGPGTSES AGCGCTACTCCGGAATCCGGTCCAGGTACTTCTCCGAGCGGT ATPESGPGTSPSGES GAATCTTCTACCGCACCAGGTTCTACTAGCTCTACCGCTGAAT STAPGSTSSTAESPG CTCCGGGCCCAGGTACTTCTCCGAGCGGTGAATCTTCTACTGC PGTSPSGESSTAPGS TCCAGGTTCTACTAGCGAATCCCCGTCTGGTACTGCTCCAGGT TSESPSGTAPGTSPS ACTTCCCCTAGCGGTGAATCTTCTACTGCTCCAGGTTCTACCA GESSTAPGSTSSTA GCTCTACCGCAGAATCTCCGGGTCCAGGTAGCTCTACTCCGTC ESPGPGSSTPSGAT AACCGGTTCCCCAGGTAGCTCTACCCCTTCTGGTGCA GSPGSSTPSGATGS ACCGGCTCCCCAGGTAGCTCTACCCCTTCTGGTGCAAACTGG PGSSTPSGANWLS CTCTCC LCW462_r88 GGTAGCCCTGCTGGCTCTCCGACTTCTACTGAGGAAGGTAGC GSPAGSPTSTEEGSP CCGGCTGGTTCTCCGACTTCTACTGAGGAAGGTACTTCTACCG AGSPTSTEEGTSTEP AACCTTCCGAAGGTAGCGCTCCAGGTACCTCTACTGAACCTT GTSTEPSE GCAGCGCTCCAGGTACCTCTACCGAACCGTCCGAGG GSAPGTSTEPSEGS GCAGCGCACCAGGTACTTCTGAAAGCGCAACCCCTGAATCCG APGTSESATPESGP GTCCAGGTGCATCTCCTGGTACCAGCTCTACCGGTTCTCCAGG GASPGTSSTGSPGS TAGCTCTACTCCTTCTGGTGCTACTGGCTCTCCAGGTGCTTCC STPSGATGSPGASP CCGGGTACCAGCTCTACCGGTTCTCCAGGTAGCTCTACCCCGT SPGSSTPSG CTGGTGCTACTGGTTCTCCAGGTACTCCGGGCAGCGGTACTG ATGSPGTPGSGTAS CTTCTTCCTCTCCAGGTAGCTCTACCCCTTCTGGTGCTACTGG SSPGSSTPSGATGSP LCW462_r89 GGTAGCTCTACCCCGTCTGGTGCTACTGGTTCTCCAGGTACTC GSSTPSGATGSPGT CGGGCAGCGGTACTGCTTCTTCCTCTCCAGGTAGCTCTACCCC PGSGTASSSPGSSTP TTCTGGTGCTACTGGCTCTCCAGGTAGCCCGGCTGGCTCTCCT SGATGSPGSPAGSP ACCTCTACTGAGGAAGGTACTTCTGAAAGCGCTACTCCTGAG TSTEEGTSESATPES TCTGGTCCAGGTACCTCTACTGAACCGTCCGAAGGTAGCGCT GPGTSTEPSEGSAP CCAGGTACCTCTGAAAGCGCAACTCCTGAGTCTGGCCCAGGT GTSESATPESGPGS CCTGCTACCTCCGGCTCTGAGACTCCAGGTACCTCT EPATSGSETPGTSES GAAAGCGCAACCCCGGAATCTGGTCCAGGTACTTCTACTGAA ATPESGPGTSTEPSE CCGTCTGAAGGTAGCGCACCAGGTACTTCTGAAAGCGCAACC GSAPGTSESATPES CCGGAATCCGGCCCAGGTACCTCTGAAAGCGCAACCCCGGAG GPGTSESATPESGP TCCGGCCCA ] Example 7: Construction ofXTEN_AM288 The entire y LCWO462 was dimerized as described in Example 6 resulting in a library of XTEN_AM288 clones designated LCWO463. 1512 isolates from library LCWO463 were screened using the protocol described in Example 6. 176 highly sing clones were ced and 40 preferred XTEN_AM288 ts were chosen for the uction of multifunctional proteins that n multiple XTEN segments with 288 amino acid residues.

Example 8: Construction ofXTEN_AM432 We generated a library ofXTEN_AM432 segments by recombining segments from library LCWO462 ofXTEN_AM144 segments and segments from library LCWO463 ofXTEN_AM288 segments. This new library ofXTEN_AM432 segment was designated LCWO464. Plasmids were isolated from cultures of E. coli harboring LCWO462 and LCWO463, respectively. 1512 isolates from library LCWO464 were screened using the protocol described in Example 6. 176 highly expressing clones were sequenced and 39 preferred M432 segment were chosen for the construction of longer XTENs and for the construction of multifunctional proteins that contain multiple XTEN segments with 432 amino acid residues.

In parallel we constructed library LMSOlOO ofXTEN_AM432 segments using preferred segments ofXTEN_AM144 and XTEN_AM288. ing this library yielded 4 isolates that were selected for further construction Example 9: Construction ofXTEN_AM875 The stuffer vector pCWO359 was digested with BsaI and KpnI to remove the stuffer segment and the resulting vector fragment was isolated by agarose gel purification.

] We annealed the phosphorylated oligonucleotide BsaI-AscI-KpnIforP: AGGTGCAAGCGCAAGCGGCGCGCCAAGCACGGGAGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 1652) and the non-phosphorylated oligonucleotide BsaI-AscI-KpnIrev: CCTCGAGTGAAGACGAACCTCCCGTGCTTGGCGCGCCGCTTGCGCTTGC (SEQ ID NO: 1653) for introducing the sequencing island A (SI-A) which encodes amino acids GASASGAPSTG (SEQ ID NO: 1654) and has the restriction enzyme AscI recognition nucleotide sequence GGCGCGCC inside.

The annealed oligonucleotide pairs were ligated with BsaI and KpnI digested stuffer vector pCWO359 prepared above to yield pCWO466 containing SI-A. We then generated a library ofXTEN_AM443 segments by recombining 43 preferred M432 segments from Example 8 and SI-A segments from pCWO466 at C-terminus using the same dimerization process described in e 5. This new library of XTEN_AM443 ts was designated LCWO479.

We generated a library ofXTEN_AM875 segments by recombining segments from library LCWO479 ofXTEN_AM443 ts and 43 preferred XTEN_AM432 segments from Example 8 using the same dimerization process described in e 5. This new library of M875 segment was designated LCWO481.

Example 10: uction ofXTEN_AM1318 We annealed the phosphorylated oligonucleotide BsaI-FseI-KpnIforP: AGGTCCAGAACCAACGGGGCCGGCCCCAAGCGGAGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 1655) and the osphorylated oligonucleotide BsaI-FseI-KpnIrev: CCTCGAGTGAAGACGAACCTCCGCTTGGGGCCGGCCCCGTTGGTTCTGG (SEQ ID NO: 1656) for introducing the sequencing island B (SI-B) which encodes amino acids GPEPTGPAPSG (SEQ ID NO: 1657) and has the restriction enzyme FseI recognition nucleotide ce GGCCGGCC inside.

The annealed oligonucleotide pairs were ligated with BsaI and KpnI digested stuffer vector pCWO359 as used in Example 9 to yield 7 ning SI-B. We then generated a library _AM443 ts by recombining 43 preferred XTEN_AM432 segments from Example 8 and SI-B segments from pCWO467 at C-terminus using the same dimerization process described in example 5. This new library of XTEN_AM443 segments was designated LCWO480.

] We generated a library ofXTEN_AM13 1 8 segments by recombining segments from y LCWO48O ofXTEN_AM443 segments and segments from library LCWO481 ofXTEN_AM875 segments using the same dimerization process as in Example 5. This new library ofXTEN_AM13 1 8 segment was designated LCWO487.

Example 11: Construction ofXTEN_AD864 Using the several consecutive rounds of dimerization, we assembled a collection of XTEN_AD864 sequences starting from segments ofXTEN_AD36 listed in Example 1. These sequences were assembled as described in Example 5. Several isolates from XTEN_AD864 were evaluated and found to show good expression and excellent solubility under physiological conditions. One intermediate construct of XTEN_ADS76 was sequenced. This clone was evaluated in a PK experiment in cynomolgus monkeys and a half-life of about 20 h was ed.

Example 12: Construction ofXTEN_AF864 Using the several consecutive rounds of zation, we assembled a collection of XTEN_AF864 sequences starting from segments _AF36 listed in Example 3. These sequences were assembled as described in Example 5. Several isolates from XTEN_AF864 were evaluated and found to show good expression and ent solubility under physiological conditions. One intermediate construct of XTEN_AF54O was sequenced. This clone was evaluated in a PK experiment in cynomolgus monkeys and a ife of about 20h was measured. A full length clone ofXTEN_AF864 had excellent solubility and showed half-life exceeding 60h in cynomolgus monkeys. A second set ofXTEN_AF ces was assembled including a sequencing island as described in Example 9.

Example 13: Construction ofXTEN_AG864 Using the several consecutive rounds of dimerization, we assembled a collection of XTEN_AG864 sequences starting from segments ofXTEN_AD36 listed in Example 1. These sequences were assembled as described in Example 5. Several isolates from XTEN_AG864 were evaluated and found to show good expression and excellent solubility under physiological conditions. A full length clone of XTEN_AG864 had excellent solubility and showed half-life exceeding 60h in cynomolgus monkeys.

Example 14: Methods of producing and evaluating CFXTEN with al and terminal XTEN The design, construction and evaluation of CFXTEN comprising FVIH and one or more XTEN is lished using a systematic ch. The regions suitable for XTEN insertion sites include, but are to limited to regions at or proximal to the known domain boundaries of FVHI, exon boundaries, known surface loops, s with a low degree of order, and hydrophilic s. By analysis of the foregoing, different s across the sequence of the FVHI B domain deleted (BDD) sequence have been identified as insertion sites for XTEN, non-limiting es of which are listed in Tables 5-8, and shown schematically in FIGS. 8 and 9. Initially, individual constructs are created (using methods described, below) in which DNA ng a single XTEN or XTEN fragment of a length g from 6 to 2004 amino acid residues is inserted into the FVHI sequence ponding to or near (e. g., within 6 amino acids) each of the single insertion sites fied in Table 5, Table 6, Table 7, Table 8, and Table 9, and the resulting constructs are expressed and the recovered protein then evaluated for their effects on retention of procoagulant activity using, e. g., one of the in vitro assays of Table 49. For example, using the methods bed below, constructs are made in which an XTEN sequence is inserted within the Al A2, B, A3, C1 and C2 domain sequences of FVIII, as well as linked to the C-terminus, and the resulting expressed fusion proteins are ted in a chromogenic assay of Table 49, compared to a FVIII not linked to XTEN. CFXTEN fusion proteins can be further classified acting to high, intermediate and low categories based on the activities they t. In those cases where the CFXTEN exhibits activity that is comparable or modestly reduced compared to FVIII, the insertion site is deemed favorable. In those cases where the ty is intermediate, the insertion site can be adjusted from 1-6 amino acids towards the N— or C-terminus of the insertion site and/or the length or net charge of the XTEN may be altered and the ing construct(s) re-evaluated to determine whether the activity is ed. Alternatively, the XTEN is inserted into the construct with flanking cleavage sites; preferably sites that are tible to cleavage by proteases found in clotting assays, such that the XTEN is released during the activation of the FVIII component, thereby ing additional information about the suitability of the XTEN insertion site in the fusion protein.

Once all of the individual ion sites are ted and the favorable insertion sites are identified, libraries of constructs are created with two, three, four, five or more XTEN inserted in the permutations of favorable sites. The length and net charge of the XTEN (e.g., XTEN of the AE versus AG family) are varied in order to ascertain the effects of these variables on FVIII activity and physicochemical properties of the fusion protein. CFXTEN constructs that retain a desired degree of in vitro procoagulant FVIII activity are then evaluated in vivo using mouse and/or dog models of ilia A, as described in Examples below, or other models known in the art. In on, ucts are assayed in the presence of FVIII inhibitors and other anti-FVIII antibodies to determine constructs that retain activity. In addition, CFXTEN ucts are made that incorporate cleavage sequences at or near the junction(s) of FVIII and XTEN (e. g., sequences from Table 8) designed to release the XTEN and are ted for ement of FVIII activity and effects on terminal half-life. By the iterative process of making constructs combining different insertion sites, varying the length and composition qualities of the XTEN (e. g., different XTEN families), and evaluation, the skilled artisan obtains, by the foregoing methods, CFXTEN with desired properties, such as but not limited to of procoagulant FVIII activity, reduced binding with FVIII inhibitors, enhanced pharmacokinetic properties, ability to administer to a subject by different routes, and/or enhanced pharmaceutical properties.

Example 15: Methods of producing and evaluating CFXTEN containing FVIII and A general scheme for producing and evaluating CFXTEN compositions is presented in , and forms the basis for the general description of this Example. Using the disclosed methods and those known to one of ordinary skill in the art, together with guidance provided in the illustrative examples, a d artesian can create and evaluate CFXTEN fusion proteins comprising XTEN and FVIII or variants of FVIII known in the art. The Example is, therefore, to be construed as merely rative, and not limitative of the methods in any way whatsoever; numerous variations will be apparent to the ordinarily skilled n. In this Example, a CFXTEN of a factor VIII BDD linked to an XTEN of the AE family of motifs is created.

The general scheme for producing polynucleotides encoding XTEN is presented in FIGS. 11 and 12. is a schematic flowchart of representative steps in the assembly of an XTEN polynucleotide construct in one of the embodiments of the invention. Individual oligonucleotides 501 are annealed into sequence motifs 502 such as a no acid motif (“12-mer”), which is ligated to additional sequence motifs from a library that can multimerize to create a pool that encompasses the desired length of the XTEN 504, as well as ligated to a smaller concentration of an oligo containing BbsI, and KpnI restriction sites 503. The motif libraries e specific sequence XTEN families; e. g., AD, AE, AF, AG, AM, or AQ ces of Table 3. As illustrated in , the XTEN length, in this case, is 36 amino acid residues, but longer lengths are also achieved by this l process. For example, multimerization is performed by ligation, overlap extension, PCR ly or similar cloning techniques known in the art that, in this case, result in a construct with 288 amino acid residues. The resulting pool of ligation products is gel-purified and the band with the d length ofXTEN is cut, ing in an isolated XTEN gene with a stopper sequence 505. The XTEN gene can be cloned into a stuffer vector.

In this case, the vector encodes an optional CBD sequence 506 and a GFP gene 508. Digestion is then performed with BbsI/HindIII to remove 507 and 508 and place the stop codon. The resulting product is then cloned into a BsaI/HindIII digested vector containing a gene encoding the FVIII, resulting in the gene 500 encoding a CFXTEN fusion protein with a 288 amino acid XTEN linked to the C-terminus of the factor VIII. As would be apparent to one of ordinary skill in the art, the methods are applied to create constructs in alternative configurations and with varying XTEN lengths or in multiple locations.

DNA sequences encoding FVIII are conveniently obtained by standard procedures known in the art from a cDNA library prepared from an appropriate cellular source, from a c library, or may be created synthetically (e. g., automated nucleic acid synthesis) using DNA sequences obtained from publicly available databases, patents, or literature references. In the present example, a FVIII B domain d (BDD) variant is prepared as described in Example 17. A gene or polynucleotide encoding the FVIII portion of the protein or its complement is then cloned into a construct, such as those described herein, which can be a plasmid or other vector under control of appropriate transcription and translation ces for high level n expression in a biological . A second gene or polynucleotide coding for the XTEN portion or its complement is genetically fused to the nucleotides ng the terminus of the FVIII gene by cloning it into the construct adjacent and in frame with the gene coding for the CF, through a ligation or multimerization step. In this , a chimeric DNA molecule coding for (or complementary to) the CFXTEN fusion n is generated within the construct. Optionally, a gene encoding for a second XTEN is inserted and ligated me internally to the nucleotides encoding the FVIII-encoding region. The constructs are designed in different configurations to encode various insertion sites of the XTEN in the FVIII sequence, including those of Table 5, Table 6, Table 7, Table 8, and Table 9 or those illustrated in FIGS. 8-9. ally, this chimeric DNA molecule is erred or cloned into another construct that is a more appropriate expression vector; e. g., a vector appropriate for a mammalian host cell such as CHO, BHK and the like. At this point, a host cell capable of expressing the chimeric DNA molecule is transformed with the chimeric DNA molecule, described more completely, below, or by well-known methods, depending on the type of cellular host, as bed supra.

Host cells containing the XTEN-FVIH expression vector are cultured in conventional nutrient media modified as appropriate for activating the er. The e conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan. After expression of the fusion protein, culture broth is harvested and separated from the cell mass and the resulting crude extract retained for purification of the fusion protein.

Gene expression is measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 ], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, gene sion is measured by immunological of fluorescent methods, such as immunohistochemical ng of cells to quantitate directly the expression of gene product. Antibodies useful for immunohistochemical ng and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal.

Conveniently, the antibodies may be prepared against the FVIII sequence polypeptide using a synthetic peptide based on the ces provided herein or against exogenous sequence fused to FVHI and encoding a specific antibody epitope. Examples of selectable markers are well known to one of skill in the art and include reporters such as enhanced green fluorescent protein (EGFP), beta-galactosidase (B- gal) or chloramphenicol transferase (CAT).

The CFXTEN polypeptide product is purified Via methods known in the art. Procedures such as gel filtration, affinity ation, salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxyapatite adsorption chromatography, hydrophobic interaction chromatography or gel electrophoresis are all techniques that may be used in the purification. Specific methods of purification are described in Robert K. Scopes, Protein Purification: Principles and Practice, s R.

Castor, ed., Springer-Verlag 1994, and Sambrook, et al., supra. step purification separations are also described in Baron, et al., Crit. Rev. Biotechnol. 10:179-90 (1990) and Below, et al., J. Chromatogr.

A. 679:67-83 (1994).

As illustrated in , the isolated CFXTEN fusion ns are characterized for their chemical and ty properties. An isolated fusion protein is characterized, e. g., for sequence, purity, apparent lar weight, solubility and stability using standard methods known in the art. The fusion protein meeting expected standards is ted for actiVity, which can be measured in Vitro or in Vivo by measuring one of the factor VIII-associated ters described herein, using one or more assays disclosed , or using the assays of the Examples or Table 49.

In addition, the CFXTEN FVIII fusion protein is administered to one or more animal species to determine standard pharmacokinetic parameters and pharmacodynamic properties, as described in Examples 25 and 26.

By the iterative process of producing, expressing, and recovering CFXTEN ucts, followed by their characterization using methods disclosed herein or others known in the art, the CFXTEN compositions comprising CF and an XTEN are produced and evaluated to confirm the expected properties such as enhanced solubility, enhanced stability, improved pharmacokinetics and reduced immunogenicity, g to an overall enhanced therapeutic activity compared to the ponding unfused FVIII. For those fusion proteins not possessing the desired properties, a different ce or configuration is constructed, expressed, isolated and evaluated by these methods in order to obtain a composition with such properties.

Example 16: Construction of expression plasmids for BDD FVIII I. Construction of B domain deleted FVIII (BDD FVIII) expression vectors The expression vector encoding BDD FVIII was created by cloning the BDD FVIII open reading frame into the pcDNA4 vector rogen, CA) ning a polyA to allow for optimal mammalian expression of the FVIII gene, resulting in a uct designated pBC0100. Several natural sites were identified within this construct for cloning use, including BsiWI 48, AflII 381, PshAI 1098, KpnI 1873, BamHI 1931, PflMI 3094, ApaI 3574, XbaI 4325, NotI 4437, XhoI 4444, BstEII 4449, AgeI 4500, PmeI 4527. To facilitate assay development, nucleotides encoding Myc and His tag were introduced into the FVIII open reading frame. pBC0100 was PCR amplified using the following primers: 1) F8-BsiWI-F: tattccCGTACchcgccaccATGCAAATAGAGCTCTCCACCT (SEQ ID NO: 1658); 2) F8-nostop-XhoI-R1: GGTGACCTCGAGcgtagaggtcctgtgcctcg (SEQ ID NO: 1659) to introduce BsiWI and XhoI in appropriate locations. The PCR product was digested with BsiWI and XhoI. PcDNA4-Myc-His/C was digested with Acc651 and XhoI, which generated two products of 5003 and 68 bps. The 5003bps product was ligated with the digested PCR’ed FVIII fragment and used for DH5alpha ormation. The enzymes Acc65I and BsiWI create compatible ends but this ligation ys the site for future digestion. The resulting construct was designated pBC0102 (pcDNA4- FVIII_3-Myc-His). To facilitate the design and execution of future cloning strategies, especially ones involving the creation of BDD FVIII expression constructs that contain multiple XTEN insertions, we selected onal unique restriction enzyme sites to incorporate, including BsiWI 908, NheI 1829 and ClaI 3281. The introduction of these sites was done via the QuikChange method (Agilent, CA) individually. The resulting uct was designated pBC0112 (pcDNA4-FVIII_4-Myc-His). To avoid problems that may arise from the linker peptides that connects n Myc/His and FVIII/Myc, and to remove restriction enzyme sites that are preferred for future XTEN ion, we mutated the sequences encoding the peptide sequences from ARGHPF (SEQ ID NO: 1660) to GAGSPGAETA (SEQ ID NO: 178) (between FVIII and Myc), NMHTG (SEQ ID NO: 1661) to SPATG (SEQ ID NO: 1662) en Myc and His) via the QuikChange method. The construct was designated pBC0114 4-FVIII_4- GAGSPGAETA—Myc-SPATG-His ('GAGSPGAETA' and 'SPATG' disclosed as SEQ ID NOS 178 and 1662, respectively)) (sequence in Table 21), which was used as the base vector for the design and creation of other expression vectors incorporating XTEN sequences. Expression and FVIII activity data for this construct are presented in II. Construction of B domain deleted FVIII (BDD FVIII) expression vectors The gene ng BDD FVIII is sized by ts (Regensburg, y) in the g vector pMK (pMK-BDD FVIII). The BDD FVIII proteins contain 1457 amino acids at a total molecular weight of 167539.66. There are 6 domains within the wild-type FVIII protein, the A1, A2, B, A3, C1 and C2 domains. In the BDD FVIII protein, most of the B domain has been deleted as it was shown to be an unstructured domain and the removal of the domain does not alter critical functions of this protein. The pMK vector used by GeneArts contains no promoter, and can not be used as an expression vector. Restriction enzyme sites NheI on the 5’ end and SfiI, SalI and XhoI on the 3’ end are introduced to tate subcloning of the DNA sequence encoding BDD FVIII into expression vectors, such as CET1019-HS (Millipore). Several unique restriction enzyme sites are also introduced into the FVIII sequence to allow further manipulation (e. g., insertion, mutagenesis) of the DNA sequences.

Unique sites listed with their cut site include, but are not limited to: SacI 391, AfiII 700, SpeI 966, PshAI 1417, Acc6512192, KpnI 2192, BamHI 2250, HindIII 2658, PfoI 2960, PflMI 3413, ApaI 3893, Bsp1201 3893, SwaI 4265, OliI 4626, XbaI 4644, and BstBI 4673. . The HindIII site resides at the very end of the A2 domain and can potentially be used for modification of the B domain. The synthesized pMK-BDD FVIII from GeneArts does not contain a stop codon. The stop codon is uced by amplifying a 127 bp fragment of FVIII using the following primers: 5’- TCTCTAGACCCACCG-3’ (SEQ ID NO: 1663); 5’- CTCCTCGAGGTCGACTCAGTAGAGGTCCTGTGCCTCG-3’ (SEQ ID NO: 1664). The fragment is digested with XbaI and SalI, and ligated to XbaI /SalI digested pMK-BDD FVIII. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened by DNA miniprep and the desired ucts are confirmed by DNA sequencing. The construct named pBCOO27 (pMK-BDD FVIII-STOP) contains coding sequences that encode the BDD FVIII protein. The pBC0027 construct is then digested with NheI /SalI, and ligated with alI digested CET1019-HS vector (Millipore). The CET1019-HS vector contains a human CMV promoter and a UCOE sequence to facilitate gene expression. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened by DNA miniprep and the desired constructs are confirmed by DNA sequencing. The final construct is designated pBCOO25 19-HS-BDD STOP), which encodes the BDD FVIII protein under the control of a human CMV promoter. Introduction of the pBCOO25 construct into mammalian cells is expected to allow expression of the BDD FVIII protein with procoagulant activity.

Example 17: Construction of expression plasmids for BDD FVIII Containing XTEN ] 1. B domain AE42 ion ] Two PCR reactions were run in parallel to insert XTEN_AE42 into the remaining B domain region of the BDD FVIII constructs. The PCR reactions involved the following primers: cgaaagcgctacgcctgagaGTGGCCCTGGCTCTGAGCCAGCCACCTCCGGCTCTGAAACCCCTGCCTC GAGCccaccagtcttgaaacgcc (SEQ ID NO: 1665); TGATATGGTATCATCATAATCGATTTCCTCTTGATCTGACTG (SEQ ID NO: 1666); agcttgaggatccagagttc (SEQ ID NO: 1667); tctcaggcgtagcgctttchTTGTCCCCTCTTCTGTTGAGGTGGGGGAGCCAGCAGGAGAACCTGGCG CGCCgttttgagagaagcttcttggt (SEQ ID NO: 1668). The PCR products then served as templates, and a second PCR was performed to uce the XTEN_AE42 into the FVIII encoding nucleotide sequences flanked by BamHI and ClaI. This PCR product was digested with BamHI and ClaI simultaneously with the ion of PBCOl 14 with the same two enzymes. The PCR product was ligated to the digested vector. This construct was designated 5 (pcDNA4-FVIII_4XTEN_AE42-GAGSPGAETA—Myc- His) ('GAGSPGAETA' and 'SPATG' sed as SEQ ID NOS 178 and 1662, respectively), and encodes the BDD FVIII with an AE42 XTEN incorporated within the residual B-domain. 2. AE42 Insertion and R1648A on The QuikChange method nt, CA) was employed to introduce an R1648A mutation into PBC0135. This construct was designated pBC0149 (pcDNA4-FVIII_4XTEN_AE42-GAGSPGAETA- Myc-SPATG-His_R1648A) ('GAGSPGAETA' and 'SPATG' disclosed as SEQ ID NOS 178 and 1662, respectively), eliminating that FVIII processing site. 3. B domain AE288 Insertion XTEN_AE288 was PCR amplified using the following primers: tctcaaaacGGCGCGCCAggtacctcagagtctgctacc (SEQ ID NO: 1669) and tggtggGCTCGAGGCtggcgcactgccttc (SEQ ID NO: 1670). PBCOO75 was used as the template for this PCR reaction. The PCR product was digested with AscI and XhoI, and PBC0135 was digested with the same enzymes. The PCR product was ligated to the PBC0135 fragment. This construct was designated pBC0136 (pcDNA4-FVIII_4XTEN_AE288-GAGSPGAETA—Myc-SPATG-His) ('GAGSPGAETA' and 'SPATG' disclosed as SEQ ID NOS 178 and 1662, respectively), and encodes the BDD FVIII with an AE288 XTEN incorporated within the residual B-domain. ] 4. AE288 Insertion and R1648A mutation XTEN_AE288 was PCR amplified using the ing primers: tctcaaaacGGCGCGCCAggtacctcagagtctgctacc (SEQ ID NO: 1671) and tggtggGCTCGAGGCtggcgcactgccttc (SEQ ID NO: 1672). Construct pBCOO75 was used as the template for this PCR on. The PCR product was digested with AscI and XhoI, and pBC0149 was digested with the same enzymes. The PCR product was ligated to the pBC0149 fragment. This construct was designated pBC0137 (pcDNA4-FVIII_4XTEN_AE288-GAGSPGAETA—Myc-SPATG-His R1648A) ('GAGSPGAETA' and 'SPATG' disclosed as SEQ ID NOS 178 and 1662, tively) and ns an AE288 XTEN sequence internal to the B domain, with the R1648A on eliminating that FVIII processing site. 3. B domain AE144 AG144 AG288 Insertions with and without R1648A mutations Select XTEN fragments were PCR amplified to introduce AscI and XhoI sites to the 5’ and 3’ end respectively. The PCR product was digested with AscI and XhoI, and pBC0135 (for R1648) or pBC0149 (for A1648) were digested with the same enzymes. The PCR product was ligated to the or pBC0149 vector. These constructs were designated pSDOOOS, 6, 7, 8, 17 and 18. uction of expression plasmids for BDD FVIII with XTEN insertion at the C terminus 1. C terminal AE288 insertion XTEN_AE288 was PCR amplified using the following primers: ggggccgaaacggccggtacctcagagtctgctacc (SEQ ID NO: 1673) and tgttcggccgtttcggcccctggcgcactgccttc (SEQ ID NO: 1674). The construct pBC0075 was used as the template for this PCR reaction. The PCR product was digested with SfiI, and pBC0114 was ed with the same enzyme. The PCR product was ligated to the digested pBC0114 fragment. This construct was designated pBC0145 (pcDNA4- FVIII_4-XTEN_AE288-GAGSPGAETA—Myc-SPATG-His) PGAETA' and 'SPATG' disclosed as SEQ ID NOS 178 and 1662, respectively), and encodes an AE288 sequence at the C-terminus of the BDD FVIII. 2. C terminal AG288 insertion XTEN _AG288 was designed and synthesized by DNA2.0 (Menlo Park, CA). The synthesized gene was PCR amplified using the following primers: ggggccgaaacggccccgggagcgtcacc (SEQ ID NO: 1675) and tgttcggccgtttcggcccctgacccggttgcccc (SEQ ID NO: 1676). The PCR product was digested with SfiI, and PBC0114 based vector was digested with the same enzyme. The PCR product was ligated to the digested PBC0114 fragment. This construct was designated pBC0146 (pcDNA4-FVIII_4- XTEN_AG288-GAGSPGAETA—Myc-SPATG-His) ('GAGSPGAETA' and 'SPATG' disclosed as SEQ ID NOS 178 and 1662, tively), and encodes an AG288 sequence at the C-terminus of the BDD FVIII. 3. C terminal AE/AG144 288 864 insertions AscI and XhoI sites were introduced into the PBC0114 based vector via QuikChange methods using the primers: 5037-PBC0114-AscI-XhoI-F: CAGGACCTCTACGGCGCgccagcctcgaGCGAACAAAAACTCATCTCAGAAGAGG (SEQ ID NO: 1677); 503 8-PBC01 I-XhoI-R: CCTCTTCTGAGATGAGTTTTTGTTCGthgaggctgchCGCCGTAGAGGTCCTG (SEQ ID NO: 1678). Various XTEN nts were PCR amplified with AscI and XhoI introduced into the 5’ and 3’ end respectively. The PCR product was d to the digested PBC0114 vector. These constructs were designated pSD0013, pSD0014, pSD0015, pSD0016, pSD0019 and 0.

Construction of sion plasmids for BDD FVIII with inter- and intra- domain XTEN insertions 1. AE7 AE42 and AEl44 Insertions Four ct strategies are used for insertion of AE42 into the designated sites (e.g., the natural or introduced restriction sites BsiWI 48, AflII 381, PshAI 1098, KpnI 1873, BamHI 1931, PflMI 3094, ApaI 3574, XbaI 4325, NotI 4437, XhoI 4444, BstEII 4449, AgeI 4500, PmeI 4527, BsiWI 908, NheI 1829 and ClaI 3281) within the BDD FVIII encoding sequence, each buting to the creation of l constructs. By design, these ions of AE42 create AscI and XhoI sites flanked on either side of the insertion ng for introduction/substitution of longer XTENs, as well as XTEN with different sequences or incorporated cleavage sequences, as needed. Specifically, the constructs that contain XTEN_144 insertions are listed in Table 21. These insertions were created by replacing either AE7 or AE42 with a PCRed XTEN_144 fragment flanked by AscI and XhoI sites. 2. Double PCR-mediated method Two PCR reactions are run in parallel to insert XTEN_AE42 into the designated site. The two PCR reactions introduce XTEN on either the 3’ or the 5’ end via use of a long primer that contains partial XTEN. The PCR products then serve as templates, and a second PCR is performed to introduce the XTEN_AE42 into the FVIH encoding nucleotide sequences flanked by select restriction enzyme sites.

This PCR product is digested with the appropriate enzymes simultaneously with the digestion of PBC0114 using the same two s. The PCR product is ligated to the digested vector. Using this method, constructs are created designated pBC0126, pBC0127, pBC0128, and pBC0129, resulting in AE42 ions at the R3, R3, P130, L216 ons respectively. The sequences are listed in Table 21.

Select 44 ces can then be PCRed to introduce Ascl and Xhol sites on either end of the fragment, and ligate to digested FVHI-XTEN_AE42 construct. For instance, pSD0053 was created by replacing the AE42 of pBC0129 with XTEN_AE144. Other XTEN_l44 constructs were created via the same strategy and are listed in Table 21. 3. QuikChange mediated two step cloning method The QuikChange method is employed to introduce XTEN_AE7 encoding sequences that are flanked by Ascl and Xhol into designated sites. The ing intermediate construct is then digested with Ascl and Xhol. XTEN_AE42 or XTEN_AE144 is PCR amplified to introduce the two sites and digested accordingly. The vector and insert are then ligated to create the final constructs. The sequences are listed in Table 21. 4. Three PCR type II restriction enzyme mediated ligation method Three PCR reactions are med to create two pieces of FVIII encoding nts flanked by one type I restriction enzyme that correlates with a unique site within the FVHI_4 gene and one type II enzyme (e. g. Bsal, Bbsl, Bqul), the third PCR reaction created the XTEN_AE42 flanked by two type II ction enzyme sites. The three PCR fragments are digested with appropriate enzymes and ligated into one linear piece that contains the XTEN_AE42 insertion within a fragment of FVIII encoding sequences. This product is then digested with appropriate unique enzymes within the FVIII ng sequences and ligated to the PBC0114 construct digested with the same enzymes, and result in constructs designated pBC0130 (with XTEN insertion at e P333), pBC0132 (with XTEN insertion at e D403), pBC0133 (with XTEN insertion at residue R490). The sequences are listed in Table 21. Select XTEN_l44 sequences can then be PCRed to uce Ascl and Xhol sites on either end of the fragment, and ligate to digested FVIH-XTEN_AE42 construct. For instance, pSD0001 and pSD0003 were created by replacing the AE42 of pBC0132 with XTEN_AE144 and XTEN_AG144 respectively. Other 44 constructs listed in Table 21 were created via the same gy. 5. Custom gene synthesis Custom gene sis is performed by GeneArt (Regensburg, Germany). The genes are designed so that they include nucleotides ng the XTEN_AE42 inserted in the designated site(s) and the genes are flanked by two unique restriction enzyme sites selected within the FVHI_4 gene. The synthesized genes and PBC0114 are digested with appropriate enzymes and ligated to create the final product with the BDD FVIII incorporating the XTEN_AE42 between the restriction sites. Select XTEN_144 sequences can then be PCRed to introduce Ascl and Xhol sites on either end of the fragment, and ligate to digested FVIII-XTEN_AE42 construct.

Construction of expression ds with dual XTEN insertions in the B domain and at the C terminus ] The construct 6, which encodes the BDD FVIII with an AE288 XTEN incorporated within the residual B-domain, is digested with BamHI and Clal, and the resulting 1372bps nt from this digestion is the insert. The construct 6 is digested with BamHI and Clal, and the 9791bps piece from this digestion is the vector. The vector and insert are ligated together to create 9, containing an AE288 insertion within the B domain and an AG288 on the C us. The same strategy is utilized to create constructs ning two AE288 insertions in the B domain and at the C terminus, tively, using PBC0145 as the vector.

Construction of expression plasmids with multiple XTEN insertions The construct pBC0127, which encodes an AE42 XTEN at the R3 position of FVHI, is ed with BsiWI and Aflll, and the resulting 468bps fragment from this digestion is the . The construct pBC0209 is digested with BsiWI and Aflll, the 10830bps piece from this digestion is the vector. The vector and insert are ligated together to create a uct designated pBC0210, containing an AE42 insertion in the A1 , an extra three ATR amino acid to restore the signal cleavage sequence, an AE288 XTEN insertion within the B domain and an AG288 on the C terminus. The same methodology is used to create constructs encoding multiple XTEN at the natural and introduced restriction sites; e. g., BsiW148, Aflll 381, PshAl 1098, KpnI 1873, BamHI 1931, PflMI 3094, ApaI 3574, Xba14325, NotI 4437, Xhol 4444, BstEII 4449, AgeI 4500, PmeI 4527, BsiWI 908, NheI 1829 and Clal 3281.

Construction of BDD FVHI-Internal-XTEN_AE288 expression vectors Two Bsal restriction enzyme sites are introduced into the PBC0027 pMK-BDD FVIII construct between the base pair 2673 and 2674 using the QuikChange method following manufacturer’s protocol (Agilent Technologies, CA). The inserted DNA sequences are gggtctcccgcgccagggtctccc, and the resulting construct is designated pBC0205 (sequence in Table 21). The DNA sequence encoding AE288 (or other variants and lengths of XTEN; e. g. AE42, AG42, AG288, AM288) is then PCR’ed with primers that introduce Bsal sites on both the 5’ and 3’. The pBC0205 vector and the insert (XTEN_288) are then digested with Bsal and ligated to create pBC0206, which encodes the FVIII gene with an XTEN_AE288 insertion within the B domain (sequence in Table 21). The pBC0206 construct is then ed with Nhel /SalI, and ligated with NheI/Sall digested CET1019-HS vector (Millipore). The CET1019-HS vector contains a human CMV promoter and a UCOE sequence to facilitate gene expression. The d DNA mixture is used to transform DH5a bacterial cells. Transformants are screened by DNA miniprep and the desired constructs are confirmed by DNA sequencing. The final construct is designated pBC0207 (CET1019-HS-BDD FVHI-STOP), which s the BDD FVIII protein under the control of a human CMV promoter nce in Table 21). Introduction of the pBC0207 construct into mammalian cells is expected to allow expression of the BDD FVIII protein with an internal XTEN_AE288. The same protocol is used to introduce, transform and express constructs containing other variants and s of XTEN; e. g. AE42, AG42, AG288, AM288, AE864, AG864, or other XTEN of Table 4.

Construction of BDD FVIII-/—XTEN_AE864 expression vectors The BDD FVIII fragment with NheI and SfiI flanking the 5’ and 3’ end is generated by ing the pBC0025 construct. This digested fragment is then ligated to a NheI/SfiI digested pSecTag vector (pBCOO48 pSecTag-FVIII-/—XTEN_AE864) encoding the FVIII ed by the XTEN_AE864 sequence. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened by DNA miniprep and the desired constructs are ed by DNA sequencing. The final construct is pBCOO60, which encodes the BDD /—XTEN_AE864 protein under the control of a human CMV promoter. Introduction of the pBCOO6O construct into mammalian cells is ed to express the FVIII protein with a C terminal XTEN fusion (BDD FVIII-/—XTEN_AE864) with procoagulant activity.

Construction of BDD FVIII-/FXI/—XTEN_AE864 expression vectors The BDD FVIII fragment with NheI and SfiI flanking the 5’ and 3’ end is generated by digesting the pBC0025 construct. This digested fragment is then ligated to a NheI/SfiI digested pSecTag vector 47 pSecTag-FVIII-/FXI/—XTEN_AE864) encoding the FVIII ed by the FXI cleavage sequence (/FXI/) and XTEN_AE864. The ligated DNA mixture is used to transform DHSa bacterial cells. ormants are screened by DNA miniprep and the desired constructs are confirmed by DNA sequencing. The final construct is pBCOOS l, which encodes the BDD FVIII-/FXI/— XTEN_AE864 protein under the control of a human CMV promoter. Introduction of the pBCOOSl construct into mammalian cells is expected to express the FVIII protein with a C terminal XTEN fusion (BDD FVIII-/FXI/—XTEN_AE864), which could be subsequently cleaved by FXI, therefore liberating the BDD FVIII n with procoagulant activity.

Construction of BDD FVIII-/FXI/—XTEN expression vectors comprising AE288 or AG288 The fused AE864 XTEN sequence in pBCOO6O is replaced by digesting the XTEN sequences AE288 and AG288 with BsaI and HindIII. A subsequent ligation step using the respective AE288 or AG288 XTEN fragment and BsaI/HindIII digested pBCOOS 1 allows the exchange of the AE288 or AG288 sequences into the BDD FVIII expression vector. The resulting final constructs are pBCOO6l for BDD FVIII-AE288 and pBCOO62 for BDD FVIII-AG288. Introduction of the pBCOO6l construct into mammalian cells is expected to express the FVIII protein with a C-terminal AE288 XTEN fusion (BDD FVIII-/—XTEN_AE288) with gulant activity. Introduction of the pBCOO62 construct into mammalian cells is expected to express the FVIII protein with a C-terminal AG288 XTEN fusion (BDD FVIII-/—XTEN_AG288) with gulant activity.

Construction of BDD FVIII-/FXI/—XTEN sion vectors with alternate XTEN The fused XTEN sequence in pBCOOS l is replaced by digesting DNA encoding other XTEN sequences (e. g. other variants and lengths of XTEN; e.g. AE42, AG42, AG288, AM288) with BsaI and HindIII. A ligation using the XTEN fragment and indIII digested pBCOOS 1 allows the ge of the various XTEN-encoding sequences into the BDD FVIII expression vector, ing the alternate ucts. Introduction of the alternate constructs into mammalian cells is expected to express the FVIII protein with a C-terminal XTEN (BDD FVIII-/FXI/—XTEN) that can be subsequently cleaved by FXI, releasing the FVIII, resulting in gulant FVIII fusion with procoagulant activity.

Example 18: Construction of expression plasmids for FVIII signal peptide-XTEN-/FXI/— BDD FVIII Construction of sion vectors for FVIII signal peptide-XTEN_AE864 The coding sequences for the FVIII signal e is generated by annealing the following two oligos: 5’- CTAGCATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGTG GGTCTCC-3’ (SEQ ID NO: 1679); 5’- ACCTGGAGACCCACTAAAGCAGAATCGCAAAAGGCACAGAAAGAAGCAGGTGGAGAGCTC TATTTGCATG-3’ (SEQ ID NO: 1680). The ed oligos are flanked by the NheI and BsaI restriction enzyme sites on either end, and is ligated to NheI/BsaI digested pCWO645 vector which encodes the FVII-XTEN_AE864. The ligated DNA mixture is used to transform DH5a bacterial cells.

Transformants is screened by DNA miniprep and the desired constructs are confirmed by DNA sequencing. The final construct is designated pBC0029, which encodes the signal peptide- E864 protein under the control of a human CMV promoter. This construct is used as an ediate construct for creating an expression construct with XTEN fused on the N-terminus of the FVIII protein, and can also be used as a master plasmid for creating expression constructs that allow XTEN fusion on the N-terminus of a ed protein.

Construction of signal peptide-XTEN_AE864-/FXI/—BDD FVIII expression vectors An l800bp fragment within the FVIII coding region is ed using primers that introduce NheI-BbsI-/FXI/—AgeI sites on the 5’ and endogenous KpnI restriction enzyme on the 3’ end. The NheI/KpnI digested FVIII fragment is ligated with NheI/KpnI digested 7 vector. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened by DNA miniprep and the desired constructs are confirmed by DNA sequencing. The resulting uct is designated pBC0052, which contains sequences that encode the /FXI/—FVIII protein without the FVIII signal peptide. This uct is used as an ediate construct for creating an expression uct with XTEN fused on the N-terminus of the FVIII protein.

The pBC0052 vector is digested with hoI enzymes, and is used to ligate with Bbsi/XhoI digested pBC0029. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened by DNA ep and the desired constructs are confirmed by DNA sequencing. The final construct is ated pBCOOS3, which encodes the signal peptide-XTEN_AE864-/FXI/—BDD FVIII protein under the control of a human CMV promoter. Introduction of the pBCOOS3 construct into mammalian cells is expected to express the FVIII protein with an N-terminal XTEN fiJsion (signal peptide-XTEN_AE864-/FXI/—BDD FVIII), which could be subsequently cleaved by FXI, therefore liberating the BDD FVIII protein.

Construction of signal peptide-XTEN -/FXI/—BDD FVIII sion vectors The fused XTEN sequence in 3 can be replaced by digesting other XTEN fragments (e. g. AM, AF, AG) with Bsal and Bbsl. A ligation using the XTEN fragment and BsaI/Bbsl digested pBC0053 allows the exchange of various XTEN pieces (e. g. AM, AF, AG) into the BDD FVIII expression vector. Various XTEN s can increase the half lives of these proteins differently, allowing modification of the properties (e. g. efficacy, potency) of these proteins. Introduction of any of these fusion constructs into mammalian cells is expected to express the FVIII protein with an N-terminal XTEN fusion (signal peptide-XTEN-/FXl/-BDD FVHI), in which the fused XTEN peptide can be subsequently cleaved by FXI, generating the BDD FVIII protein.

Example 19: Construction of BDD FVIII with interdomain XTEN insertion Construction of BDD FVIII expression vectors with an XTEN insertion at the A2-B domain boundaries The pBC0027 construct DD FVIll-STOP) is a cloning vector designed to contain the BDD FVIH protein coding sequences, but not a promoter oned to initiate the expression of BDD FVHI. This construct is used for manipulation of the coding sequences of BDD FVIII as the vector backbone contains very few ction enzyme sites. therefore allowing easy cloning strategies. The BDD FVIH proteins contain 1457 amino acids at a total molecular weight of 167539.66. There are 6 domains within the wild-type FVIII protein, the Al, A2, B, A3, C1 and C2 s. In the BDD FVIII protein, most of the B domain has been deleted as it is believed to be an unstructured domain and the removal of the domain does not alter critical functions of this n. However, the B domain boundaries seem to be excellent positions for creating XTEN fusions to allow extension of the protein half lives.

Within the 7 construct, there is a unique Hindlll restriction enzyme site at the boundary of A2-B junction. The XTEN (e.g., ces of Tables 4, or 13-17) are amplified using primers that introduce a Hindlll and FXI cleavage site on either end of the XTEN coding sequence. The fused XTEN sequence can be altered by amplifying various XTEN fragments. Various XTEN fusions can increase the half lives of these proteins ently, allowing modification of the properties (e.g. efficacy, potency) of these ns. The Hindlll-/FXl/-XTEN-/FXl/-Hindlll fragment is digested with Hindlll and ligated with Hindlll digested pBC0027. The ligated DNA mixture is used to transform DH5a bacterial cells.

Transformants are screened by DNA miniprep and the desired constructs are confirmed by DNA sequencing. The final construct is designated pBC0054, which encodes the BDD FVIII protein with an interdomain XTEN fusion (FVlll(Al FXl/-XTEN-/FXl/-FVHI(Cl-C2)) but not a promoter to initiate gene expression.

The pBC0054 construct is digested with Nhel /Sall, and ligated with Nhel/Sall digested 9-HS vector (Millipore). The CETlOl9-HS vector ns a human CMV promoter and a UCOE sequence to facilitate gene expression. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened by DNA miniprep and the desired constructs are ed by DNA sequencing. The final construct is ated pBCOOSS l9-HS- FVIII(Al-A2)-/FXl/- XTEN-/FXl/-FVHI(Cl-C2)), which encodes the BDD FVIII protein with an interdomain (inter-A2/B domain) XTEN fusion (A1-A2)-/FXI/—XTEN-/FXI/—FVIII(C1-C2)) under the control of a human CMV promoter. Introduction of the pBC0055 construct into mammalian cells is expected to express the BDD FVIII protein with an omain XTEN fusion (A1 -A2)-/FXI/-XTEN-/FXI/-FVIII(C1- C2)), which could be subsequently cleaved by FXI, therefore liberating the BDD FVIII n.

Construction of BDD FVIII expression vectors with an XTEN insertion at the A1-A2 domain boundaries The pBC0027 construct is designed as a template for two PCR reactions using the following four primers: (Reaction I) 5’-ATGATGGCATGGAAGCCTAT-3’ (SEQ ID NO: 1681); 5’- ATCCCTCACCTTCGCCAGAACCTTCAGAACCCTCACCTTCAGAACCTTCACCAGAACCTTCA CCATCTTCCGCTTCTTCATTATTTTTCAT-3’ (SEQ ID NO: 1682).

(Reaction II) 5’- TTCTGGCGAAGGTGAGGGATCTGAAGGCGGTTCTGAAGGTGAAGGTGGCTCTGAGGGTTCC GAATATGATGATGATCTTACTGATTCTGAAAT-3’ (SEQ ID NO: 1683); 5 ’- TATTCTCTGTGAGGTACCAGC-3’ (SEQ ID NO: 1684).

The PCR products generated are 150bps and 800 bps respectively. The 800 bp product is used as the template for the next round of PCR on with the 150bp product as one primer and 5 ’- TATTCTCTGTGAGGTACCAGC-3’ (SEQ ID NO: 1685) as the other. The product for the second round of PCR is 930 bps and is digested with PshAI and ACC651 restriction enzymes. This PshAI/Acc651 flanked DNA nt is ligated with PshAI/Acc651 ed pBC0027. The d DNA mixture is used to transform DH5a ial cells. Transformants is screened by DNA miniprep and the desired constructs are confirmed by DNA sequencing. The final construct is designated pBC0058 (pMK-BDD FVIII-D345-XTEN_Y36), which encodes the BDD FVIII protein with an interdomain (inter-A1/A2 domain) XTEN fusion after the D345 residue.

] The pBC0058 construct is digested with NheI /SalI, and ligated with NheI/SalI digested CET1019-HS vector (Millipore). The CETlOl9-HS vector contains a human CMV promoter and a UCOE sequence to facilitate gene expression. The ligated DNA mixture is used to transform DH5a bacterial cells. Transformants are screened by DNA miniprep and the desired constructs are confirmed by DNA sequencing. The final construct is designated pBC0059 19-HS-BDD FVIII D345- XTEN_Y36), which encodes the BDD FVIII protein with an interdomain (inter-A1/A2 ) XTEN fusion after the D345 residue under the control of a human CMV promoter. Introduction of the pBC0059 construct into mammalian cells is expected to express the BDD FVIII protein with an interdomain XTEN fusion (BDD FVIII D345-XTEN_Y36).

Example 20: Construction of FVIII with omain XTEN insertion Construction of BDD FVIII expression vectors with an XTEN insertion after P598 n the A2 domain) The coding sequences for XTEN_Y36 is amplified using PCR techniques with the following primers: 5 ’- GAAGCTGGTACCTCACAGAGAATATACAACGCTTTCTCCCCAATCCAGGTGAAGGTTCTGGT 3’ (SEQ ID NO: 1686) ’-AACTCTGGATCCTCAAGCTGCACTCCAGCTTCGGAACCCTCAGAGCC-3’ (SEQ ID NO: 1 687).

The 184 bp PCR product is flanked by the KpnI and BamHI restriction enzyme sites on either end, and is ligated to KpnII/BamHI digested pBC0027 vector which encodes the BDD FVIII gene. The ligated DNA e is used to transform DH5a bacterial cells. Transformants are screened by DNA miniprep and the desired constructs are confirmed by DNA sequencing. The final construct is designated 6, which contains DNA sequences encoding the FVIII protein with an XTEN_Y36 fusion after the P598 residue. This cloning strategy is used to introduce various forms ofXTEN into the BDD FVIII protein by altering the template for the PCR on and changing the primers accordingly.

The pBC0056 construct is digested with NheI /SalI, and ligated with NheI/SalI digested CET1019-HS vector (Millipore). The CET1019-HS vector contains a human CMV promoter and a UCOE sequence to tate gene expression. The d DNA mixture is used to transform DH5a bacterial cells. ormants are screened by DNA miniprep and the desired constructs are confirmed by DNA sequencing. The final uct is designated 7 (CET1019-HS- FVIII P598- XTEN_Y32), which encodes the BDD FVIII protein with an intradomain (within A2 domain) XTEN fusion under the control of a human CMV promoter. Introduction of the pBC0057 construct into ian cells is expected to express the BDD FVIII protein with an intradomain XTEN fusion (FVIII P598-XTEN_Y32).

Construction of BDD FVIII expression vectors with other intradomain XTEN insertions To introduce various XTEN segments into other intradomain sites within BDD FVIII (e. g., the XTEN of Tables 4, or 13-17), primers are ed that amplify XTEN with an overhang that can anneal with BDD FVIII. The coding sequence of FVIII (pMK-BDD FVIII) is designed with various unique restriction enzyme sites to allow these ic insertions. The unique restriction enzymes are listed below with their cut site: NheI 376, SacI 391, AfiII 700, SpeI 966, PshAI 1417, Acc6512192, KpnI 2192, BamHI 2250, HindIII 2658, PfoI 2960, PflMI 3413, ApaI 3893, Bsp1201 3893, SwaI 4265, CHI 4626, XbaI 4644, BstBI 4673, SalI4756, and XhoI 4762. The NheI and SalI sites on either end of the coding sequence are used to insert the DNA fragment into a human CMV promoter driven , the CET1019-HS (Millipore) for expression in mammalian cells. These constructs s the BDD FVIII protein with an XTEN fusion with sequences listed in Table 21.

Example 21: Construction of FVIII with XTEN insertions CFXTEN with two XTEN: To obtain CFXTEN with two XTEN insertions in various regions (from N-termini to C- termini: A1 -R1, A1-R2, A2-R1, A2-R2, B domain, a3, A3-R1, A3-R2, C-termini), contructs that expressed s with single-XTEN insertions that retained FVIII activity were utilized. The coding ce of FVIII (pBC0114 pcDNA4-FVIII_4-X10-Myc-SPATG-His extra RE) ('SPATG' disclosed as SEQ ID NO: 1662) was designed with various unique restriction enzyme sites to allow these specific combinations. The unique ction enzymes are listed in Table 18 below with their relative sites between different regions: BsiWI (between N-termini and A1 -R1), AflII (between A1 -R1 and A1-R2), Nhel (between A1-R2 and A2-R1), KpnI (between A2-R1 and A2-R2), BamHl (between A2-R2 and B domain), Clal (between a3 and A3-R1), PflMI (between A3 -R1 and A3-R2), Xbal (between A3-R2 and ini), Agel (between FVIII C-termini and stop codon). Building blocks and ction s for cloning the libraries were chosen, as listed in the table below. The chosen components in each region were mixed at molar ratio of 1 :1, and two sets of DNA mixtures were digested with unique restriction enzymes. DNA fragments were separated with 1% agarose gel and purified by Qiagen gel extraction kit.

DNA with XTEN insertion in the first desired region was regarded as the insert (the smaller DNA fragment in agarose gel), while DNA with XTEN insertion in the second desired region was regarded as vector (the bigger DNA fragment in agarose gel). The insert and vector were d in order to reconstitute the plasmid. The ligated DNA mixture was used to transform DH50L E. coli competent host cells. Transformants were screened by rolling circle amplification (RCA) and Sanger sequencing to cover approximately 3-4 times the potential library size. Unique clones were identified and minipreped. Two distinct restriction ions were then used to further confirm the integrity ofXTEN in each region. The amino acid and the encoding DNA sequences for the ing CFXTEN fusion proteins are listed in Table 21.

CFXTEN with one or two XTEN insertions within the B/a3 domain and C terminus: The B/a3 domain and C-terminus of FVIII are unstructured regions that tolerated XTEN insertions well. The B/a3 domain further mediated ctions with other cofactors, including the von Willibrand Factor. To investigate the optimal XTEN insertions at the B/a3 domain, select deletions and mutations of the region were made via PCR-based mutagenesis methods. Select PCR reactions and the vectors were digested with unique restriction enzymes as listed in Table 18. DNA fragments were separated with 1% e gel and purified by Qiagen gel extraction kit. DNA with XTEN insertion in the first desired region was regarded as the insert (the smaller DNA fragment in agarose gel), while DNA with XTEN insertion in the second d region was regarded as vector (the bigger DNA fragment in agarose gel). The insert and vector were ligated in order to titute the plasmid. The ligated DNA e was used to transform DH50t E. coli competent host cells. Transformants were screened by colony PCR and Sanger sequencing to cover approximately 8X the potential library size. Unique clones were fied and minipreped. One three-enzyme restriction digestion was then used to further confirm the integrity ofXTEN in each region. The amino acid and the encoding DNA sequences for the resulting CFXTEN fusion proteins are listed in Table 21.

Table 18. Clonin desi n for FVIII libraries with two XTEN ions pSD0005, pSD0006, pSD0007, pSD0008, LSD0001 pSD0017, pSD0018, pBC0136, pBC0137 pSD0013 (C-termini) NheI -- ClaI (B-domain) , pSD0006, pSD0007, pSD0008, LSD0002 pSD0017, 8, pBC013 6, pBC013 7 pSD0014 (C-termini) NheI -- ClaI (B-domain) Library . Vector components Restriction Insert components (XTEN region) ID QETEN re ion; en mes pSD0005, pSD0006, pSD0007, pSD0008, LSD0003 pSD0017,pSD0018,pBC0136,pBC0137 pSD0019 mini) NheI--C1aI (B-domain) pSD0005, pSD0006, 7, pSD0008, LSD0004 pSD0017,pSD0018,pBC0136,pBC0137 pSD0020 (C-termini) C1aI (B-domain) pSD0045,pSD0046,pSD0048,pSD0049, LSD0005 1(A2-R1) Bs1WI AflII. __ pSD0050,pSD0051,pSD0052 (A1-R1) pSD0045,pSD0046,pSD0048,pSD0049, LSD0006 pSD0002(A2-R1) Bs1WI AflII. __ pSD0050,pSD0051,pSD0052 (A1-R1) pSD0045,pSD0046,pSD0048,pSD0049, LSD0007 pSD0003 (A2 R1) . _ Bs1WI AflII pSD0050,pSD0051,pSD0052 (A1-R1) pSD0045,pSD0046,pSD0048,pSD0049, LSD0008 pSD0004(A2 R1) Bs1WI AflII. __ pSD0050,pSD0051,pSD0052 (A1-R1) pSD0045, pSD0046, pSD0049, pSD0050, BsiWI--AflII LSD0037 pSD0032 (AZ-R1) pSD0051, pSD0052 (A1-R1) pSD0045, pSD0046, pSD0049, LSD0038 9 (a3) BamHI__ClaI pSD0050,pSD0051,pSD0052 (A1-R1) LSD0039 pSD0039 (a3) pSD0032, pSD0001, pSD0003 ) BamHI--C1aI pSD0045, pSD0046, pSD0049, LSD0040 0,pSD0010,pSD0041 (A3 R1) __ _ ClaI XbaI pSD0050,pSD0051,pSD0052 (A1-R1) LSD0041 pSD0040, pSD0010, pSD0041 ) pSD0032, pSD0001, pSD0003 (AZ-R1) C1aI--XbaI pSD0062, pSD0063, pSD0043, pSD0044 pSD0045, pSD0046, pSD0049, LSD0042 CM Xbal__ (A3-R2) pSD0050,pSD0051,pSD0052(Al-R1) LSD0043 F213???” pSD0063’ pSD0043’ pSD0044 pSD0032, pSD0001, pSD0003 (A2-R1) C1aI--XbaI LSD0044 F213???” 3’ pSD0043’ 4 pSD0040, pSD0010, pSD0041 (A3-R1) PflMI+XbaI LSD0045 pSD0039 (a3) pSD0040, 0, pSD0041 (A3-R1) BamHI--C1aI pSD0062, pSD0063, pSD0043, LSD0046 pSD0039 (a3) BamHI__ClaI pSD0044(A3_R2) 7 pSD0046(A1-R1) pSD0001,pSD0003 (AZ-R1) BsiWI—-AflII LSD0048 pSD0045,pSD0051(A1-R1) 3 (AZ-R1) BsiWI--AflII pNL0006 PCR product “13990300603 Domam andC PflMI termim) pNL0007 PCR product ”13990300603 Domam andC C1aI--PflMI termim) pNL0008 PCR product ”139903009 (B Domam andC PflMI termim) pNL0009 PCR product pSD0039 (a3 Domain) BamHI+AscI pNL0010 LSD0003.006 (B Domain and C termini) 9 (a3 Domain) XbaI+AgeI Example 22: Construction of BDD FVIII expression vectors with 3-5 XTEN insertions at sites 18/26 403 745/1656 1720 1900 or 2332 FVHI-fusion ucts With XTEN insertions at sites 18/26, 403, 745/1656, 1720, 1900 or 2332 were chosen to recombine and generate constructs with 3, 4, 5 or 6 XTEN insertions.

] Construction of BDD FVIII expression vectors with 3-5 XTEN insertions at sites 26, 403, 1656, 1720, or 1900 The chosen constructs with single XTEN at the desired sites were: O, pSDOOOl, pSDOO39, pSDOOlO, and pSDOO62. Constructs with double XTENs at the d sites included LSD0005.002, LSDOO38.001, LSDOO40.002, LSDOO42.013, LSDOO39.010, LSDOO41.008, LSDOO43.008, LSDOO45.002, 6.002, and LSDOO44.002. ng blocks and restriction enzymes for cloning the constructs were chosen, as listed in Table 19 below. Chosen components were digested with unique ction enzymes. DNA of inserts and vectors were separated with 1% agarose gel and purified by Qiagen gel extraction kit. The insert and vector were ligated, and then transformed into DH50t E. coli competent host cells. Four colonies for each construct were analyzed by RCA and DNA sequencing. Clones with desired XTEN insertions were minipreped. Restriction digestions were then used to further m the integrity ofXTEN in each region. The amino acid and the encoding DNA sequences for the resulting CFXTEN fusion proteins are listed in Table 21. The resulting constructs were numbered pSDOO77 to pSDOO92.

Construction of BDD FVIII expression vectors with 4-6 XTEN insertions at sites 18, 403, 1656, 1720, 1900 or 2332 Constructs pSDOO77 to pSDOO92 served as building blocks to generate 4- to 6-XTEN constructs with ions at 18, 403, 1656, 1720, 1900 and 2332. Building block ucts and restriction enzymes for cloning the constructs were chosen, as listed in Table 19 below. Chosen components were digested with unique restriction enzymes. DNA of inserts and vectors were separated with 1% agarose gel and purified by Qiagen gel extraction kit. The insert and vector were ligated, and then transformed into DH50t E. coli competent host cells. Eight colonies for each construct were analyzed by colony PCR and DNA sequencing. Clones with d XTEN insertions were minipreped.

Restriction ions were then used to further confirm the integrity ofXTEN in each . The amino acid and the encoding DNA sequences for the resulting CFXTEN fusion proteins are listed in Table 21.

The resulting constructs were numbered pBCO247 to pBCO257, pNLOO22, 23, 24, 25, and 30 uction of BDD FVIII expression vectors with 4-6 XTEN insertions at sites 18, 403, 745, 1720, 1900 or 2332 Constructs pBCO247 to pBCO252, pBCO255, pNLOO22 to pNLOO25 served as building blocks to generate 4- to 6-XTEN constructs with insertions at 18, 403, 745, 1720, 1900 and 2332. Building block constructs and ction enzymes for cloning the constructs were chosen, as listed in Table 19 below. Chosen components were digested with unique restriction enzymes. DNA of inserts and vectors were separated with 1% agarose gel and purified by Qiagen gel extraction kit. The insert and vector were d, and then transformed into DH50t E. coli ent host cells. Eight colonies for each construct were analyzed by colony PCR and DNA sequencing. Clones with desired XTEN insertions were minipreped. Restriction digestions were then used to further m the integrity ofXTEN in each region. The amino acid and the ng DNA sequences for the resulting CFXTEN fusion proteins are listed in Table 21. The resulting constructs were numbered pBCO25 8 to pBCO268.

Table 19: Cloning design for FVIII libraries with 3-5 XTEN insertions at sites 26, 403, 1656, 1720, or 1900 Cfiljgza Insert components (XTEN region) Vi§¥E§T§22:31ts RSStrigign pSDOO77 pSDOOSO (Al-R1) LSDOO39.010 (AZ-R1, a3) BsiWI+AflII pSDOO78 O (A3-R1) LSD0005.002 (Al-R1, A2-R1) ClaI--Xba1 pSDOO79 pSDOO62 ) LSD0005.002 (Al-R1, A2-R1) ClaI--Xba1 pSDOO8O pSDOOSO (Al-R1) LSDOO45.002 (a3, A3-R1) BsiWI—-Afl11 pSDOO81 O (Al-R1) LSDOO46.002 (a3, A3-R2) -AflII pSDOO82 pSDOOSO (Al-R1) LSDOO44.002 (A3-R1, A3-R2) BsiWI--AflII pSDOO83 pSDOOlO (A3-R1) LSDOO39.010 (AZ-R1, a3) ClaI--Xba1 pSDOO84 pSDOO62 (A3-R2) LSDOO39.010 (AZ-R1, a3) ClaI--Xba1 pSDOO85 pSDOO62 (A3-R2) LSDOO41.008 (AZ-R1, A3-R1) PflMI--Xba1 pSDOO86 pSDOO62 ) LSDOO45.002 (a3, A3-R1) Xba1 pSDOO87 LSDOO39.010 (AZ-R1, a3) LSDOO40.002 (Al-R1, A3-R1) NheI--Cla1 pSDOO88 LSDOO39.010 , a3) 2.013 (Al-R1, A3-R2) NheI--C1a1 pSDOO89 LSDOO44.002 (A3-R1, A3-R2) LSD0005.002 , A2-R1) ClaI--Xba1 pSDOO9O LSDOO44.002 (A3-R1, A3-R2) LSDOO38.001 (Al-R1, a3) ClaI--Xba1 pSDOO91 LSDOO44.002 (A3-R1, A3-R2) LSDOO39.010 (AZ-R1, a3) ClaI--Xba1 pSDOO92 LSDOO44.002 (A3-R1, A3-R2) pSDOO77 (Al-R1, A2-R1, a3) ClaI--Xba1 pBC0247 pSDOO77 LSD0050.003 \heI--BstBI pBC0248 pSDOO78 LSD0050.003 \heI--BstBI pBC0249 pSDOO79 LSD0050.003 \heI--BstBI pBC0250 pSDOO8O LSD0050.003 \heI--BstBI pBC0251 pSDOO82 0.003 BstBI pBC0252 pSDOO8O LSD0050.003 \heI--BstBI pBC0253 7 LSD0050.003 BstBI pBC0254 pSDOO88 LSD0050.003 \heI--BstBI pBC0255 pSDOO89 LSD0050.003 \heI--BstBI pBC0256 O LSD0050.003 \heI--BstBI pBC0257 pSDOO92 LSD0050.003 \heI--BstBI pNL0022 LSDOOO3.009 pSDOO83 Agel pNL0023 LSDOOO3.009 pSDOO84 XbaI--Agel pNL0024 LSDOOO3.009 pSDOO85 XbaI--Agel pNL0025 LSDOOO3.009 6 XbaI--Agel pNLOO3O LSDOOO3.009 pSDOO91 XbaI--Agel pBC0258 LSDOOO3.006 pBC0247 BamHI--Cla1 pBC0259 LSDOOO3.006 pBC0248 BamHI--Cla1 nggza Insert ents (XTEN region) veg§¥Ec§?§:gfinlts sttrigign pBC026O LSDOOO3.006 pBC0249 -Cla1 Example 23: uction of CFXTEN expression vectors with three or four XTENs: the first XTEN in the B domain, the second XTEN at the C-terminus, and the third or fourth XTEN insertion within the A1 or A2 or A3 domains ies of CFXTEN fusion proteins were constructed with three XTEN insertions by ing coagulation-active clones with XTEN insertions in the Al, A2, or A3 domains and clones with XTEN inserted within the B domain and at the C-terminus. Additional libraries were constructed with a fourth XTEN added in the Al, A2, or A3 domains to select members of the 3 XTEN libraries. The design of the cloning scheme is summarized in the table below. DNA was prepared for the inserts and vectors by restriction enzyme digestion and agarose gel purification. After ligating the inserts with the corresponding vectors, the ligated DNA mixture was used to transform DH50t competent E. coli host cells. Transformants were screened by RCA and sequencing to cover imately 3-4 times the potential library size. Unique clones were identified and mini-prepped. Three distinct restriction digestions were then used to further confirm the integrity of each XTEN. The amino acid and the ng DNA ces for the ing CFXTEN fusion proteins are listed in Table 21.

Table 20: Clonin desi n for FVIII ies with 3 XTEN insertions at sites B domain C-termini and A1/A2/A3 domain Library Insert components (XTEN Vector 0011119011911“ Restriction ID region) (XTEN region) enzymes LSD0003.006 (B domain and C- pSD0045, pSD0046, pSD0049, pSD0050, “130049 BamHI Ag“__ pSD0051, pSD0052 (Al-R1) LSD0003.009 (B domain and C- pSD0045, pSD0046, pSD0049, pSD0050, LSDOOSO BamHI Ag“__ pSD0051, pSD0052 (Al-R1) LSD0051 igfig3‘006 (B domam and C' pSD0032, pSD0001, pSD0003 (AZ-R1) BamHI--Agel pSD0062, pSD0063, pSD0043, LSD0056 LSD0003.009 (B domam and lm). . . C1aI+XbaI pSD0044 (A3-R2) pBC0274 pSD0009 (A3_R1) LSD0003.006 (B domain and C-teimini) C1aI--XbaI pBC0275 4 (A3_R2) LSD0003.006 (B domain and ini) C1aI--XbaI pBC0276 0 (A1_R1) LSD0003.006 (B domain and C-teimini) pBC0277 pBC0281 (A2_R1) LSD0003.006 (B domain and C-teimini) pBC0278 pBC0282 (A3_R1) LSD0003.006 (B domain and C-teimini) C1aI--XbaI .BC0279 .BC0283 A3 R2 LSD0003.006 B domain and C-teimini C1aI--XbaI L09_01 pBC0284 (CT) 39135013555:: :fgffgé89’ 290’ 291’ 292’ XbaI--AgeI L09_01 pSD0014 (CT) 39135013555:: :fgffgé89’ 290’ 291’ 292’ AgeI pBC0285, 286, 287, 288, 289, 290, 291, 292, L09_01 pSD0020 (CT) XbaI AgeI__ 293 (B domain and A3_R2) Table 21: DNA and Amino Acid Se uences of FVIII-XTEN Constructs Construct Amino ”Cid sequence disclosed as DNA sequence disclosed as Name SEQ ID N0= SEQ ID NO: pBC01 14 595 596 pBC0126 597 598 pBC0127 599 600 pBC0165 601 602 pBC0183 603 604 pBC0184 605 606 pBC0166 607 608 pBC0185 609 610 pBC0167 61 1 612 pBCO 128 613 614 pBC0168 615 616 pBCO 129 617 618 pBC0169 619 620 pBC013 0 621 622 pBC013 1 623 624 pBC0132 625 626 pBC0170 627 628 pBC013 3 629 63 0 pBC0171 63 1 632 4 63 3 634 pBC0172 63 5 63 6 pBC013 5 63 7 63 8 pBC0149 639 640 pBC013 6 641 642 pBC013 7 643 644 pBC013 8 645 646 pBC0139 647 648 0 649 650 pBC0173 651 652 pBC0174 653 654 pBC0175 655 656 pBC0176 657 65 8 pBC0177 659 660 pBC0178 661 662 pBCO 141 663 664 pBC0179 665 666 pBC0180 667 668 pBC0142 669 670 pBC0143 671 672 pBC0181 673 674 pBC0182 675 676 pBC0144 677 678 pBC0145 679 680 pBC0146 681 682 pSD0001 683 684 pSD0002 685 686 pSD0003 687 688 pSD0004 689 690 pSD0005 691 692 pSD0006 693 694 7 695 696 pSD0008 697 698 pSD0009 699 700 pSD0010 701 702 pSD0011 703 704 2 705 706 pSD0013 707 708 4 709 710 pSD0017 711 712 pSD0018 713 714 pSD0019 715 716 pSD0020 717 718 pSD0015 719 720 pSD0016 721 722 pSD0021 723 724 pSD0022 725 726 pSD0023 727 728 pSD0024 729 730 pSD0025 731 732 pSD0026 733 734 pSD0027 735 736 pSD0028 737 738 pSD0029 739 740 pSD0030 741 742 pSD0031 743 744 pSD0032 745 746 pSD0033 747 748 4 749 750 pSD0035 751 752 pSD0036 753 754 pSD0037 755 756 pSD0038 757 758 pSD0039 759 760 pSD0040 761 762 pSD0041 763 764 pSD0042 765 766 pSD0043 767 768 pSD0044 769 770 2 771 772 pSD0063 773 774 pSD0045 775 776 pSD0046 777 778 pSD0047 779 780 pSD0048 781 782 pSD0049 783 784 pSD0050 785 786 pSD0051 787 788 pSD0052 789 790 pSD0053 791 792 pSD0054 793 794 795 796 pSD0056 797 798 pSD0057 799 800 pSD0058 801 802 pSD0059 803 804 pSD0060 805 806 pSD0061 807 808 LSD0001.002 809 810 1.005 811 812 1.006 813 814 LSD0001.011 815 816 1.012 817 818 LSD0001.013 819 820 LSD0001.016 821 822 LSD0001.021 823 824 LSD0002.001 825 826 LSD0002.002 827 828 LSD0002.014 829 830 LSD0003.004 831 832 LSD0003.006 833 834 LSD0003.009 835 836 LSD0003.014 837 838 LSD0004.010 839 840 LSD0004.011 841 842 LSD0004.014 843 844 LSD0004.016 845 846 LSD0004.022 847 848 LSD0003.016 849 850 LSD0005.002 851 852 LSD0005.004 853 854 LSD0005.005 855 856 LSD0005.011 857 858 LSD0005.018 859 860 LSD0006.002 861 862 LSD0006.005 863 864 LSD0006.007 865 866 LSD0006.011 867 868 LSD0007.002 869 870 LSD0007.004 871 872 LSD0007.013 873 874 LSD0008.001 875 876 LSD0008.002 877 878 LSD0008.006 879 880 LSD0008.009 881 882 LSD0008.017 883 884 LSD0002.025 885 886 LSD0002.013 887 888 LSD0003.025 889 890 LSD0004.025 891 892 LSD0003.005 893 894 LSD0007.008 895 896 LSD0044.002 897 898 LSD0044.005 899 900 LSD0044.039 901 902 LSD0044.022 903 904 LSD0044.003 905 906 LSD0044.001 907 908 LSD003 8.001 909 910 LSD003 8.003 911 912 LSD003 8.008 913 914 LSD003 8.012 915 916 LSD003 8.013 917 918 LSD003 8.015 919 920 LSD0039.001 921 922 9.003 923 924 LSD0039.010 925 926 .001 927 928 LSD0045.002 929 930 LSD0042.014 931 932 LSD0042.023 933 934 LSD0042.006 935 936 LSD0042.013 937 938 LSD0042.001 939 940 LSD0042.039 941 942 LSD0042.047 943 944 LSD0042.003 945 946 LSD0042.004 947 948 LSD0042.008 949 950 LSD0042.038 951 952 LSD0042.082 953 954 LSD0042.040 955 956 LSD0037.002 957 958 LSD0037.009 959 960 LSD0037.011 961 962 LSD0047.002 963 964 LSD0047.005 965 966 LSD0048.007 967 968 LSD0046.001 969 970 LSD0046.002 971 972 LSD0046.003 973 974 LSD0040.011 975 976 LSD0040.042 977 978 0.002 979 980 LSD0040.008 981 982 LSD0040.021 983 984 LSD0040.037 985 986 LSD0040.046 987 988 0.003 989 990 LSD0040.006 991 992 LSD0040.007 993 994 LSD0040.010 995 996 LSD0040.039 997 998 LSD0040.052 999 1000 LSD0041.001 1001 1002 LSD0041.004 1003 1004 LSD0041.006 1005 1006 LSD0041.008 1007 1008 LSD0041.010 1009 1010 LSD0041.014 1011 1012 LSD0041.016 1013 1014 LSD0041.035 1015 1016 LSD0043.001 1017 1018 LSD0043.002 1019 1020 LSD0043.005 1021 1022 3.006 1023 1024 LSD0043.007 1025 1026 LSD0043.008 1027 1028 LSD0043.015 1029 1030 LSD0043.029 1031 1032 LSD0043.043 1033 1034 pSD0077 1035 1036 pSD0078 1037 1038 pSD0079 1039 1040 pSD0080 1041 1042 pSD0081 1043 1044 pSD0082 1045 1046 pSD0083 1047 1048 pSD0084 1049 1050 pSD0085 1051 1052 pSD0086 1053 1054 pSD0087 1055 1056 pSD0088 1057 1058 pSD0089 1059 1060 pSD0090 1061 1062 pSD0091 1063 1064 pSD0092 1065 1066 LSD0049.002 1067 1068 LSD0049.008 1069 1070 LSD0049.011 1071 1072 LSD0049.012 1073 1074 LSD0049.020 1075 1076 9.021 1077 1078 LSD0050.002 1079 1080 0.003 1081 1082 LSD0050.007 1083 1084 LSD0050.010 1085 1086 LSD0050.012 1087 1088 LSD0050.014 1089 1090 LSD0051.002 1091 1092 1.003 1093 1094 2.001 1095 1096 LSD0052.003 1097 1098 LSD0053.021 1099 1100 LSD0053.022 1101 1102 53.024 1103 1104 LSD0054021 1105 1106 LSD0054025 1107 1108 LSD0054026 1109 1110 LSI)0055.021 1111 1112 LSD0055022 1113 1114 LSD0055026 1115 1116 LSD0056021 1117 1118 6024 1119 1120 LSD0056025 1121 1122 *0\10001 1123 1124 *01’ L0002 1125 1126 ’U/333/ L0003 1127 1128 L0004 1129 1130 L0005 1131 1132 L0006 1133 1134 "0’5 L0007 1135 1136 33/L0008 1137 1138 L0009 1139 1140 mm 1141 1142 chmz44 1143 1144 chmz45 1145 1146 chmz46 1147 1148 chmz47 1149 1150 pBC0248 1151 1152 chmz49 1153 1154 pBC0250 1155 1156 pBC0251 1157 1158 chmzsz 1159 1160 pBC0253 1161 1162 pBCD254 1163 1164 pBC0255 1165 1166 pBC0256 1167 1168 pBC0257 1169 1170 ch0259 1171 1172 0 1173 1174 chmzéz 1175 1176 pBC0263 1177 1178 pBCD264 1179 1180 pBC0266 1181 1182 pBC0267 1183 1184 pBC0268 1185 1186 pN10016 1187 1188 pN10017 1189 1190 pN10018 1191 1192 leoozz 1193 1194 pN10023 1195 1196 pN10024 1197 1198 leoozs 1199 1200 leooso 1201 1202 LSD0057001 1203 1204 LSD0057004 1205 1206 LSD0057005 1207 1208 LSD0057010 1209 1210 LSD0058003 1211 1212 LSD0058005 1213 1214 LSD0058006 1215 1216 LSD0059.002 1217 1218 LSD0059.003 1219 1220 LSD0059.005 1221 1222 LSD0059.006 1223 1224 LSD0060001 1225 1226 LSD0060003 1227 1228 LSD0060004 1229 1230 LSD006L002 1231 1232 LSD006L007 1233 1234 LSD006L008 1235 1236 LSD006L012 1237 1238 LSD0062001 1239 1240 LSD0062002 1241 1242 LSD0062006 1243 1244 LSD0062007 1245 1246 LSD0063001 1247 1248 LSD0063003 1249 1250 LSD0063011 1251 1252 LSD0064017 1253 1254 4018 1255 1256 LSD0064020 1257 1258 LSD0064021 1259 1260 LSD0065001 1261 1262 LSD0065007 1263 1264 5014 1265 1266 LSD0066001 1267 1268 LSD0066002 1269 1270 LSD0066009 1271 1272 LSD0066011 1273 1274 LSD0067004 1275 1276 7005 1277 1278 7006 1279 1280 LSD0067008 1281 1282 LSD0068001 1283 1284 LSD0068002 1285 1286 LSD0068005 1287 1288 LSD0068010 1289 1290 LSD0069004 1291 1292 LSD0069008 1293 1294 LSD0070.003 1295 1296 LSD0070.004 1297 1298 LSD0070.005 1299 1300 LSD007L001 1301 1302 LSD007L002 1303 1304 L008 1305 1306 LSD0072.001 1307 1308 LSD0072.002 1309 1310 LSD0072.003 1311 1312 LSI)0073.002 1313 1314 LSI)0073.004 1315 1316 LSD0073.006 1317 1318 LSD0074.007 1319 1320 LSD0074.010 1321 1322 LSD0074.01 1 1323 1324 LSD0075.003 1325 1326 LSD0075.004 1327 1328 LSD0075.007 1329 1330 LSD0076.002 1331 1332 LSD0076.003 1333 1334 pSD0093 1335 1336 pSD0094 1337 1338 pSD0095 1339 1340 pSD0096 1341 1342 pSD0097 1343 1344 pSD0098 1345 1346 pSD0099 1347 1348 pSD0100 1349 1350 pSD0101 1351 1352 pSD0102 1353 1354 pSD0103 1355 1356 pSD0104 1357 1358 pcs0001 1359 1360 pcs0002 1361 1362 pcs0003 1363 1364 pcs0004 1365 1366 pcs0005 1367 1368 pCSOOO6 1369 1370 pBC0269 1371 1372 pBC0270 1373 1374 pBC0271 1375 1376 pBC0272 1377 1378 pBC0273 1379 1380 4 1381 1382 pBC0275 1383 1384 pBC0276 1385 1386 pBC0277 1387 1388 pBC0278 1389 1390 pBC0279 1391 1392 pBC0280 1393 1394 pBC0281 1395 1396 2 1397 1398 pBC0283 1399 1400 4 1401 1402 pBC0285 1403 1404 pBC0286 1405 1406 pBC0287 1407 1408 pBC0288 1409 1410 pBC0289 1411 1412 0 1413 1414 pBC0291 1415 1416 2 1417 1418 pBC0293 1419 1420 pBC0294 1421 1422 pBC0295 1423 1424 pBC0296 1425 1426 pBC0297 1427 1428 8 1429 1430 pBC0299 1431 1432 pBC0300 1433 1434 pBC0301 1435 1436 pBC0302 1437 1438 pBC0303 1439 1440 pBC0304 1441 1442 pBC0305 1443 1444 pBC0306 1445 1446 pBC0307 1447 1448 pBC0308 1449 1450 pBC0309 1451 1452 pBC0310 1453 1454 pBC0311 1455 1456 pBC0312 1457 1458 pBC0313 1459 1460 4 1461 1462 1463 1464 pBC0316 1465 1466 pBC0317 1467 1468 pBC0318 1469 1470 pBC0319 1471 1472 0 1473 1474 pBC0321 1475 1476 PBC0322 1477 1478 PBC0323 1479 1480 pNL0040 1481 1482 pNL0041 1483 1484 pNL0042 1485 1486 pNL0043 1487 1488 Example 24: Transfection of Mammalian Cells, Expression of FVIII-XTEN and Assessment of FVIII Activity Mammalian cells, including but not limited to CH0, BHK, COS, and HEK293, are suitable for transformation with the vectors of the Examples, above, in order to express and recover FVIII-XTEN fusion protein. The following are s for methods used to express BDD FVlll and FVIll-XTEN fusion protein constructs pBCOl l4, pBCOl35, pBCOl36, pBCOl37, pBCOl45, pBCOl46, and pBCOl49 by transient transfection, which includes electroporation and chemical (PEI) transfection methods. nt HEK293 cells purchased from ATCC were revived in medium of vendor’s recommendation and passaged for a few generations before multiple vials were frozen in the medium with 5% DMSO. One vial was d and ed one more time before ection. The HEK293 cells were plated 1-2 days before transfection at a density of approximately 7X 105 per ml in one T175 per transfection, using 35 ml medium. On the day of transfection the cells were trypsinized, detached and counted, then rinsed in the medium until an even cell suspension was achieved. The cells were counted and an appropriate volume of cells (based on cell count above) were transferred to 50mL centrifuge tube, such that there were approximately 4 X 10° cells per ection. Cells were centrifuged for 5min at 500 RCF, the supernatant discarded, and the cells resuspended in 10ml of D-PBS. oporation: For electroporation, an appropriate volume of resuspension buffer was added using a ipette (supplied in the NeonTM Transfection System 100 uL Kit), such that 110 pl of buffer was available per transfection. Separate volumes of 110 pl of cell suspension were added to each Eppendorf tube containing 11 ul of plasmid DNA for each of the individual FVIII-XTEN constructs for a total of 6 ug (volume of DNA may be less, qs to 11 ul with sterile H20). A NeonTM Transfection Device was used for transfection. The program was set to electroporate at 1100v for a pulse width of 20ms, for a total of two pulses. A NeonTM Tube (supplied in the NeonTM Transfection System 100 uL Kit) was placed into NeonTM Pipette Station. A volume of 3 mL of Electrolytic Buffer E2 ied in the NeonTM Transfection System 100 uL Kit) was added to the NeonTM Tube. NeonTM Pipettes and 100 pl NeonTM Tips were used to electroporate 100 pl of cell-plasmid DNA mixture using the NeonTM Pipette Station.

The electroporation was executed and when complete, the NeonTM Pipette was removed from the Station and the pipette with the transfected cells was used to transfer the cells, with a circular motion, into a 100 mm x 20mm petri plate ning 10 ml of Opti-MEM I Reduced-Serum Medium (1X, Invitrogen), such that transfected cells were evenly distributed on plate. The cells for each transfection were incubated at 37°C for expression. On day 3 post-transfection, a 10% volume of salt solution of 10mM Hepes, 5mM CaClg, and 4M NaCl was added to each cell culture and gently mixed for 30 minutes. Each cell culture was transferred to a 50 ml conical centrifuge tube and was centrifuged at 3000 rpm for 10 minutes at 4°C.

The supematants for each culture were placed into a new 50 ml conical tube and then split into aliquots of 5x1 ml in orf and 2x15ml conical tubes for assay or were flash frozen before testing for expression of FVIII-XTEN in ELISA and performance in an FVIII activity assay, as bed herein.

Chemical transfection: Chemical transfection can be accomplished using standard methods known in the art. In the present Example, PEI is utilized, as described.

Suspension 293 Cells are seeded the day before transfection at 7 x 105 cells/mL in sufficient Freestyle 293 (Invitrogen) medium to provide at least 30 ml working volume, and incubated at 37°C. On the day of transfection, an aliquot of 1.5 ml of the transfection medium is held at room temperature, to which 90 uL of lmg/ml PEI is added and vortexed briefly. A volume of 30 ul of DNA encoding the FVIII-XTEN_AE288 construct (concentration of lmg/ml) is added to the PEI solution, which is vortexed for 30 sec. The mixture is held at room temperature for 5-15 min. The DNA/PEI e is added to the HEK293 cells and the suspension is incubated at 37°C using pre-established shake flask conditions.

About four hours after the addition of the DNA/PEI mix, a 1x volume of expansion media is added and the cells incubated at 37°C for 5 days. On the day of harvest, a 10% volume of salt solution of 10mM Hepes, 5mM CaClg, and 4M NaCl is added to the cell culture and gently mixed for 30 minutes. The cell culture is transferred to a 50 ml conical centrifuge tube and is centrifuged at 4000 rpm for 10 s at 4°C. The supernatant is placed into a new 50 ml l tube and then split into ts of 5x1 ml in Eppendorf and 2x15ml l tubes for assay or are flash frozen before testing for expression of FVIII- XTEN in ELISA and/or mance in an FVIII activity assay, as bed herein.

Generation of stable pools and cell lines that produce FVIII-XTEN ] Stable pools are generated by ing transfected cells for 3-5 weeks in medium containing selection antibiotics such as puromycin, with medium change every 2-3 days. Stable cells can be used for either production or generation of stable clones. For stable cell line selection during y screening, cells from stable pools either from on—going passaging or revived from frozen vials are seeded in 96-well plates at a target density of 0.5 cell/well. About 1 week after g spent medium from wells with single cell cluster as observed under microscope are tested for expression of FVIII by activity assay or antigen measurement.

For additional rounds of screening, normalized numbers of cells are seeded in multi-well plates.

Spent medium is harvested and tested for FVIII concentration by ELISA and FVIII activity assay. Cells would also be harvested from the plates and counted using Vi-Cell. Clones are ranked by (l) FVIII titers according to ELISA and ty; (2) ratios of ELISA titer/cell count and activity titer/cell count; and (3) integrity and homogeneity of products produced by the clones as measured by Western blots. A number of clones for each of the constructs are selected from the primary screening for additional rounds of screening.

For the second round of screening, cells in 96-well plates for the top clones ed from primary screening are first expanded in T25 flasks and then seeded in ate 24-well plates. Spent medium is collected from the plates for FVIII activity and antigen quantification and cells harvested and counted by Vi-Cell. Clones are ranked and then selected according to titers by ELISA and activity assay, ELISA titer/cell and activity cell count ratios. Frozen vials are ed for at least 5-10 clones and again these clones were screened and ranked according to titers by ELISA and activity, and ratios of ELISA titer/cell count and activity titer/cell count, and product integrity and homogeneity by Western blot, and 2-3 clones are ed for productivity evaluation in shake flasks. Final clones are selected based on specific productivity and product quality.

Production of FVIII-XTEN secreted in cell culture medium by suspension 293 stable HEK293 stable cell clones selected by the foregoing s are seeded in shake flasks at 1-2 X 105 cells/ml in expression medium. Cell count, cell viability, FVIII activity and antigen expression titers are monitored daily. On the day when FVIII activity and antigen titers and product quality are optimal, the e is harvested by either centrifugation/sterile filtration or depth filtration/sterile filtration. The filtrate is either used immediately for tangential flow filtration (TFF) processing and purification or stored in -80°C freezer for TFF processing and purification later.

Example 25: Purification and Characterization of CFXTEN Constructs Exemplary s for the purification and characterization of CFXTEN constructs with one or more XTEN follow.

] Purification of TEN AE864 by FVIII affinity chromatography CFXTEN containing supernatant is filtered using a Cuno us Biocap filter and a Cuno BioAssure capsule and subsequently concentrated by tangential flow filtration using a Millipore Pellicon 2 Mini cartridge with a 30,000 Da MWCO. Using the same tangential flow filtration cartridge the sample is diafiltered into 10 mM ine, 20 mM calcium chloride, 300 mM sodium chloride, and 0.02% Tween 80 at pH 7.0. FVIHSelect resin (GE 1701) selectively binds FVIII or B domain deleted FVIII using a 13kDa recombinant protein ligand coupled to a tography resin. The resin is equilibrated with 10 mM ine, 20 mM calcium de, 300 mM sodium chloride, and 0.02% Tween 80 at pH 7.0 and the supernatant loaded. The column is washed with 20 mM histidine, 20 mM calcium chloride, 300 mM sodium chloride, and 0.02% Tween 80 at pH 7.0, then is washed with 20 mM histidine, 20 mM calcium chloride, 1.0 M sodium chloride, and 0.02% Tween 80 at pH 7.0, and eluted with 20 mM histidine, 20 mM calcium chloride, 1.5 M sodium chloride, and 0.02% Tween 80 dissolved in 50% ethylene glycol at pH 7.0.

Concentration and Buffer Exchange by Tangential Flow Filtration and Diafiltration Supernatant batches totaling at least 10 L in , from stable CHO cells lines expressing CFXTEN are filtered using a Cuno ZetaPlus Biocap filter and a Cuno BioAssure capsule. They are subsequently trated approximately d by tangential flow filtration using a Millipore Pellicon 2 Mini cartridge with a 30,000 Da MWCO. Using the same tangential flow filtration dge the sample is diafiltered with 10 mM histidine, 20 mM m chloride, 300 mM sodium chloride, and 0.02% Tween 80 at pH 7.0 10 mM tris pH 7.5, 1 mM EDTA with 5 s worth of buffer exchange.

Samples are divided into 50 ml aliquots and frozen at -80°C.

Purification of CFXTEN by Anion Exchange Chromatography Using an Akta FPLC system the sample is purified using a -650M column. The column is equilibrated into buffer A (0.02 mol/L imidazole, 0.02 mol/L glycine ethylester hydrochloride, 0.1 5 mol/L, NaCl, 2.5% glycerol, pH 6.9) and the sample loaded. The sample is eluted using buffer B (5 mmol/L histidine HCl (His/HCI), 1.15 mol/L NaCl, pH 7.0). The 215 nm chromatogram is used to monitor the elution profile. The eluted fractions are assayed for FVIH by ELISA, SDS-PAGE or activity assay. Peak fractions are pooled and stored or subjected to thrombin activation immediately (O’Brien et al., Blood (1990) 4-1672). Fractions are assayed for FVIH activity using an aPTT based factor assay. A Bradford assay is performed to determine the total amount of protein in the load and elution fractions.

Purification of CFXTEN by Hydrophobic Interaction Chromatography CFXTEN samples in Buffer A (50 mmol/l histidine, 1 mmol/l CaCl 2, 1 M NaCl, and 0.2 g/l Tween 80®, pH 7.0) are loaded onto a toyopearl ether 650M resin equilibrated in Buffer A. The column is washed with 10 column volumes of Buffer A to remove DNA, incorrectly folded forms and FVIH, and other contaminant proteins. The CFXTEN is eluted with Buffer B (25 mmol/l histidine, 0.5 mmol/l CaCl 2 and 0.4 mol/l NaCl, pH 7.0) as a single step n (US patent 6005082). Fractions are assayed for FVIH activity using an aPTT based factor assay. A Bradford assay is performed to ine the total amount of protein in the load and elution fractions.

Removal of Aggregated protein from monomeric CFXTEN with Anion Exchange tography Using an Akta FPLC system the sample is purified using a macrocap Q column. The column is equilibrated into buffer A (20 mM MES, lmM CaCl2, pH 7.0) and the sample is loaded. The sample is eluted using a linear gradient of 30% to 80% buffer B (20 mM MES, lmM CaC12, pH 7.0 + 500 mM NaCl) over 20 column volumes. The 215 nm chromatogram is used to monitor the n profile. The fractions corresponding to the early portion of the elution contain primarily monomeric protein, while the late portion of the elution contains primarily the aggregated species. Fractions from the macrocapQ column is analyzed via size exclusion chromatography with 60 cm BioSep G4000 column to determine which to pool to create an aggregate free sample.

Activation of FVIII by Thrombin Purified FVIII in 5 mmol/L histidine HCl (His/HCI), 1.15 mol/L NaCl, pH 7.0 is treated with thrombin at a 1:4 ratio of units of human thrombin to units FVIII, and the sample is incubated at 37°C for up to 2 hours. To monitor the activation process, aliquots of this sample are then withdrawn, and acetone precipitated by the addition of 4.5 vol ice-cold acetone. The sample is incubated on ice for 10 minutes, and the itate is collected by centrifugation at 13,000 g in a microfuge for 3 minutes. The acetone is d, and the precipitate is resuspended in 30 ML SDS-PAGE reducing sample buffer and boiled for 2 minutes. Samples are then assayed by SDS-PAGE or western blot. The conversion of FVIII t0 FVIIIa is examined by looking for the conversion of the heavy chain into 40 and 50 kDa fragments and the sion of the light chain into a 70 kDa fragment (O’Brien et al., Blood (1990) 75:1664-1672).

SEC Analysis of CFXTEN ] FVII-XTEN purified by affinity and anion exchange chromatography is analyzed by size exclusion chromatography with 60 cm BioSep G4000 column. A monodispersed population with a hydrodynamic radius of ~10 nm/ apparent MW 0f~1.7 MDa (XTEN—288 fusion) or ~12 nm/ an apparent MW of 5.3 MDa (XTEN—864 ) is indicative of an ation-free sample. CFXTEN is expected to have an apparent lar weight factor up to or about 8 (for an XTEN—288 fusion with FVIII) or up to or about ~15 (for an XTEN—864 fusion with FVIII).

ELISA based tration Determination of CFXTEN The tative determination of factor VIII / CFXTEN n concentrations using the double antibody enzyme linked immuno-sorbent assay (ELISA) is med using proven antibody pairings (VisuLizeTM FVIII Antigen kit, Affinity Biologicals, Ontario Canada). Strip wells are pre- coated with sheep polyclonal antibody to human FVIII. Plasma samples are diluted and applied to the wells. The FVIII antigen that is present binds to the coated antibody. After washing away d material, peroxidase-labeled sheep detecting antibody is applied and allowed to bind to the captured FVIII. The wells are again washed and a solution of TMB (the peroxidase substrate tetramethylbenzidine) is applied and d to react for a fixed period of time. A blue color develops which changes to yellow upon quenching the reaction with acid. The color formed is measured spectrophotometrically in a microplate reader at 450 nm. The absorbance at 450 nm is directly proportional to the quantity of FVIII antigen captured onto the well. The assay is calibrated using either the calibrator plasma provided in the kit or by substituting a CFXTEN standard in an appropriate matrix.

Assessment of CFXTEN Activity via a FXa Coupled Chromogenic Substrate Assay Using the ChromogeniX Coamatic Factor VIII (ChromogeniX, cat# 82258563) the activity of FVIII or CFXTEN comprising FVIII is ed as follows. In the presence of m ions and phospholipids, factor X is ted to factor Xa by factor IXa. This activation is y stimulated by factor VIII which acts as a cofactor in this reaction. By using optimal amounts of Ca2+, olipid and factor IXa, and an excess of factor X, the rate of activation of factor X is ly related to the amount of factor VIII. Factor Xa hydrolyses the chromogenic substrate S-2765 thus liberating the chromophoric group, pNA. The color is then read spectrophotometrically at 405 nm. The generated factor Xa and thus the intensity of color is proportional to the factor VIII actiVity in the . Hydrolysis of S-2765 by thrombin formed is prevented by the addition of the synthetic thrombin inhibitor I-2581 together with the substrate. The actiVity of an unknown sample is ined by comparing final A405 of that sample to those from a standard curve constructed from known FVIII amounts. By also ining the amount of FVIII antigen present in the samples (Via A280 or ELISA), a specific actiVity of a sample is determine to understand the relative potency of a particular preparation of FVIII. This enables the relative efficiency of different isolation strategies or construct designs for CFXTEN fusions to be assessed for activity and ranked. aPTT Based Assays for CFXTEN Activity Determination ] CFXTEN acts to replace FVIII in the intrinsic or contact activated ation pathway. The ty of this coagulation pathway is assessed using an activated partial thromboplastin time assay (aPTT). FVIII activity specifically is measured as follows: a standard curve is prepared by diluting normal control plasma (Pacific Hemostasis cat# ) two-fold with FVIII deficient plasma (cat# 100800) and then conducting 6, 4-fold serial dilutions again with factor VIII deficient plasma. This creates a standard curve with points at 500, 130, 31, 7.8, 2.0, 0.5 and 0.1 IU/ml of ty, where one unit of actiVity is defined as the amount of FVIIIC actiVity in 1 ml of normal human plasma. A FVIII- deficient plasma also is included to determine the background level of actiVity in the null plasma. The sample is prepared by adding CFXTEN to FVIII deficient plasma at a ratio of 1:10 by volume. The samples is tested using an aPTT assay as follows. The samples are incubated at 37C in a molecular devices plate reader spectrophotometer for 2 minutes at which point an equal volume of aPTT reagent (Pacific Hemostasis cat# 100402) is added and an additional 3 minute 37C incubation performed. After the incubation the assay is activated by adding one volume of calcium chloride (Pacific Hemostasis cat# 100304). The turbidity is monitored at 450 nm for 5 minutes to create on profiles. The aPTT time, or time to onset of clotting activity, is defined as the first time where OD405 nm increased by 0.06 over baseline. A log — linear standard curve is created with the log of actiVity relating linearly to the aPTT time. From this the actiVity of the sample in the plate well is ined and then the actiVity in the sample is determined by lying by 11 to account for the on into the FVIII deficient plasma.

By also ining the amount of FVIII antigen present in the samples (Via A280 or ELISA), a specific activity of a sample can be determine to understand the relative potency of a particular preparation of FVIII. This enables the relative efficiency of different isolation strategies or construct designs for CFXTEN fusions to be ranked.

Western Blot Analysis of FVIII / XTEN expressed proteins Samples were run on a 8% homogeneous SDS gel and subsequently transferred to PVDF membrane. The samples in lanes l-15 were: MW Standards, FVIII(42.5 ng), pBC0100B, pBC0114A, pBC0100, pBC0114, pBC0126, pBC0127 (8/5/11; #9), pBC0128, pBC0135, pBC0136, pBC0137, , pBC0149, and pBC0146, respectively. The ne was lly blocked with 5% milk then probed with anti-FVIII monoclonal antibody, GMA—012, specific to the A2 domain of the heavy chain (Ansong C, Miles SM, Fay PJ.J Thromb Haemost. 2006 Apr;4(4):842-7). Insertion ofXTEN288 in the B-domain was observed for pBC0136 (lane 8, ) and pBC0137 (lane 9, ), whereas XTEN288 insertion at the C-terminus was observed for 6 (lane 12, ). All of the assayed FVIII-XTEN proteins revealed the presence of single chain protein with molecular weight of at least 21 kDa higher than that of pBC01 14 base construct or FVIII standard. In addition, AE42 insertion was observed for pBC0135 (lane 7, ) and pBC0149 (lane 11, ) with the single chain running NS kDa higher than that of pBC01 14 base protein and heavy chain running at NS kDa higher than 90 kDa band of the base protein.

Assay of Expressed FVIII by ELISA To verify and quantitate the expression of FVIII-XTEN fusion proteins of the constructs by cell e, an ELISA assay was established. Capture dies, either SAF8C-AP (Affinity Biologicals), or GMA—8002 (Green in Antibodies), or GMA011 antibodies (Green Mountain Antibodies) for LC ELISA) or by GMA016 antibodies were immobilized onto wells of an ELISA plate. The wells were then incubated with ng buffer (lx PBS/3% BSA) to prevent non-specific binding of other proteins to the anti-FVIII antibody. FVIII standard dilutions (~50 ng-0.024 ng range), quality controls, and cell culture media samples were then incubated for 1.5 h in the wells to allow binding of the expressed FVIII protein to the coated antibody. Wells were then washed extensively, and bound protein is incubated with anti-FVIII detection antibody, SAF8C-Biotinylated (Affinity Biologicals). Then streptaVidin-HRP, which binds the biotin conjugated to the FVIII detection antibody, is added to the well and incubated for l h. Finally, OPD substrate is added to the wells and its hydrolysis by HRP enzyme is monitored with a plate reader at 490 nm wavelength. Concentrations of FVIII-containing samples were then calculated by comparing the colorimetric response at each culture dilution to a standard curve. The results, in Table 22, below, show that XTEN of the various constructs are expressed at 0.4 - l [Lg/ml in the cell culture media. The results obtained by ELISA and the actiVity data indicate that FVIII- XTEN fusion proteins were very well expressed using the bed transfection methods. Furthermore, under the mental conditions, the s demonstrate that the specific ty values of the FVIII- XTEN proteins were similar or greater than that of pBC01 14 base construct (expressing BDD FVIII) and t that XTEN insertion into the C-terminus or B-domain of FVIII s in preservation of FVIII protein function.

Chromogenic Activity Assay for CFXTEN fusion protein BDD FVIII and CFXTEN fusion protein ucts pBC0114, pBC0135, pBC0136, pBC0137, pBC0145, pBC0146, and pBC0149, in various configurations, including XTEN AE288 and AG288 ed at the C-terminus of the FVIII BDD ce and FVIII-XTEN fusion proteins with AE42 and AE288 inserted after e 745 (or residue 743) and before residue 1640 (or residue 1638) of the B- domain (including constructs with the P1648 sing site mutated to alanine), were expressed in transiently transfected Freestyle 293 cells, as described above, and tested for procoagulant activity. The procoagulant activity of each of the FVIII-XTEN ns present in cell culture medium was assessed using a Chromogenix Coamatic® Factor VIII assay, an assay in which the activation of factor X was linearly related to the amount of factor VIII in the sample. The assay was performed according to manufacturer’s instructions using the end-point method, which was ed ophotometrically at OD405 nm. A standard curve was created using purified FVIII protein at concentrations of 250, 200, 150, 100, 75, 50, 37.5, 25, 12.5, 6.25, 3.125 and 1.56 mU/ml. Dilutions of factor VIII standard, quality controls, and samples were prepared with assay buffer and PEI culture medium to account for the effect of the medium in the assay performance. Positive controls consist of purified factor VIII n at 20, 40, and 80 mU/ml concentrations and cell e medium of pBC01 14 FVIII base construct, lacking the XTEN insertions. Negative controls consisted of assay buffer or PEI culture medium alone. The cell culture media of the FVIII-XTEN constructs were obtained as described, above, and were tested in replicates at 1:50, 1:150, and 1:450 dilutions and the actiVity of each was calculated in U/ml. Each FVIII- XTEN construct exhibited procoagulant actiVity that was at least comparable, and in some cases greater than that of the base construct positive control, and support that under the conditions of the experiments, the linkage of XTEN, ing AE288 or AG288, at the C-terminus of FVIII or insertion of XTEN, including AE42 or AE288 within the B-domain resulted in retention or even enhancement of FVIII procoagulant actiVity= Table 22: Results of ELISA and genic FVIII activity assays FVIII- Act1v1ty. . tration. Specrfic Actmty. . .

XTEN ption of Construct (IU/ml) (IU/mg) Construct BDD FVIII base construct used for FVIII construct with XTE\I AG288 pBC0146 -“ 12759 inserted at the inus of FVIII FVIII construct with XTE\I AE288 pBC0145 -“ 4844 inserted at the C-terminus of FVIII FVIII construct with XTE\I AE42 inserted between residue 745 and 1640 pBC0149 4.9 55 81 inserted between residue 745 and 1640 and with Ar1648 to Ala mutation FVIII construct with XTE\I AE288 inserted between residue 745 and 1640 pBC0137 1.9 0.3 6013 inserted between e 745 and 1640 and with Arg1648 to Ala mutation Coatest Assay for Cell Culture Sample Activity Assay containing CFXTEN fusion protein Using the Coatest assay, the activity of FVIII or CFXTEN comprising FVIH is assessed as follows.

Assay Matrix: All wells in the same plate were adjusted to the same percentage of media to control for matrix effects. The test samples were diluted such that the OD405 g would fall within the linear range of the standard. The range of concentrations for the FVIH rd was 100 mU/mL to 0.78 mU/mL, prepared by four-fold serial dilutions of the FVlll rd in 1X t buffer (DiaPharma) plus the pre-determined percentage of culture media.

The Coatest SP FVIII arma) reagent package includes the 10X Coatest buffer stock solution, factor lXa + factor X, phospholipid, CaClgand substrate. The 1x Coatest solution was prepared by adding 9X volume of cold dngO to 1X volume of the stock. The cell culture media was then added to the prepared 1X solution at a pre-determined ratio to normalize the percentage of matrix in all test wells. Factor lXa + factor X, phospholipid, and substrate were reconstituted according to manufacturer’s recommendations. t Assay Procedure: Assay reagents were prepared and kept on ice until needed. 25 [ll of the diluted test samples and standards were added to a 96 well plate in duplicate. 50 pl of phospholipid/factor lXa/factor X was added to each well and mixed by gently tapping the side of the plate. Plates were incubated at 37°C for 5 min on a 37°C plate heater. 25 pl of C3C12 was added to each well and mixed. The plates were incubated at 37°C for 5 min on a plate heater. 50 ul of substrate was then added to each well, mixed, and the plates incubated at 37°C for an additional 5-10 min until the top standard developed an OD405 g of about 1.5. 25 [ll of 20% acetic acid was added to each well with mixing to stop the reaction and wells were read at OD405 using a SpectraMAX® plus ular Devices) spectrophotometer. Data is was performed using the SoftMax program (verion 5.2). The LLOQ varied per assay, but was generally 0.0039 lU/ml.

RLults: The data are presented in Tables 23-26. Table 23 presents results from CFXTEN fusion proteins wih XTEN inserted in single sites chosed on the basis of criteria described herein, ing Example 34. The pBC00114 FVIII positive control showed good expression and FVIII activity.

Of the 106 -XTEN fusion proteins assayed, 68% retained able FVIH activity, with 30% exhibiting 3+ to 4+ activity in the coagulation assay. Thirty-one percent of the fusion proteins assayed had results below the limits of quantitation (which may be due to poor expression, reflected in the corresponding expression ELISA results). All four B-domain insertion constructs exhibited good activity, as did the C-terminal linked constructs, ting that these are likely favorable insertion sites The s of the single insertion site data guided the creation ofXTEN constructs with 2 XTEN insertions, the results of which are presented in Table 24. l, the positivity rate was 67%, with 31% of fusion proteins exhibiting 3+ to 4+ activity in the coagulation assay.

The results of the foregoing data guided the creation of XTEN constructs With 3 XTEN insertions, the s of Which are presented in Table 25. Overall, 92% of the samples had measurable FVIII activity, With fully 79% exhibiting 3+ to 4+ activity in the coagulation assay.

A d number of constructs With 4 XTEN inserted in the Al, A2 and A3 domains were d and assayed, With 4 of 5 exhibiting FVIH actiVity (Table 26), suggesting that insertion of multiple XTEN does not compromise the ability of the resulting fusion ns to retain FVIII actiVity.

Conclusions: Under the ions of the experiments, the results support that the criteria used to select XTEN insertion sites are valid, that insertion of one or more XTEN into the selected sites of FVIII is more likely than not to result in retention of procoagulant actiVity of the resulting CFXTEN molecule, and that insertion of three XTEN appears to result in a greater proportion of fusion proteins retaining high levels of FVIII procoagulant actiVity compared to single or double XTEN ion constructs.

Table 23: Results of Coagulation Activity Assays for CFXTEN comprising one XTEN 11188652011 Domain uct Activity 13%;???“ pBC01 14 +——+ +——+ 3 A1 pBC0126 LLOQ* LLOQ 3 A1 pBC0127 —— —— 18 A1 5 22 A1 pBC0183 +——+ 26 A1 pBC0184 40 A1 pBC0166 60 A1 pBC0185 LLOQ LLOQ 1 16 A1 7 LLOQ LLOQ 130 A1 pBC0128 LLOQ LLOQ 188 A1 pBC0168 216 A1 pBCO 129 230 A1 pBC0169 LLOQ LLOQ 333 A1 pBC0130 3 75 A2 pBC013 1 LLOQ +++ 403 A2 pBC0132 442 A2 pBC0170 490 A2 pBC0133 —— 518 A2 pBC0171 LLOQ -- 599 A2 pBC0134 ++ 713 A2 pBC0172 —— 745 B pBC0135 745 B pBC0149 745 B pBC0136 ++ ++ 745 B pBC0137 1720 A3 pBC013 8 1796 A3 pBC0139 —— 1802 A3 pBCO 140 —— 1827 A3 pBC0173 LLOQ LLOQ 1861 A3 pBC0174 LLOQ LLOQ 1896 A3 pBC0175 LLOQ LLOQ 1900 A3 pBC0176 +——+ +——+ 1904 A3 pBC0177 —— -- Inserfion Expression Domain Construct Activity Site ELISA 1937 A3 pBC0178 LLOQ LLOQ 2019 A3 pBC0141 LLOQ + 2068 C1 chnl79 ++ ++ 2111 C1 chnlso LLOQ LLOQ 2120 C1 chnl42 LLOQ 2171 C2 pBC0143 2188 C2 pBC0181 2227 C2 chnlsz 2277 C2 chn144 2332 CT chnl45 2332 CT chn146 403 A2 pSD0001 599 A2 pSD0002 403 A2 pSD0003 599 A2 pSD0004 745 B pSD0005 745 pSD0006 745 pSD0007 745 UUUUUU pSD0008 1720 A3 pSD0009 1720 A3 pSD0010 2171 C2 pSD0011 2171 C2 pSD0012 2332 CT‘ pSD0013 2332 CT‘ 4 745 7 745 0303 pSD0018 2332 CT‘ 9 2332 CT‘ pSD0020 2332 CT‘ 5 2332 CT‘ pSD0016 0 1J4enn pSD0021 32 A1 pSD0022 65 A1 pSD0023 81 A1 pSD0024 119 A1 pSD0025 211 A1 pSD0026 220 A1 pSD0027 224 A1 pSD0028 336 A1 pSD0029 339 A1 pSD0030 378 A2 pSD0031 LLOQ 399 A2 pSD0032 409 A2 pSD0033 416 A2 pSD0034 487 A2 pSD0035 LLOQ 494 A2 pSD0036 LLOQ 500 A2 pSD0037 LLOQ 603 A2 pSD0038 1656 A3 pSD0039 +——+ 1656 A3 pN1009** ++++ Insse£2011 Domain uct Activity Eprfigion 1711 A3 pSD0040 ++ + 1725 A3 pSD0041 LLOQ 1749 A3 pSD0042 LLOQ LLOQ 1905 A3 pSD0043 1910 A3 pSD0044 —— + 1900 A3 pSD0062 1900 A3 pSD0063 18 A1 pSD0045 18 A1 pSD0046 22 A1 pSD0047 LLOQ LLOQ 22 A1 8 LLOQ LLOQ 26 A1 pSD0049 26 A1 pSD0050 40 A1 pSD0051 40 A1 pSD0052 216 A1 pSD0053 LLOQ LLOQ 216 A1 pSD0054 LLOQ LLOQ 375 A2 pSD0055 LLOQ + 442 A2 6 LLOQ LLOQ 442 A2 pSD0057 LLOQ LLOQ 1796 A3 pSD0058 LLOQ LLOQ 1796 A3 pSD0059 -- -- 1802 A3 pSD0060 -- -- 1802 A3 pSD0061 LLOQ LLOQ *LLOQ: below the limits of quantitation ** pNL009 includes a deletion of 745-1656 Table 24: Results of Coagulation Activity Assays for CFXTEN comprising two XTEN Insertion 1 Insertion 2 Insertion Domain Insertion Domain Construct Activity Site Site 745 B 2332 CT 1.002 +++ 745 B 2332 CT LSD0001.005 +++ 745 B 2332 CT LSD0001.006 +++ 745 B 2332 CT LSD0001.011 +++ 745 B 2332 CT LSD0001.012 +++ 745 B 2332 CT LSD0001.013 +++ 745 B 2332 CT LSD0001.016 +++ 745 B 2332 CT LSD0001.021 +++ 745 B 2332 CT 2.001 +++ 745 B 2332 CT LSD0002.002 +++ 745 B 2332 CT LSD0002.014 +++ 745 B 2332 CT LSD0003.004 +++ 745 B 2332 CT LSD0003.006 +++ 745 B 2332 CT 3.009 +++ 745 B 2332 CT LSD0003.014 + 745 B 2332 CT LSD0004.010 +++ 745 B 2332 CT LSD0004.011 LLOQ 745 B 2332 CT LSD0004.014 +++ 745 B 2332 CT LSD0004.016 +++ Insertion 1 ion 2 11156111011 Domain Insnrnon Domain Construct Activity 8116 Sam 745 B 2332 CT LSD0004.022 +++ 745 B 2332 CT LSD0003.016 +++ 0745 B 2332 CT pNLOO6 0745 B 2332 CT pNLOO7 0745 B 2332 CT pNLOO8 ++ 1656 213 2332 CT pNLOlO 26 A1 403 A2 LSD0005.002 ++ 26 A1 403 A2 5.004 ++ 40 A1 403 A2 LSD0005.005 ++ 40 A1 403 A2 LSD0005.011 ++ 18 A1 403 A2 LSD0005.018 ++ 26 A1 599 A2 LSD0006.002 + 40 A1 599 A2 LSD0006.005 ++ 40 A1 599 A2 LSD0006.007 ++ 40 A1 599 A2 LSD0006.011 +++ 40 A1 403 A2 7.002 + 40 A1 403 A2 LSD0007.004 + 26 A1 403 A2 LSD0007.013 ++ 26 A1 599 A2 LSD0008.001 ++ 40 A1 599 A2 LSD0008.002 ++ 26 A1 599 A2 LSD0008.006 + 18 A1 599 A2 LSD0008.009 ++ 40 A1 599 A2 LSD0008.017 + 745 B 2332 CT LSD0002.025 +++ 745 B 2332 CT LSD0002.013 +++ 745 B 2332 CT LSD0003.025 +++ 745 B 2332 CT LSD0004.025 +++ 745 B 2332 CT LSD0003.005 ++ 26 A1 403 A2 LSD0007.008 ++ 1720 A3 1900 A3 LSD0044.002 LLOQ 1725 A3 1900 A3 LSD0044.005 LLOQ 1720 A3 1900 A3 LSD0044.039 LLOQ 1711 A3 1905 A3 LSD0044.022 LLOQ 1720 A3 1905 A3 4.003 LLOQ 1725 A3 1905 A3 LSD0044.001 LLOQ 1656 A3 26 A1 LSD003 8.001 ++ 1656 A3 18 A1 LSD003 8.003 ++ 1656 A3 18 A1 LSD0038.008 +++ 1656 A3 40 A1 LSD0038.012 ++ 1656 A3 40 A1 LSD0038.013 ++ 1656 A3 26 A1 LSD0038.015 ++ 1656 A3 399 A2 LSD0039.001 + 1656 A3 403 A2 LSD0039.003 ++ 1656 A3 403 A2 LSD0039.010 ++ 1656 A3 1725 A3 LSD0045.001 + 1656 A3 1720 A3 LSD0045.002 ++ 1900 A3 18 A1 LSD0042.014 + 1900 A3 18 A1 LSD0042.023 + 1900 A3 26 A1 LSD0042.006 + 1900 A3 26 A1 LSD0042.013 ++ 1900 A3 40 A1 LSD0042.001 + 1900 A3 40 A1 LSD0042.039 + Insertion 1 Insertion 2 Insertion Domain Insertion Domain Construct Activity Site Site 1900 A3 26 A1 LSD0042.047 + 1905 A3 18 A1 LSD0042.003 + 1905 A3 40 A1 LSD0042.004 LLOQ 1905 A3 26 A1 LSD0042.008 LLOQ 1905 A3 26 A1 LSD0042.038 LLOQ 1905 A3 40 A1 LSD0042.082 LLOQ 1910 A3 26 A1 LSD0042.040 LLOQ 18 A1 399 A2 LSD0037.002 ++ 26 A1 399 A2 LSD0037.009 + 40 A1 399 A2 LSD0037.011 ++ 18 A1 403 A2 LSD0047.002 ++ 18 A1 403 A2 7.005 + 18 A1 403 A2 8.007 + 1656 A3 1900 A3 LSD0046.001 ++ 1656 A3 1900 A3 LSD0046.002 + 1656 A3 1905 A3 LSD0046.003 + 1711 A3 40 A1 LSD0040.011 LLOQ 1711 A3 26 A1 LSD0040.042 LLOQ 1720 A3 26 A1 LSD0040.002 + 1720 A3 40 A1 LSD0040.008 + 1720 A3 18 A1 LSD0040.021 + 1720 A3 26 A1 LSD0040.037 LLOQ 1720 A3 18 A1 LSD0040.046 + 1725 A3 26 A1 LSD0040.003 LLOQ 1725 A3 40 A1 LSD0040.006 LLOQ 1725 A3 26 A1 LSD0040.007 LLOQ 1725 A3 18 A1 LSD0040.010 LLOQ 1725 A3 40 A1 0.039 LLOQ 1725 A3 18 A1 LSD0040.052 + 1720 A3 403 A2 LSD0041.001 + 1720 A3 399 A2 LSD0041.004 LLOQ 1711 A3 403 A2 LSD0041.006 LLOQ 1720 A3 403 A2 LSD0041.008 LLOQ 1725 A3 403 A2 LSD0041.010 LLOQ 1725 A3 403 A2 LSD0041.014 LLOQ 1725 A3 399 A2 LSD0041.016 LLOQ 1711 A3 403 A2 1.035 LLOQ 1900 A3 399 A2 LSD0043.001 LLOQ 1900 A3 403 A2 LSD0043.002 LLOQ 1905 A3 403 A2 LSD0043.005 LLOQ 1900 A3 399 A2 LSD0043.006 LLOQ 1900 A3 403 A2 LSD0043.007 LLOQ 1900 A3 403 A2 LSD0043.008 LLOQ 1905 A3 399 A2 LSD0043.015 LLOQ 1905 A3 403 A2 LSD0043.029 LLOQ 1910 A3 403 A2 LSD0043.043 LLOQ Table 25: s of Coagulation Activity Assays for CFXTEN comprising three XTEN Insertion 1 Insertion 2 ion 3 Insertion . Insertion . Insertion . . .

. Domain Site Site.

Domain Domain Construct ActiVity Site.

Insertion 1 ion 2 Insertion 3 Insertion Domain 332011 Domain Insertion Domain Construct Site Activity Site 26 A1 403 A2 1656 A3 pSD0077 +++ 26 A1 403 A2 1720 A3 pSD0078 ++ 26 A1 403 A2 1900 A3 pSD0079 ++ 26 A1 1656 A3 1720 A3 pSD0080 +++ 26 A1 1656 A3 1900 A3 pSD0081 LLOQ 26 A1 1720 A3 1900 A3 pSD0082 403 A2 1656 A3 1720 A3 pSD0083 +++ 403 A2 1656 A3 1900 A3 pSD0084 +++ 403 A2 1720 A3 1900 A3 pSD0085 1656 A3 1720 A3 1900 A3 pSD0086 +++ 18 A1 745 B 2332 CT LSD0049.002 +++ 26 A1 745 B 2332 CT LSD0049.008 +++ 26 A1 745 B 2332 CT LSD0049.011 +++ 40 A1 745 B 2332 CT LSD0049.012 +++ 40 A1 745 B 2332 CT LSD0049.020 +++ 18 A1 745 B 2332 CT 9.021 +++ 40 A1 745 B 2332 CT LSD0050.002 +++ 18 A1 745 B 2332 CT LSD0050.003 +++ 26 A1 745 B 2332 CT 0.007 LLOQ 18 A1 745 B 2332 CT 0.010 +++ 26 A1 745 B 2332 CT LSD0050.012 +++ 40 A1 745 B 2332 CT LSD0050.014 +++ 403 A2 745 B 2332 CT LSD0051.002 +++ 399 A2 745 B 2332 CT LSD0051.003 +++ 403 A2 745 B 2332 CT LSD0052.001 +++ 399 A2 745 B 2332 CT LSD0052.003 +++ 1725 A3 745 B 2332 CT LSD0053.021 LLOQ 1720 A3 745 B 2332 CT LSD0053.022 +++ 1711 A3 745 B 2332 CT LSD0053.024 +++ 1720 A3 745 B 2332 CT LSD0054.021 +++ 1711 A3 745 B 2332 CT LSD0054.025 ++ 1725 A3 745 B 2332 CT LSD0054.026 +++ 1900 A3 745 B 2332 CT LSD0055.021 +++ 1905 A3 745 B 2332 CT LSD0055.022 +++ 1900 A3 745 B 2332 CT LSD0055.026 +++ 1900 A3 745 B 2332 CT 6.021 +++ 1900 A3 745 B 2332 CT LSD0056.024 +++ 1910 >w 745 B 2332 CT LSD0056.025 +++ 0745 1900 A3 2332 CT pBC0294* 0745 A3 1900 2332 CT ch0295* 0745 UUUUUUUUUUUUUUUUUUUUUUUUUUUU 1900 A3 2332 CT ch0296* 0745 1900 A3 2332 CT ch0297* 0745 1900 A3 2332 CT ch0298* 0745 1900 A3 2332 CT ch0299* 0745 1900 A3 2332 CT pBCO300* 0745 1900 A3 2332 CT pBCO301* 0745 1900 A3 2332 CT pBCO302* 0745 1900 A3 2332 CT pBCO303* 0745 1900 A3 2332 CT pBCO304* 0745 1900 A3 2332 CT pBCO305* 0745 1900 A3 2332 CT pBCO306* 0745 1900 A3 2332 CT pBCO307* Insertion 1 Insertion 2 Insertion 3 Ingeitelon Domain Insseitgon Domain 31161011 Domain Construct Activity 0745 B 1900 A3 2332 CT pBC0308* 0745 B 1900 A3 2332 CT pBC0309* 0745 B 1900 A3 2332 CT pBC0310* 0745 B 1900 A3 2332 CT pBC0311* 0745 B 1900 A3 2332 CT pBC0312* 0745 B 1900 A3 2332 CT pBC0313* 0745 B 1900 A3 2332 CT pBC0314* 0745 B 1900 A3 2332 CT pBC0315* 0745 B 1900 A3 2332 CT 6* 0745 B 1900 A3 2332 CT pBC0317* 0745 B 1900 A3 2332 CT pBC0318* 0745 B 1900 A3 2332 CT pBC0319* 0745 B 1900 A3 2332 CT pBC0320* 0018 A1 0745 B 2332 CT pBC0269* 0403 A2 0745 B 2332 CT pBC0270* 1720 A3 0745 B 2332 CT pBC0271* 1900 A3 0745 B 2332 CT pBC0272* 0403 A2 0745 B 2332 CT 3* 1720 A3 0745 B 2332 CT pBC0274* 1900 A3 0745 B 2332 CT pBC0275* 0018 A1 0745 B 2332 CT pBC0276* 0403 A2 0745 B 2332 CT pBC0277* 1720 A3 0745 B 2332 CT 8* 1900 A3 0745 B 2332 CT pBC0279* *Construct with R1648A mutation Table 26: Results of Coagulation Activity Assays for CFXTEN comprising four XTEN XTEN XTEN XTEN XTEN XTEN XTEN Construct ID Activity Insert 1 Insert 2 Insert 3 Insert 4 Insert 5 Insert 6 26 403 1656 1720 - - pSD0087 26 403 1656 1900 - - pSD0088 26 1656 1720 1900 - - pSD0090 403 1656 1720 1900 - - pSD0091 0040 0403 745 2332 - - LSD0058.006* 0018 0409 745 2332 - - LSD0059.002* -- 0040 0409 745 2332 - - LSD0059.006* -- 0040 0409 745 2332 - - LSD0060.001* -- 0018 0409 745 2332 - - 0.003* -- 0040 1720 745 2332 - - LSD0061.002* -- 0026 1720 745 2332 - - LSD0061.007* 0018 1720 745 2332 - - LSD0061.008* 0018 1720 745 2332 - - LSD0061.012* 0018 1720 745 2332 - - LSD0062.001* 0026 1720 745 2332 - - LSD0062.002* 0018 1720 745 2332 - - LSD0062.006* 0018 1900 745 2332 - - LSD0063.001* 0018 1900 745 2332 LSD0064.017* 0026 1900 745 2332 4.020* 0040 1900 745 2332 LSD0064.021* 0040 1905 745 2332 LSD0065.001* 0018 1905 745 2332 LSD0065.014* 0040 1905 745 2332 LSD0066.001* 0026 1905 745 2332 LSD0066.002* 0018 1905 745 2332 LSD0066.009* 0018 1905 745 2332 6.011* 0018 1910 745 2332 LSD0067.004* 0018 1910 745 2332 LSD0067.005* 0040 1910 745 2332 LSD0067.006* 0026 1910 745 2332 LSD0067.008* 0018 1910 745 2332 8.001* 0026 1910 745 2332 LSD0068.002* 0040 1910 745 2332 LSD0068.005* 0018 1910 745 2332 LSD0068.010* 0409 1720 745 2332 LSD0069.004* 0403 1720 745 2332 LSD0069.008* 0409 1720 745 2332 LSD0070.003* 0403 1720 745 2332 LSD0070.004* 0403 1720 745 2332 LSD0070.005* 0403 1900 745 2332 LSD0071.001* 0403 1900 745 2332 LSD0071.002* 0409 1900 745 2332 LSD0071.008* 0403 1900 745 2332 LSD0072.001* 0403 1900 745 2332 LSD0072.002* 0409 1900 745 2332 LSD0072.003* 0409 1905 745 2332 LSD0073.002* 0403 1905 745 2332 LSD0073.004* 0403 1905 745 2332 LSD0073.006* 0403 1905 745 2332 LSD0074.007* 0409 1905 745 2332 LSD0074.010* 0403 1905 745 2332 LSD0074.011* 0409 1910 745 2332 LSD0075.004* 0403 1910 745 2332 5.007* 0403 1910 745 2332 LSD0076.002* 0403 1910 745 2332 LSD0076.003* 0403 1910 745 2332 pSD0093* 1720 1900 745 2332 pSD0094* 1720 1905 745 2332 pSD0095* 1720 1910 745 2332 pSD0097* 1720 1910 745 2332 pSD0098* 0403 1656 1720 2332 pNL0022 0403 1656 1900 2332 pNL0023 0403 1720 1900 2332 pNL0024 1656 1720 1900 2332 p1JL0025 0018 0403 1656 2332 pBC0247 0018 0403 1720 2332 pBC0248 0018 0403 1900 2332 9 0018 1656 1720 2332 pBC0250 0018 1656 1900 2332 pBC0251 0018 1720 1900 2332 2 0018 0403 0745 2332 LSD57005 0018 0745 1720 2332 LSD62001 0018 0745 1900 2332 pBC0262 0403 0745 1720 2332 LSD70004 0403 0745 1900 2332 pBC0266 0745 1720 1900 2332 pBC0268 0188 1900 0745 2332 pcs0001* 0599 1900 0745 2332 pcs0002* 2068 1900 0745 2332 pcs0003* 2171 1900 0745 2332 pcs0004* 2227 1900 0745 2332 pcs0005* 2277 1900 0745 2332 pCS0006* 0403 1656 1720 1900 pNL0030 0018 0403 1656 1720 pBC0253 0018 0403 1656 1900 pBC0254 0018 0403 1720 1900 chozss 0018 1656 1720 1900 pBC0256 0018 0403 0745 1720 pBC0259* 0018 0403 0745 1900 pBC0260* 0018 0745 1720 1900 pBC0263 0403 0745 1720 1900 pBC0267 0018 0403 1656 1720 pBC0257 0018 0403 0745 1720 pBC0264 *Construct with R1648A mutation Example 26: ination ofXTEN Radii and d parameters In order to quantify the hydrodynamic radii of the XTEN components of CFXTEN fusion proteins and how the value of multiple XTEN versus single XTEN varies, a series of formulae were created based on empirically-derived data from size exclusion tography assays of various fusion proteins comprising one or more XTEN. It is ed that the incorporation of multiple XTEN into a CFXTEN provides a higher total hydrodynamic radius of the XTEN component compared to CFXTEN with fewer XTEN yet having approximately the same total ofXTEN amino acids. The maximum radius of a single XTEN ptide is calculated (hereinafter “XTEN Radius”) according to the formula given by Equation 11: XTEN Radius = («/XTEN length 0.2037) + 3.4627 11 The sum of the maximum of the XTEN Radii for all XTEN segments in a CFXTEN is calculated (hereinafter “Sum XTEN Radii”) according to the formula given by Equation III: Z XTEN Radius1- i =1 Sum XTEN Radii = 111 wherein: n = the number ofXTEN segments and i is an iterator The ratio of the SUM XTEN Radii of a CFXTEN sing multiple XTEN to that of an XTEN Radius for a single XTEN of an equivalent length (in total amino acid residues to that of the CFXTEN) is calculated (hereinafter “Ratio XTEN Radii”) according to the formula given by Equation [1.21 XTEN Radiusi ;;1XTEN Lengthg * 0.2037) + 3.4627 Ratio XTEN Radii = (1 IV wherein: n = the number ofXTEN segments and i is an iterator RLults: on 11 was d to XTEN of lengths 144, 288, 576 and 864. The results are presented in Table 27. Equation IV was applied to various CFXTEN fusion proteins described herein with two, three, or four XTEN. The Ratio ofXTEN Radii has a value of l for all CFXTEN that contain a single XTEN. The Ratio XTEN Radii are presented in Table 28. The Ratio ofXTEN Radii for pSDOO92, which contains 5 XTEN insertions, has a value of 3.31. Collectively, the results indicate that the inclusion of le XTEN increases the Ratio XTEN Radii to values greater than 2, with four insertions resulting in higher values than three insertions.

Table 27: Results of Radii Calculations for CFXTEN comprising XTEN XEN Len; XTEN Radius Table 28: Results of Radii ations for CFXTEN comprising XTEN Ratio Insert . Insert . Insert Domain Domain Domam. Insert XTEN Site Site Site Site Domain Construct Radii -_—_ -——-—-—— -——-—-—— Insertion 1 Insertion 2 Insertion 3 Insertion 4 Ratio Insert Insert . Insert Domain Domam Domain Insert XTEN Site Site Site Site Domain uct Radii 745 B 2332 CT LSD0001.006 1.71 745 B 2332 CT LSD0001.011 1.71 745 B 2332 CT LSD0001.012 1.71 745 B 2332 CT LSD0001.013 1.67 745 B 2332 CT 1.016 1.67 745 B 2332 CT LSD0001.021 1.67 745 B 2332 CT LSD0002.001 1.67 745 B 2332 CT LSD0002.002 1.67 745 B 2332 CT LSD0002.004 1.71 745 B 2332 CT LSD0002.008 1.67 745 B 2332 CT LSD0002.014 1.67 745 B 2332 CT LSD0003.001 1.67 745 B 2332 CT LSD0003.004 1.66 745 B 2332 CT LSD0003.006 1.67 745 B 2332 CT LSD0003.009 1.67 745 B 2332 CT LSD0003.014 1.66 745 B 2332 CT LSD0003.018 1.67 745 B 2332 CT LSD0004.010 1.66 745 B 2332 CT LSD0004.011 1.67 745 B 2332 CT LSD0004.014 1.66 745 B 2332 CT LSD0004.016 1.66 745 B 2332 CT LSD0004.022 1.66 745 B 2332 CT LSD0003.016 1.67 26 A1 403 A2 LSD0005.002 1.71 26 A1 403 A2 LSD0005.004 1.71 40 A1 403 A2 LSD0005.005 1.71 40 A1 403 A2 LSD0005.011 1.71 18 A1 403 A2 LSD0005.018 1.71 26 A1 599 A2 LSD0006.002 1.71 40 A1 599 A2 LSD0006.005 1.71 40 A1 599 A2 LSD0006.007 1.71 40 A1 599 A2 LSD0006.011 1.71 40 A1 403 A2 LSD0007.002 1.71 40 A1 403 A2 LSD0007.004 1.71 26 A1 403 A2 LSD0007.013 1.71 26 A1 599 A2 LSD0008.001 1.71 40 A1 599 A2 LSD0008.002 1.71 26 A1 599 A2 LSD0008.006 1.71 18 A1 599 A2 LSD0008.009 1.71 40 A1 599 A2 8.017 1.71 745 B 2332 CT LSD0002.025 1.71 745 B 2332 CT LSD0002.013 1.67 745 B 2332 CT 3.025 1.67 745 B 2332 CT LSD0004.025 1.67 745 B 2332 CT LSD0003.005 1.66 26 A1 403 A2 LSD0007.008 1.71 1720 A3 1900 A3 LSD0044.002 1.71 1725 A3 1900 A3 LSD0044.005 1.71 1720 A3 1900 A3 LSD0044.039 1.71 171 1 A3 1905 A3 LSD0044.022 1.71 1720 A3 1905 A3 LSD0044.003 1.71 Insertion 1 Insertion 2 Insertion 3 Insertion 4 Ratio Insert Insert . Insert Domain Domam Domain Insert XTEN Site Site Site Site Domain Construct Radii 1725 A3 1905 A3 LSD0044.001 1.71 1656 A3 26 A1 LSD003 8.001 1.71 1656 A3 18 A1 LSD003 8.003 1.71 1656 A3 18 A1 LSD003 8.008 1.71 1656 A3 40 A1 LSD003 8.012 1.71 1656 A3 40 A1 LSD003 8.013 1.71 1656 A3 26 A1 LSD003 8.015 1.71 1656 A3 399 A2 LSD0039.001 1.71 1656 A3 403 A2 LSD0039.003 1.71 1656 A3 403 A2 LSD0039.010 1.71 1656 A3 1725 A3 5.001 1.71 1656 A3 1720 A3 LSD0045.002 1.71 1900 A3 18 A1 LSD0042.014 1.71 1900 A3 18 A1 LSD0042.023 1.71 1900 A3 26 A1 LSD0042.006 1.71 1900 A3 26 A1 2.013 1.71 1900 A3 40 A1 LSD0042.001 1.71 1900 A3 40 A1 LSD0042.039 1.71 1900 A3 26 A1 LSD0042.047 1.71 1905 A3 18 A1 LSD0042.003 1.71 1905 A3 40 A1 LSD0042.004 1.71 1905 A3 26 A1 LSD0042.008 1.71 1905 A3 26 A1 LSD0042.038 1.71 1905 A3 40 A1 LSD0042.082 1.71 1910 A3 26 A1 LSD0042.040 1.71 18 A1 399 A2 LSD0037.002 1.71 26 A1 399 A2 LSD0037.009 1.71 40 A1 399 A2 LSD0037.011 1.71 18 A1 403 A2 LSD0047.002 1.71 18 A1 403 A2 LSD0047.005 1.71 18 A1 403 A2 LSD0048.007 1.71 1656 A3 1900 A3 LSD0046.001 1.71 1656 A3 1900 A3 6.002 1.71 1656 A3 1905 A3 LSD0046.003 1.71 1711 A3 40 A1 LSD0040.011 1.71 1711 A3 26 A1 0.042 1.71 1720 A3 26 A1 LSD0040.002 1.71 1720 A3 40 A1 LSD0040.008 1.71 1720 A3 18 A1 LSD0040.021 1.71 1720 A3 26 A1 LSD0040.037 1.71 1720 A3 18 A1 LSD0040.046 1.71 1725 A3 26 A1 LSD0040.003 1.71 1725 A3 40 A1 LSD0040.006 1.71 1725 A3 26 A1 LSD0040.007 1.71 1725 A3 18 A1 LSD0040.010 1.71 1725 A3 40 A1 0.039 1.71 1725 A3 18 A1 LSD0040.052 1.71 1720 A3 403 A2 LSD0041.001 1.71 1720 A3 399 A2 LSD0041.004 1.71 1711 A3 403 A2 LSD0041.006 1.71 1720 A3 403 A2 LSD0041.008 1.71 Insertion 1 Insertion 2 Insertion 3 Insertion 4 Ratio Insert . Insert . Insert Domain Domaln Domaln.

Site Site Site Insert XTBN Slte Domaln. Construct Radn 1725 A3 403 A2 LSD0041.010 1.71 1725 A3 403 A2 1.014 1.71 1725 A3 399 A2 LSD0041.016 1.71 1711 A3 403 A2 1.035 1.71 1900 A3 399 A2 LSD0043.001 1.71 1900 A3 403 A2 LSD0043.002 1.71 1905 A3 403 A2 LSD0043.005 1.71 1900 A3 399 A2 LSD0043.006 1.71 1900 A3 403 A2 LSD0043.007 1.71 1900 A3 403 A2 LSD0043.008 1.71 1905 A3 399 A2 LSD0043.015 1.71 1905 A3 403 A2 LSD0043.029 1.71 1910 A3 403 A2 LSD0043.043 1.71 26 A1 403 A2 1656 A3 pSD0077 2.30 26 A1 403 A2 1720 A3 pSD0078 2.30 26 A1 403 A2 1900 A3 9 2.30 26 A1 1656 A3 1720 A3 0 2.30 26 A1 1656 A3 1900 A3 pSD0081 2.30 26 A1 1720 A3 1900 A3 pSD0082 2.30 403 A2 1656 A3 1720 A3 pSD0083 2.30 403 A2 1656 A3 1900 A3 pSD0084 2.30 403 A2 1720 A3 1900 A3 pSD0085 2.30 1656 A3 1720 A3 1900 A3 pSD0086 2.30 26 A1 403 A2 1656 A3 1720 A3 pSD0087 2.83 26 A1 403 A2 1656 A3 1900 A3 pSD0088 2.83 26 A1 403 A2 1720 A3 1900 A3 pSD0089 2.83 26 A1 1656 A3 1720 A3 1900 A3 pSD0090 2.83 403 A2 1656 A3 1720 A3 1900 A3 pSD0091 2.83 26 A1 403 A2 1656 A3 1720 A3 pSD0092 2.83 18 A1 745 B 2332 CT 9.002 2.24 26 A1 745 B 2332 CT LSD0049.008 2.24 26 A1 745 B 2332 CT LSD0049.011 2.24 40 A1 745 B 2332 CT LSD0049.012 2.24 40 A1 745 B 2332 CT LSD0049.020 2.24 18 A1 745 B 2332 CT LSD0049.021 2.24 40 A1 745 B 2332 CT LSD0050.002 2.24 18 A1 745 B 2332 CT LSD0050.003 2.24 26 A1 745 B 2332 CT LSD0050.007 2.24 18 A1 745 B 2332 CT LSD0050.010 2.24 26 A1 745 B 2332 CT LSD0050.012 2.24 40 A1 745 B 2332 CT LSD0050.014 2.24 403 A2 745 B 2332 CT LSD0051.002 2.24 399 A2 745 B 2332 CT LSD0051.003 2.24 403 A2 745 B 2332 CT LSD0052.001 2.24 399 A2 745 B 2332 CT LSD0052.003 2.24 1725 A3 745 B 2332 CT LSD0053.021 2.24 1720 A3 745 B 2332 CT LSD0053.022 2.24 1711 A3 745 B 2332 CT LSD0053.024 2.24 1720 A3 745 B 2332 CT LSD0054.021 2.24 1711 A3 745 B 2332 CT LSD0054.025 2.24 1725 A3 745 B 2332 CT LSD0054.026 2.24 Insertion 1 Insertion 2 Insertion 3 Insertion 4 Ratio Insert Insert Site Domain.

XTEN Construct Radn 1900 B 1905 B 1900 B 1900 B 1900 B 1910 B Exam le 27: Bindin Interference of FVIII-XTEN t0 anti-FVIII Antibod The ability ofXTEN inserted into different locations of CFXTEN fusion proteins to affect the binding of anti-FVIII antibodies was determined by sandwich ELISA assays. Two anti-FVIII antibodies; i.e. GMA—8021 (Green Mountain Antibodies, Burlington, VT) and ESH8 (American Diagnostica Inc., rd, CT), that bind to the A2 and C2 domains, respectively were utilized as capture antibodies. A non-XTEN containing FVIII-His-Myc n was used as a calibration standard and positive control for all ELISAs. Ten CFXTEN fusion proteins with single XTEN ions in either the Al, A2 or A3 domains were created that onally contained His and Myc affinity tags. The protein concentrations of each test sample was ized to 100% based on an anti-His capture-anti-Myc detection ELISA run concurrently on the same plate as the anti-FVIII antibody capture-anti-Myc detection ELISA.

Briefly, appropriate wells on a l plate were coated with GMA—8021, ESH8 or anti-His antibody overnight at 4°C, then were washed and blocked with BSA. Equal volumes of the respective control or fusion proteins were introduced into duplicate wells and allowed to interact with coated GMA- 8021, ESH8 or anti-His antibody for 2h at room ature. After tion, d material was washed away and a rabbit anti-Myc detection antibody was added and incubated for an additional h at room temperature. The plate was then washed and a peroxidase-conjugated donkey anti-rabbit secondary antibody was introduced and incubated for 1h at room temperature. The plate was washed again, followed by the addition of TMB ate and the reaction was allowed to proceed for 5-20 min.

H2SO4 was introduced to stop the reaction and absorbance was read by spectrophotometer at 450nm.

RLults: The results are presented in Table 29. Collectively, the results demonstrate that the two antibodies against the CFXTEN fusion proteins with XTEN ed into the A2 domain exhibited reduced g of FVIII compared to CFXTEN with XTEN inserted into the Al or A3 domain when the VIII e antibody was GMA—8021 (with binding affinity to the A2 domain). In contrast, there was no discernible pattern of inhibition or enhancement of binding by any of the CFXTEN when the anti- FVIII capture antibody was ESH8, with binding affinity to the C2 domain.

Table 29: Bindin Interference of FVIII-XTEN t0 anti-FVIII Antibod XTEN insertion Concentration on aFVIII/Myc + concentration on aHis/Myc Sample Tested (Domain, site, His/M GMA—SOZl/Myc (A2 ESHS/Myc XTEN) yc domain) ((12 domain) His-Myc 100% 104% FVIII-XTEN—His- A2, 403, AE144 100% 103%: 1% 141%:24% Myc A2, 403, AG144 100% 104% :: 6% 129% :: 12% A2, 399, AE144 100% 100% :: 8% 140% :: 18% A3, 1656, AG144 100% 153% 158% A1, 18, AE144 100% 129% 130% A1,18, AG144 100% 150% 131% A1, 26, AE144 100% 155% 87% A1, 26, AG144 100% 157% 147% A1, 40, AE144 100% 137% 147% A1, 40, AG144 100% 164% :: 0% 153% :: 18% aFVIII/Myc = GMA—8021/Myc or yc antibody condition; aHis/Myc = anti-His/Myc antibody condition Example 28: Activity Assay of CFXTEN fusion proteins in the ce of FVIII inhibitors Inhibitor Testing Titration Procedure: Select antibodies inhibiting FVIII procoagulant activity were purchased from commercial sources. The antibodies target select s of FVIII (e. g. A2, A3, C1, C2) and inhibit FVIII- dependent procoagulant activity. In order to establish the optimal tration of FVIII inhibitors to utilize in the assay, an initial ion experiment was performed using varying amounts of each inhibitory antibody ted at 37°C for 2 hrs with the base vector expressing wild-type FVIII with a His/Myc double tag, and a second sample with antibody and at least one CFXTEN fusion protein. The samples were then utilized in a coagulation assay to determine the FVIII activity. The activity was measured by the Coatest assay procedure described herein. The concentration that resulted in optimal inhibition of FVIII activity was determined for each antibody individually.

Inhibitor Testing Procedure: The FVIII inhibitor antibodies were then used at their optimal concentration for assay of test samples. CFXTEN and positive control s were individually incubated with each antibody at 37°C for 2 hrs and the samples were then collected and ed in the Coatest activity assay, along with untreated aliquots of the CFXTEN and positive control. In some cases, CFXTEN constructs with a R1648A mutation were tested to determine the effect, if any, of this mutation on resistance to inhibitors as ed by the retention of FVIII activity.

Res—ults: The results of the titration experiment are shown in . The data indicate a right-shift of approximately 0.7 order of ude in the amount of antibody ed to inhibit the procoagulant ty of the CFXTEN LSD0049.002 to the 50% level, compared to FVIII positive control, indicating that the CFXTEN with three XTEN insertions (at insertion points corresponding to amino acid e 18, 745 and 2332 of the BDD-FVIII) had lower binding with the antibody compared to FVIII, reflected in the retention of coagulation activity.

The results of the t assays are presented in Tables 30 and 31, for the FVIII inhibitor antibodies GMA8008 and GMA8021, respectively. All of the untreated CFXTEN fusion protein constructs tested exhibited procoagulant activity, as did the pBCOOl 14 FVIII positive control. The ve control sample pre-incubated with FVIH inhibitor antibodies resulted in a sharp decrease in the measured coagulation activity to 0.05-O.15 (5-15%) relative to the untreated sample, as did the majority of the CFXTEN constructs treated with the GMA8008 antibody to the C2 domain. However, three CFXTEN fusion proteins ed at least twice the relative remaining activity compared to the FVIII l; LSDOO49.020, LSD0053.024, and LSD0056.025, each with three XTEN inserts.

] The CFXTEN samples showed a lower degree of inhibition with the GMA8021 antibody to the A2 domain compared to untreated samples that was further reduced by either the additional numbers of XTEN inserts (tabular data shown in Table 30). shows the graph of median values of the ratio to control of retained activity showing a linear relationship between s ofXTEN inserted and reduced inhibition to the GMA8021 antibody relative to the inhibition of the FVIII l. Similarly, the means i S.E. for the ratio to control values were 2.26i0.l2 for l XTEN, 3.48+O.26 for 2 XTEN and 5.70i0.29 for 3 XTEN insertions. CFTXEN with at least three XTEN inserts treated with the GMA8021 dy had at least 4.5 to ld greater retention of FVIII activity compared to FVIII control. In addition, in those CFXTEN with three XTEN insertions, constructs with a higher degree of separation (in numbers of amino acid residues) between any two ions appeared to result in a higher degree of procoagulant activity and, hence, less binding by the FVIH inhibitor antibody, compared to insertions clustered more closely; e.g. on the C-terminal side of the B-domain. The assay results of constructs with the R1648A mutation appeared to be comparable to those without the mutation. sions: The results support that, under the conditions of the experiments, insertion of XTEN into FVIH resulted in protection against binding by FVIII inhibitors, with retention of procoagulant activity, and that ion of multiple XTEN s increased resistance to, in particular, the A2 domain inhibitor antibody. Lastly, there appears to be an effect by having spatial separation between the XTEN inserts.

Table 30: Results of Coagulation Assay with CFXTEN treated with antibody GMA8008 to C2 Domain Construct Y" Ratio t0 XTEN XTEN XTEN Remaining .

. . . Mutations Name Actmty. .

Control Insertlon 1 Insertion 2 Insertion 3 pBC0114 CT 0.05-0.15 l pBC0149 0.1 0.8 E42_1 E144_5 pSD0045 0.3 l . l A pSD0046 0.3 1.0 0018_AG144_F pSD0050 0.2 0.9 0026_AG144_F 0040_AE144_5 pSD0051 0.3 1.3 A pSD0052 0.2 1.0 0040_AG144_F 0403_AE144_2 pSD0001 0.2 0.9 A pBC0136 0.2 1.2 0745_AE288_1 pBC0137 0.2 1.1 0745_AE288_1 R1648A Construct R9131”? Ratio to XTEN XTEN XTEN .

Name Remaining Mutations Control Insertion 1 Insertion 2 Insertion 3 Actmty pSD0013 0.1 0.9 2332_AE144_6B 4 0.1 0.8 2332_AG144_1 pSD0019 0.1 0.5 2332_AE288_1 pBC0146 0.1 0.7 2332_AG288_1 pSD0015 0.1 0.8 2332_AE864 LSD0038.008 0.1 0.9 0018_AG144_F 1656_AG144_C LSD003 8.013 0.1 0.6 0040_AG144_F G144_C LSD003.09 0.1 0.9 0745_AE144_3B 2332_AE288_1 LSD003.06 0.0 0.8 0745_AE144_3B E288_1 R1648A LSD0046.001 0.0 0.6 1656_AG144_C 1900_AG144_C 0403_AE144_2 G144_ PSD077 0.1 1.0 0026_AG144_F A C 1720_AG144_ PSD080 0.1 1.0 0026_AG144_F 1656_AG144_C C 0403_AE144_2 1720_AG144_ PSD083 0.1 0.8 A 1656_AG144_C C 0403_AE144_2 E144_ PSD084 0.1 0.9 A 1656_AG144_C 4A 0018_AE144_5 2332_AE288_ LSD0050.010 0.1 0.7 A 0745_AE144_3B 1 0018_AE144_5 2332_AE288_ LSD0049.021 0.0 0.6 A 0745_AE144_3B 1 R1648A 2332_AE288_ LSD0049.002 0.1 0.9 0018 AG144 F 0745 AE144 3B 1 R1648A 0026_AE144_5 2332_AE288_ LSD0049.008 0.1 0.9 A 0745_AE144_3B 1 R1648A 2332_AE288_ LSD0049.011 0.1 0.9 0026_AG144_F 0745_AE144_3B 1 R1648A 0040_AE144_5 2332_AE288_ LSD0049.020 0.2 2.6 A 0745_AE144_3B 1 R1648A 2332_AE288_ LSD0050.002 0.0 0.2 G144_F 0745_AE144_3B 1 1711_AE144_4 2332_AE288_ LSD0053.024 0.2 2.5 A 0745_AE144_3B 1 2332_AE288_ LSD0054.021 0.2 1.5 1720_AG144_C 0745_AE144_3B 1 1900_AE144_4 2332_AE288_ LSD0055.021 0.2 1.6 A 0745_AE144_3B 1 R1648A E288_ LSD0056.021 0.2 1.6 1900_AG144_C 0745_AE144_3B 1 2332_AE288_ LSD0056.025 0.3 2.0 1910_AG144_C 0745_AE144_3B 1 proportion of activity remaining relative to ponding ted sample The ratio of the relative remaining activity (relative to its own control) compared to FVIII pBC0114 positive control Table 31: Results of Coagulation Assay with CFXTEN treated with antibody GMA8021 to A2 Domain AetiVity Control pBC0114 0.05-0.15 1 pBCO 149 0.2 1.3 0745_AE42_1 pSD0045 0.3 2.7 0018_AE144_5A pSD0046 0.2 2.1 0018_AG144_F pSD0050 0.2 2.4 0026_AG144_F pSD0051 0.3 3.1 0040_AE144_5A pSD0052 0.3 2.7 0040_AG144_F pSD0001 0.2 1.6 0403 AE144 2A pBC0136 0.3 2.4 0745_AE288_1 pBC0137 0.3 2.4 E288_1 R1648A pSD0013 0.2 1.8 2332_AE144_6B pSD0014 0.2 2.1 2332_AG144_1 pBC0145 0.3 2.1 2332_AE288_1 pSD0019 0.3 2.3 2332_AE288_1 pBC0146 0.3 2.1 2332_AG288_1 pSD0015 0.3 2.8 2332_AE864 LSD0038.008 0.4 3.0 0018_AG144_F 1656_AG144_C LSD0038.013 0.4 3.0 0040_AG144_F 1656_AG144_C LSD003.09 0.3 3.6 E144_3B 2332_AE288_1 LSD003.06 0.3 3.4 0745 AE144 3B 2332 AE288 1 R1648A LSD0046.001 0.2 4.4 1656_AG144_C G144_C PSD077 0.4 5.8 0026_AG144_F 0403_AE144_2A 1656_AG144_C PSD080 0.4 5.7 0026_AG144_F 1656_AG144_C 1720_AG144_C PSD083 0.3 5.0 0403_AE144_2A 1656_AG144_C 1720_AG144_C PSD084 0.3 4.5 E144_2A 1656_AG144_C 1900_AE144_4A LSD0050.010 0.4 6.7 0018_AE144_5A 0745_AE144_3B 2332_AE288_1 LSD0049.021 0.4 6.7 0018_AE144_5A 0745_AE144_3B 2332_AE288_1 R1648A LSD0049.002 0.5 9.2 0018_AG144_F 0745_AE144_3B 2332_AE288_1 R1648A LSD0049.008 0.4 5.9 0026_AE144_5A 0745_AE144_3B 2332_AE288_1 R1648A LSD0049.011 0.4 5.6 0026_AG144_F 0745_AE144_3B 2332_AE288_1 R1648A 9.020 0.3 5.0 0040_AE144_5A 0745_AE144_3B E288_1 R1648A 0.002 0.3 6.2 0040 AG144 F 0745 AE144 3B 2332 AE288 1 LSD0053 .024 0.3 4.5 1711_AE144_4A 0745_AE144_3B 2332_AE288_1 LSD0054.021 0.5 5.2 G144_C 0745_AE144_3B E288_1 .021 0.5 5.4 1900_AE144_4A 0745_AE144_3B 2332_AE288_1 R1648A LSD0056.021 0.5 5.1 1900_AG144_C 0745_AE144_3B 2332_AE288_1 LSD0056.025 0.5 4.8 1910_AG144_C 0745_AE144_3B 2332_AE288_1 proportion of activity remaining relative to ponding untreated sample The ratio of the relative remaining activity (relative to its own control) ed to FVlll pBC0114 positive control Example 29: Protein ation of CFXTEN fusion proteins 5 and pBC0146 Two CFXTEN constructs with C-terminal XTEN were utilized to establish a purification method. For both pBC0145 with a C-terminal XTEN of 288 amino acids of the AE family (see sequence in Table 21) and pBC0146 with a C-terminal XTEN of 288 amino acids of the AG family (see sequence in Table 21), a tangential flow filtration (TFF) step was used to buffer exchange the clarified conditioned media from cell culture. Products were then captured using a strong anion exchange chromatography resin, and then further d using VIIIS elect affinity tography (GE Healthcare). An additional size exclusion chromatography (GE Healthcare) was d to FVIII-pBC0146 as a third polish step to remove high molecule weight species. The purity of both fusion proteins was deemed acceptable by HPLC-SEC and was further confirmed by SDS-PAGE analysis of the two CFXTEN constructs showing CFXTEN products at expected sizes. The specific activity of both molecules was comparable to B- domain deleted FVIII, as measured by aPTT ation assay and ELISA determination of FVIII concentration.

Example 30: Pharmacokinetics of CFXTEN fusion proteins pBC0145 and pBC0146 in HemA and WF DKO mice Male FVIII knock-out (HemA) mice or FVIII/VWF double knock-out (DKO) mice, 8-12 weeks old, were treated with a single intravenous administration of either recombinant BDD-FVIII, the CFXTEN pBC0145 0r pBC0146 fusion purified proteins (from Example 23) at 200 IU/kg dose ime point). At select time points, blood samples were collected via vena cava sampling. In HemA mice, blood samples were collected at 5 min, 1 4, 8, 16, 20, 24, 32, and 48 hrs post-dosing for rBDD-FVIII, and at 5 min, 8, 16, 24, 32, 48, 55 and 72 hrs post-dosing for pBC0145 and pBC0146 fusion proteins. In the VWF DKO mice, blood samples were collected at 5min, 30 min and 1hr post-dosing for rBDD-FVIII, and at 5 min, 4, 8, 16 and 24 hr post-dosing for the pBC0145 and pBC0146 fusion ns.

Plasma FVIII activity was measured by FVIII chromogenic assay and the PK profile was analyzed by the WinNonlin program. ts: As show in Table 32 and , CFXTEN with the AE C-terminus XTEN insertion (pBC0145) exhibited ld and 14.1-fold FVIII half-life (Tl/2) ion compared to rBDD FVIII in HemA mice and FVIII/VWF DKO mice, respectively. The CFXTEN with the AG C-terminus XTEN insertion (pBC0146) had 1.4-fold and 14.4-fold extended half-life ed to rBDD-FVIII in the HemA mice and FVIII/VWF DKO mice, tively. The magnitude of the FVIII half-life extension conferred by XTEN insertion was much more pronounced in the FVIII/VWF DKO mice compared to the HemA mice, demonstrated by the 14-fold longer FVIII half-life from both FVIII-AE-XTEN and FVIII-AG- XTEN compared to rBDD-FVIII. In addition, in comparison to rBDD-FVIII, FVIII with inal AE 0r AG-XTEN insertion also had cantly improved FVIII recovery at the 5 min interval, reduced clearance and volume of distribution, and increased AUC in the DKO mice. Under the conditions of the experiment, CFXTEN with C-terminus XTEN insertions demonstrated great potential on FVIII half-life extension, and, when combined with other FVIII intra-domain insertions could potentially further extend FVIII half-life.

Table 32: Pharmacokinetic ters of CFXTEN in HemA and FVIIINWF DKO mice min AUC D Mouse Tm MRT C1 Vss — Tm Fold Mouse Strain.

Treatment Recovery (hrkngU/m (hr) (hr) (mL/hr/kg) (mL/kg) Increase Strain.

(%) L/mIU) pBC0145 73 11.88 16.47 3.81 62.74 0.26 1.6 HemA 6 64 10.54 13.31 5.66 75.34 0.18 1.4 HemA ; 89 7.58 11.02 4.33 47.68 0.23 FVIII/ pBC0145 74 3.38 3.76 13.06 63.68 0.0765 13.9 VWF FVIII/ pBC0146 61 3.45 3.61 17.40 86.63 0.0575 14.2 ; 23 0.24 0.24 460.62 161.51 0.0022 ed to rBDD-FVIII Example 31: Cell culture and concentration of cell culture media for CFXTEN fusion proteins pSD0050 and pSD0062 CFXTEN construct variants pSD0050 with an intradomain AG XTEN of 144 amino acids inserted after amino acid residue 26 of BDD FVIII with an intradomain AE XTEN of 144 , pSDOO62 amino acids inserted after residue 1900 of BDD FVIII (Note: amino acid numbering based full length , as well as a construct encoding rBDD-FVIII, were transfected into HEK293F cells (Invitrogen, Carlsbad, CA) using polyethyleneimine (PEI, Polysciences Inc. Warrington, PA). The transiently transfected cells were grown in 293 Free Style medium media (Invitrogen, Carlsbad, CA) for 4 days and 50-100 ml cell culture media were then concentrated 10- to 20-fold by Centricon Spin Column (100 kDa MW cut-oft) to reach 10-30 IU/ml FVIII activity. The concentrated materials were then flash-frozen and stored at -80°c for future in vitro analysis and in vivo pharmacokinetic studies.

Example 32: Pharmacokinetics of CFXTEN fusion proteins pSD0050 and pSD0062 in HemA and FVIIINWF DKO mice Male HemA or FVIII/VWF double knock-out (DKO) mice, 8-12 weeks old, were treated with a single intravenous administration of cell culture concentrates from Example 31 ning either recombinant BDD-FVIII, the CFXTEN pBDOOSO or pBD062 at 100-300 IU/kg (n=3/group). At select time points, blood samples were collected Via retro orbital bleeds from the same set of mice. In HemA mice, blood samples were collected at 5 min, 24 hr and 48 hr post-dosing, while in FVIII/VWF DKO mice blood s were collected at 5 min, 8 hr and 16 hr. The FVIII activity of plasma samples and cell culture concentrates were analyzed by a FVIII chromogenic assay, and the PK profile of rBDD FVIII and FVIII-XTEN variants were analyzed using the WinNonlin program.

Res—ults: The PK profiles of the two CFXTEN intradomain insertion variants pSD0050 and pSDOO62 and rBDD-FVIII in HemA mice and FVIII/VWF DKO mice are shown in and Table 33. In HemA mice, a comparable initial recovery at the 5 min interval was observed for the three test FVIII molecules. Both CFXTEN fusion proteins trated ld longer half-life compared to wild-type BDD-FVIII. In FVIII-VWF DKO mice, because of the loss ofVWF protection, rBDD-FVIII had only a 15 min plasma half-life. In the case of the two CFXTEN, however, half-life were extended to 3.15 hr and 3.83 hr, respectively; values that are comparable to the CFXTEN with 288 C-terminus XTEN insertions (Example 24), suggesting that r extension of the XTEN length at a given insertion point may not be necessary. Under the experimental conditions, the study results clearly demonstrate that intradomain insertion of an XTEN with 144 amino acid residues not only preserved FVIII ty, but also provided similar FVIII half-life benefit as the C-terminus 288 amino acid XTEN insertion variants, suggesting that the combination of the FVIII intradomain and C-terminus insertions may allow further extension of FVIII half-life.

Table 33: Pharmacokinetic parameters of CFXTEN in HemA and FVIIINWF DKO mice Mouse Treatment Rejcbriigry Tm MRT Cl VSS lrrcilfi?mL Tm POM Stram (hr) (hr) /kg) (mL/kg) Increase (%) /mIU) pSD0050 40 14.12 14.25 5.27 75.03 0.19 2.3 HemA pSD0062 43 12.96 14.79 4.24 62.67 0.24 2.1 11131_ 47 6.19 2.62 6.35 16.62 0.16 pSD0050 34 3.15 2.59 21.73 56.28 0.05 ~12 FVIII/ VWF pSD0062 35 3.83 3.71 18.51 68.69 0.05 ~15 DKO T331? 23 ~0.25 Compared to rBDD-FVIII ] Example 33: Pharmacokinetic analysis of CFXTEN fusion polypeptides in rats ] The pharmacokinetics of various CFXTEN fusion proteins, compared to FVIII alone, are tested in Sprague-Dawley rats. CFXTEN and FVIII are administered to female e-Dawley rats (n=3) IV through a jugular vein catheter at 3-10 ug/rat. Blood s (0.2 mL) are collected into pre-chilled heparinized tubes at predose, 0.08, 0.5, 1, 2, 4, 8, 24, 48, 72 hour time points, and sed into plasma.

Quantitation of the test es is performed by ELISA assay using an anti-FVIII antibody for both capture and detection. A non-compartmental analysis is performed in WinNonLin with all time points included in the fit to ine the PK parameters. Results are expected to show increased terminal half- life and area under the curve, and a reduced volume of distribution for the CFXEN compared to FVIII alone, and the results are used in conjunction with results from coagulation and pharmacodynamic assays to select those fusion protein configurations with desired properties.

Example 34: Analysis of FVIII for XTEN insertion sites The selection ofXTEN insertion sites within the factor VIII molecule was performed by predicting the locations of permissive sites within loop structures or otherwise flexible surface exposed structural elements. For these analyses, the atomic coordinates of two independently determined X-ray crystallographic structures of FVIII were use (Shen BW, et al. The tertiary structure and domain organization of coagulation factor VIII. Blood. (2008) Feb 1;111(3):1240-1247; Ngo JC, et al. Crystal structure of human factor VIII: ations for the formation of the factor IXa-factor VIIIa complex.

Structure (2008)16(4):597-606), as well as those of factor VIII and factor VIIIa derived from molecular dynamic simulation (MDS) teswarlu, D. Structural investigation of zymogenic and activated forms of human blood coagulation factor VIII: a computational molecular dynamics study. BMC Struct Biol. (2010) 10:7). Atomic coordinates in Protein Data Bank (PDB) format were analyzed to identify regions of the FVIII/FVIIIa predicted to have a high degree solvent accessible surface area using the algorithms ASAView (Ahmad S, et al. ASAView: database and tool for solvent accessibility representation in proteins. BMC Bioinformatics (2004) 5:51) and GetArea (Rychkov G, Petukhov M.

Joint neighbors approximation of macromolecular solvent accessible surface area. J Comput Chem (2007) 28(12):1974-1989). The resulting set of sites was then further prioritized on the basis of high predicted atomic positional fluctuation based on the basis of the published results of the MDS study.

Sites within the acidic peptide regions g the A1, A2, and A3 domains, as well as those that appeared by visual inspection to be in areas other than surface exposed loops were ritized. The resulting set of potential sites was evaluated on the basis of interspecies sequence vation, with those sites in regions of high sequence conservation among 20 vertebrate species being ranked more favorably. Additionally, putative clearance receptor g sites, FVIII interaction sites with other molecules (such as vWF, FIX), domain and exon boundaries were also considered in fusion site selection. Finally, sites within close proximity to mutations implicated in hemophilia A listed in the Haemophilia A Mutation, Search, Test and Resource Site (HAMSTeRS) database were eliminated (Kemball-Cook G, et al. The factor VIII Structure and on Resource Site: HAMSTeRS version 4. c Acids Res. (1998) 216-219). Based on these criteria, the construction of 42 FVIII-XTEN variants was proposed for XTEN ions. Of these, three represent XTEN insertions within the residual B domain sequence, two ent extensions to the C-terminus of the factor VIII molecule, and 37 represent XTEN insertions within structurally def1nedinter- and omain structural elements; i.e., residues 3, 18, 22, 26, 40, 60, 116, 130, 188, 216, 230, 333, 375, 403, 442, 490, 518, 599, 713, 745, 1720, 1796,1802,1827, 1861,1896, 1900,1904,1937, 2019, 2068, 2111, 2120, 2171, 2188, 2227, 2277, and 2332.

Example 35: onal is of FVIII-XTEN constructs Two FVIII-XTEN fusion proteins, FVIII-AE288 (F8X-40) and FVIII-AG288 (F8X-41), contain an AE288_1 XTEN or an 1 XTEN, respectively, fused at the C-terminus of FVIII C2 . To determine if FVIII activity was ed after XTEN fusion, HEK293 cells were transfected separately with these two FVIII-XTEN fusion constructs by using polyethylenimine (PEI) in serum-free medium. At 3 or 5 days post-transfection, the cell culture supernatant was tested for FVIII activity by a two-stage chromogenic assay. Purified recombinant FVIII, ated against WHO international standard, was used to establish the standard curve in the chromogeinic assay. The fusion n products of both F8X-40 and F8X-41 constructs were expressed at levels comparable to those of wild- type BDD-FVIII constructs. (Table 34).

Table 34. FVIII Titer of FVIII-XTEN fusion roteins in transient transfection cell culture FVIII Molecules FVIII O66a pBC 0114a F8X~40 F8X~41 awn B a. Both FVIII 066 and pBC 0114 contain B-domain deleted FVIII without XTEN fusion. b. The F8X-4lsample was from a 3-day transfection while other samples were from a 5-day transient transfection.

Example 36: Functional analysis of FVIII-XTEN constructs: FVIII activity and PK ties The half-life extension potential of the F8X-4O and F8X-4l constructs was evaluated in FVIII and von Willebrand factor double out mice by hydrodynamic plasmid DNA ion, with a FVIIIFc DNA construct serving as a positive control. Mice were randomly d into 3 groups with 4 mice per group. Plasmid DNA encoding BDD FVIIIFc fusion protein, F8X-4O or F8X-4l, all sharing the same DNA vector ne, was administered to mice in the tive groups. Approximately 100 micrograms of the appropriate plasmid DNA was injected into each mouse via hydrodynamic injection, and blood plasma samples were collected at 24 hours and 48 hours post-injection. The plasma FVIII activity was measured by a two-stage chromogenic assay using calibrated recombinant FVIII as a standard. As shown in , s from the F8X-4O and F8X-4l groups showed higher plasma FVIII titers than did those from the BDD C, suggesting FVIII fusion with XTEN gs the half- life of FVIII in viva. Taken together, these data support the sion that FVIII-XTEN fusion proteins retained FVIII activity in transient transfection and exhibited prolonged circulating half-life in an animal model.

Example 37: Pharmacodynamic evaluation of CFXTEN in animal models The in vivo pharmacologic activity of CFXTEN fusion proteins are assessed using a y of preclinical models of bleeding including but not limited to those of hemophilia, surgery, trauma, thrombocytopenia/platelet dysfunction, clopidogrel/heparin—induced bleeding and hydrodynamic injection. These models are developed in multiple species including mice, rat, rabbits, and dogs using methods equivalent to those used and published for other FVIII approaches. CFXTEN compositions are ed in an aqueous buffer compatible with in vivo administration (for example: phosphate-buffered saline or Tris-buffered saline). The compositions are administered at appropriate doses, dosing frequency, dosing schedule and route of administration as optimized for the particular model. Efficacy determinations include measurement of FVIII ty, age clotting assay, FVIII chromogenic assay, activated partial prothrombin time (aPTT), ng time, whole blood clotting time (WBCT), thrombelastography (TEG or ROTEM), among others.

In one example of a PD model, CFXTEN and FVIII are administered to cally-deficient or experimentally-induced HemA mice. At s time points post-administration, levels of FVIII and CFXTEN are measured by ELISA, activity of FVIII and CFXTEN is measured by commercially- available FVIII activity kits and clotting time is measured by aPTT assay. Overall, the results can indicate that the CFXTEN constructs may be more efficacious at inhibiting bleeding as compared to FVIII and/or equivalent in potency to able dosage of FVIII with less frequent or more convenient dosing intervals.

In a mouse ng challenge PD model CFXTEN and FVIII are administered to cally- deficient or experimentally-induced HemA mice and effect on hemostatic challenge is measured.

Hemostatic challenge can include tail transaction challenge, hemarthropthy challenge, joint bleeding or saphenous vein challenge among others. At various time points post-administration levels of FVIII and CFXTEN are measured by ELISA, activity of FVIII and CFXTEN are measured by cially available FVIII activity kit, bleeding time is measured and clotting time is measured by aPTT assay.

Overall the results are expected to indicate that the CFXTEN ucts are more efficacious at inhibiting bleeding as compared to FVIII and/or equivalent in potency to comparable dosage of FVIII With less frequent or more convenient dosing intervals, and the results are used in ction With results from coagulation and other assays to select those fusion protein configurations With desired properties.

In a dog PD model, CFXTEN and FVIII are administered to cally-deficient hemophiliac dogs. At various time points post administration, levels of FVIII and CFXTEN are measured by ELISA, activity of FVIII and CFXTEN are measured by commercially available FVIII activity kit and clotting time is ed by aPTT assay. Overall the results indicates that the CFXTEN constructs may be more efficacious at inhibiting bleeding as compared to FVIII and/or equivalent in potency to comparable dosage of FVIII With less frequent or more convenient , and the s are used in conjunction With results from coagulation and other assays to select those fusion protein configurations With desired properties.

In a dog bleeding challenge PD model CFXTEN and FVIII are administered to cally deficient hemophiliac dogs and effect on hemostatic challenge is measured. Hemostatic challenge includes cuticle bleeding time among others. At various time points post-administration levels of FVIII and CFXTEN are ed by ELISA, activity of FVIII and CFXTEN are measured by commercially available FVIII activity kit, bleeding time is measured and clotting time are measured by aPTT assay.

Overall the results indicate that the CFXTEN constructs may be more efficacious at inhibiting bleeding as compared to FVIII and/or equivalent in y to comparable dosage of FVIII With less frequent or more convenient dosing als, and the results are used in conjunction With results from coagulation and other assays to select those fusion protein configurations With desired properties.

Additional preclinical models of bleeding include but are not limited to those of hemophilia, surgery, trauma, thrombocytopenia/platelet dysfunction, clopidogrel/heparin—induced bleeding and hydrodynamic injection. These models can developed in multiple species ing mice, rat, rabbits, and dogs using methods equivalent to those used and published for other FVIII ches. l the results indicate that the CFXTEN constructs may be more efficacious at inhibiting bleeding as compared to FVIII and/or equivalent in potency to comparable dosage of FVIII with less nt or more convenient dosing als, and the results are used in conjunction With results from coagulation and other assays to select those filSiOIl protein configurations With desired properties. e 38: CFXTEN with cleavage sequences ] C-terminal XTEN releasable by FXIa A CFXTEN fusion protein consisting of an XTEN protein fused to the C-terminus of FVIII is created with an XTEN release site cleavage sequence placed in between the FVIII and XTEN components, as depicted in . Exemplary sequences are provided in Table 51. In this case, the release site cleavage sequence is incorporated into the CFXTEN that contains an amino acid sequence that is recognized and d by the FXIa protease (EC 3.4.21.27, Uniprot P03951). Specifically the amino acid sequence KLTRAET (SEQ ID NO: 1688) is out after the arginine of the sequence by FXIa protease. FXI is the procoagulant protease located immediately before FVIII in the intrinsic or contact activated coagulation pathway. Active FXIa is produced from FXI by proteolytic cleavage of the n by FXIIa. Production of FXIa is tightly controlled and only occurs when coagulation is necessary for proper hemostasis. Therefore, by incorporation of the KLTRAET cleavage sequence (SEQ ID NO: 1688), the XTEN domain is only be removed from FVIII concurrent with activation of the intrinsic coagulation y and when coagulation is ed physiologically. This creates a situation where the CFXTEN fusion protein is sed in one additional manner during the activation of the intrinsic pathway.

C-terminal XTEN releasable by FIIa [thrombin] A CFXTEN fusion n consisting of an XTEN n fused to the C-terminus of FVIII is created with an XTEN release site cleavage sequence placed in between the FVIII and XTEN components, as depicted in . In this case, the release site contains an amino acid sequence that is recognized and cleaved by the FIIa protease (EC 3.4.21.5, Uniprot P00734). Specifically the sequence LTPRSLLV (SEQ ID NO: 1618) [Rawlings N.D., et a1. (2008) Nucleic Acids Res, 36: D320], is out after the arginine at position 4 in the sequence. Active FIIa is produced by cleavage of FII by FXa in the presence of phospholipids and calcium and is down stream from factor IX in the coagulation pathway.

Once activated its natural role in coagulation is to cleave fibrinogen (, which then in turn, begins clot formation. FIIa activity is tightly controlled and only occurs when coagulation is necessary for proper hemostasis. ore, by incorporation of the LTPRSLLV ce (SEQ ID NO: 1618), the XTEN domain is only removed from FVIII concurrent with activation of either the extrinsic or intrinsic coagulation pathways, and when coagulation is required physiologically. This creates a situation where CFXTEN fusion is processed in one onal manner during the activation of coagulation.

C-terminal XTEN releasable by Elastase-2 A CFXTEN fusion protein consisting of an XTEN n fused to the C-terminus of FVIII is created with an XTEN release site cleavage sequence placed in between the FVIII and XTEN components, as ed in . Exemplary sequences are provided in Table 51. In this case, the release site contains an amino acid sequence that is recognized and cleaved by the e1astase-2 protease (EC 3.4.21.37, Uniprot P08246). Specifically the sequence VP (SEQ ID NO: 1689) ngs N.D., et a1. (2008) Nucleic Acids Res, 36: D320], is out after position 4 in the sequence. Elastase is constitutively expressed by phils and is present at all times in the circulation. Its activity is tightly controlled by serpins and is therefore minimally active most of the time. Therefore as the long lived CFXTEN circulates, a fraction of it is cleaved, creating a pool of shorter-lived FVIII to be used in coagulation. In a desirable feature of the inventive composition, this s a circulating ug depot that constantly releases a prophylactic amount of FVIII.

C-terminal XTEN releasable by MMP-12 A CFXTEN fusion protein consisting of an XTEN protein fused to the C-terminus of FVIII is created with an XTEN release site cleavage sequence placed in n the FVIII and XTEN components, as depicted in . Exemplary sequences are provided in Table 51. In this case, the release site contains an amino acid sequence that is recognized and d by the MMP-12 protease (EC 3.4.24.65, Uniprot P39900). Specifically the sequence GPAGLGGA (SEQ ID NO: 1690) [Rawlings N.D., et al. (2008) c Acids Res., 36: D320], is cut after position 4 of the sequence. MMP-12 is constitutively expressed in whole blood. Therefore as the long lived CFXTEN circulates, a fraction of it is cleaved, creating a pool of shorter-lived FVIII to be used in coagulation. In a desirable feature of the inventive ition, this creates a circulating pro-drug depot that ntly releases a prophylactic amount of FVIII.

C-terminal XTEN releasable by MMP-13 A CFXTEN fusion protein consisting of an XTEN protein fused to the inus of FVIII is created with an XTEN release site ge sequence placed in between the FVIII and XTEN components, as ed in . Exemplary sequences are provided in Table 51. In this case, the release site ns an amino acid sequence that is recognized and d by the MMP-13 protease (EC 3424-, Uniprot P45452). Specifically the sequence GPAGLRGA (SEQ ID NO: 1691) [Rawlings N.D., et al. (2008) Nucleic Acids Res., 36: D320], is cut after position 4. MMP-13 is constitutively expressed in whole blood. Therefore as the long lived CFXTEN circulates, a fraction of it is d, creating a pool of shorter-lived FVIII to be used in coagulation. In a desirable feature of the inventive composition, this s a circulating pro-drug depot that constantly releases a prophylactic amount of FVIII.

C-terminal XTEN releasable by MMP-17 A CFXTEN fusion protein consisting of an XTEN protein fused to the C-terminus of FVIII is created with an XTEN release site cleavage sequence placed in between the FVIII and XTEN components, as depicted in . Exemplary sequences are provided in Table 51. In this case, the release site contains an amino acid sequence that is ized and cleaved by the MMP-2O protease (EC.3.4.24.-, Uniprot Q9ULZ9). Specifically the sequence APLGLRLR (SEQ ID NO: 1692) [Rawlings N.D., et al. (2008) Nucleic Acids Res., 36: D320], is cut after position 4 in the sequence. MMP-17 is constitutively sed in whole blood. Therefore as the long lived CFXTEN circulates, a fraction of it is cleaved, creating a pool of shorter-lived FVIII to be used in coagulation. In a desirable feature of the inventive composition, this creates a circulating pro-drug depot that constantly releases a prophylactic amount of FVIII.

C-terminal XTEN releasable by MMP-20 A CFXTEN fusion protein consisting of an XTEN protein fused to the C-terminus of FVIII is created with an XTEN release site cleavage sequence placed in between the FVIII and XTEN components, as depicted in . Exemplary sequences are provided in Table 51. In this case, the release site contains an amino acid sequence that is recognized and cleaved by the MMP-2O protease (EC.3.4.24.-, Uniprot 060882). Specifically the sequence PALPLVAQ (SEQ ID NO: 1693) [Rawlings N.D., et a1. (2008) c Acids Res., 36: D320], is cut after position 4 (depicted by the arrow). MMP- is constitutively expressed in whole blood. Therefore as the long lived CFXTEN circulates, a fraction of it is cleaved, creating a pool of shorter-lived FVIII to be used in coagulation. In a desirable feature of the ive composition, this creates a circulating pro-drug depot that constantly releases a prophylactic amount of FVIII.

Optimization of the release rate ofXTEN ts of the ing Examples can be created in which the release rate ofXTEN incorporated at the C-terminus, the N—terminus, or internal XTEN is altered. As the rate ofXTEN release by an XTEN release protease is dependent on the sequence of the XTEN release site, by varying the amino acid sequence in the XTEN release site one can control the rate ofXTEN release. The sequence specificity of many proteases is well known in the art, and is documented in several data bases.

In this case, the amino acid specificity of ses is mapped using combinatorial libraries of ates [Harris, J. L., et a1. (2000) Proc Natl Acad Sci U S A, 97: 7754] or by following the cleavage of substrate es as illustrated in [Schellenberger, V., et a1. (1993) Biochemistry, 32: 4344]. An alternative is the fication of optimal protease cleavage sequences by phage y [Matthews, D., et a1. (1993) Science, 260: 1113]. Constructs are made with variant sequences and assayed for XTEN release using standard assays for detection of the XTEN polypeptides.

Example 39: Human Clinical Trial Designs for Evaluating CFXTEN comprising FVIII te® FS is recombinant human coagulation factor VIII, intended for promoting asis in hemophilia A subjects. Due to its short half— life, te is dosed intravenously every other day for prophylaxis and 8 to every 12 h in treatment of bleeds until hemostasis is achieved. It is believed that fusion of one or more XTEN to FVIII improves the half-life of the protein, enabling a reduced dosing frequency using such -containing fusion protein compositions.

Clinical trials are ed such that the efficacy and advantages of CFXTEN, relative to Kogenate or other commercially available FVIII preparations, can be verified in humans. Such studies comprises three phases. First, a Phase I safety and pharmacokinetics study in adult patients is conducted to determine the m tolerated dose and pharmacokinetics and pharmacodynamics in humans (either normal subjects or ts with hemophilia), as well as to define potential toxicities and adverse events to be tracked in future studies. The Phase I studies are conducted in which single rising doses of CFXTEN itions are administered by the route (e.g., aneous, intramuscular, or intravenously) and biochemical, PK, and clinical parameters are measured at defined als. This permits the determination of the minimum effective dose and the maximum tolerated dose and establishes the threshold and maximum concentrations in dosage and circulating drug that constitute the therapeutic window for the respective components, as well as bioavailability when administered by the intramuscular or subcutaneous routes. From this information, the dose and dose le that permits less frequent stration of the CFXTEN compositions, yet retains the pharmacologic response, is obtained. fter, clinical trials are conducted in patients with the ion, verifying the effectiveness of the CFXTEN compositions under the dose conditions, which can be conducted in comparison to a positive control such as Kogenate to establish the enhanced properties of the CFXTEN compositions.

Phase II and III clinical trials are conducted in patients suffering from any disease in which factor VIII may be ed to e clinical . For example, the CFXTEN is used in clinical trials for treatment of indications approved for use of factor VIII; such indications include bleeding episodes in hemophilia A, ts with inhibitors to factor VIII, prevention of bleeding in surgical interventions or invasive procedures in hemophilia A patients with inhibitors to factor VIII, treatment of ng episodes in patients with congenital factor VIII deficiency, and prevention of bleeding in al interventions or invasive procedures in patients with congenital factor VIII deficiency.

CFXTEN may also be indicated for use in additional patient populations. A phase II dosing study is conducted in hemophilia A patients where pharmacodynamic, coagulation, bleeding and other physiologic, PK, safety and clinical ters and clinical endpoints appropriate for trials are measured as a function of the dosing of the fusion ns compositions, yielding dose-ranging information on doses that is appropriate for a subsequent Phase III trial, in addition to collecting safety data related to adverse events. The PK parameters are correlated to the physiologic, clinical and safety parameter data to establish the therapeutic window and the therapeutic dose regimen for the CFXTEN composition, ting the clinician to establish the appropriate dose ranges for the composition. In one trial, hemophilia A patients with factor VIII inhibitors would be evaluated to establish doses and dose regimen of CFXTEN pharmaceutical compositions that result in achieVing and ining hemostasis and ting or attenuating bleeding episodes. Finally, a phase III efficacy study is ted wherein patients are administered the CFXTEN pharmaceutical composition and a ve control (such as a commercially-available Kogenate) are administered using a dosing schedule deemed appropriate given the pharmacokinetic and pharmacodynamic properties of the respective compositions derived from the Phase II findings, with all agents administered for an appropriately extended period of time to e the study endpoints. Parameters that are monitored include aPTT assay, one- or two-stage clotting assays, control of bleeding episodes, or the occurrence of spontaneous bleeding episodes; parameters that are tracked ve to the placebo or positive control groups. Efficacy outcomes are determined using standard statistical methods. Toxicity and e event markers are also be followed in this study to verify that the compound is safe when used in the manner described. In another phase III trial, hemophilia A patients with factor VIII inhibitors would be evaluated to establish the effectiveness of CFXTEN ceutical compositions in achieVing and maintaining hemostasis and preventing or attenuating bleeding episodes.

Example 40: Analytical size exclusion chromatography ofXTEN fusion proteins with diverse payloads Size exclusion chromatography analyses were performed on fusion proteins containing various therapeutic ns and unstructured recombinant proteins of increasing . An exemplary assay used a TSKGel-G4000 SWXL (7.8mm X 300m) column in which 40 ug of purified glucagon fusion protein at a concentration of 1 mg/ml was separated at a flow rate of 0.6 ml/min in 20 mM phosphate pH 6.8, 114 mM NaCl. Chromatogram s were monitored using OD214nm and OD280nm. Column calibration for all assays were performed using a size exclusion calibration standard from BioRad; the markers e thyroglobulin (670 kDa), bovine gamma-globulin (158 kDa), chicken ovalbumin (44 kDa), equine myoglobuin (17 kDa) and vitamin B12 (1.35 kDa). Representative chromatographic profiles of Glucagon-Y288, Glucagon-Y144, on-Y72, Glucagon-Y36 are shown as an y in . The data show that the apparent lar weight of each compound is proportional to the length of the attached XTEN sequence. However, the data also show that the apparent molecular weight of each construct is significantly larger than that expected for a globular protein (as shown by comparison to the standard proteins run in the same assay). Based on the SEC analyses for all ucts evaluated, including a CFXTEN composition, the apparent lar weights, the apparent molecular weight factor (expressed as the ratio of nt molecular weight to the calculated molecular weight) and the hydrodynamic radius (RH in nm) are shown in Table 35. The results indicate that incorporation of different XTENs of 576 amino acids or greater confers an apparent molecular weight for the fusion protein of approximately 339 kDa to 760, and that XTEN of 864 amino acids or greater s an apparent molecular weight greater than approximately 800 kDA. The results of proportional ses in apparent molecular weight to actual lar weight were consistent for fusion proteins created with XTEN from several different motif families; i.e., AD, AE, AF, AG, and AM, with increases of at least four-fold and ratios as high as about 17-fold. onally, the incorporation ofXTEN fusion partners with 576 amino acids or more into fusion proteins with the various payloads (and 288 residues in the case of glucagon fused to Y288) resulted with a hydrodynamic radius of 7 nm or greater, well beyond the glomerular pore size of approximately 3-5 nm. Accordingly, it is expected that fusion proteins comprising growth and XTEN have reduced renal nce, contributing to increased terminal half-life and improving the therapeutic or biologic effect relative to a corresponding ed biologic payload protein.

Table 35: SEC analysis of various polypeptides Apparent KEEN or Apparent Construct Therapeutic Molecular 1181011 . MW Name PI‘OtelIl Weight partner (kDa) Factor uct XTEN or Therapeutic Actual Apparent Sgibiliflgi‘ RH mm“ MW Name Protein MW (kDa) Weight (11m) r (kDa) Factor AC89 AF120 Glucagon 14.1 76.4 5.4 4.3 AC88 AF108 Glucagon 13.1 61.2 4.7 3.9 AC73 AF144 Glucagon 16.3 95.2 5.8 4.7 AC53 AG576 GFP 74.9 339 4.5 7.0 AC39 AD576 GFP 76.4 546 7.1 7.7 AC41 AE576 GFP 80.4 760 9.5 8.3 AC52 AF576 GFP 78.3 526 6.7 7.6 AC398 AE288 FVII 76.3 650 8.5 8.2 AC404 AE864 FVII 129 1900 14.7 10.1 AC85 AE864 Exendin-4 83.6 938 11.2 8.9 AC114 AM875 Exendin-4 82.4 1344 16.3 9.4 AC143 AM875 hGH 100.6 846 8.4 8.7 AC227 AM875 IL-1ra 95.4 1103 11.6 9.2 AC228 AM1318 IL-1ra 134.8 2286 17.0 10.5 Example 41: Pharmacokinetics of extended polypeptides fused to GFP in cynomolgus monkeys The pharmacokinetics of GFP-L288, 76, GFP-XTEN_AF576, GFP-XTEN_Y576 and XTEN_AD836-GFP were tested in cynomolgus monkeys to determine the effect of composition and length of the unstructured polypeptides on PK parameters. Blood samples were analyzed at various times after injection and the tration of GFP in plasma was measured by ELISA using a polyclonal antibody against GFP for capture and a biotinylated preparation of the same polyclonal dy for detection. Results are summarized in . They show a surprising increase of ife with increasing length of the XTEN sequence. For example, a half-life of 10 h was determined for GFP- XTEN_L288 (with 288 amino acid residues in the XTEN). Doubling the length of the unstructured polypeptide fusion partner to 576 amino acids increased the half-life to 20-22 h for multiple fusion protein constructs; i.e., GFP-XTEN_L576, GFP-XTEN_AF576, GFP-XTEN_Y576. A further increase of the unstructured polypeptide fusion partner length to 836 residues ed in a half-life of 72-75 h for XTEN_AD836-GFP. Thus, increasing the r length by 288 residues from 288 to 576 residues increased in Vivo half-life by about 10 h. However, increasing the polypeptide length by 260 residues from 576 residues to 836 residues increased half-life by more than 50 h. These results show that there is a surprising threshold of ctured polypeptide length that results in a r than proportional gain in in vivo half-life. Thus, fusion proteins comprising extended, unstructured polypeptides are ed to have the property of enhanced pharmacokinetics compared to polypeptides of shorter s.

Example 42: Serum ity ofXTEN A fusion protein containing XTEN_AE864 fused to the N—terminus of GFP was incubated in monkey plasma and rat kidney lysate for up to 7 days at 37°C. Samples were withdrawn at time 0, Day 1 and Day 7 and analyzed by SDS PAGE followed by detection using Western analysis and detection with antibodies t GFP as shown in . The sequence of XTEN_AE864 showed negligible signs of degradation over 7 days in plasma. However, XTEN_AE864 was rapidly degraded in rat kidney lysate over 3 days. The in Vivo stability of the fusion protein was tested in plasma samples wherein the GFP_AE864 was immunoprecipitated and analyzed by SDS PAGE as described above. Samples that were awn up to 7 days after injection showed very few signs of degradation. The results demonstrate the resistance of CFXTEN to degradation due to serum ses; a factor in the enhancement of pharmacokinetic properties of the CFXTEN fusion proteins.

Example 43: Increasing solubility and ity of a peptide payload by linking to XTEN In order to evaluate the ability ofXTEN to enhance the physicochemical properties of solubility and stability, fusion proteins of glucagon plus shorter-length XTEN were prepared and evaluated. The test articles were prepared in Tris-buffered saline at neutral pH and characterization of the Gog-XTEN solution was by reverse-phase HPLC and size ion chromatography to affirm that the protein was homogeneous and non-aggregated in solution. The data are ted in Table 36. For comparative purposes, the lity limit of unmodified glucagon in the same buffer was measured at 60 uM (0.2 mg/mL), and the result trate that for all lengths ofXTEN added, a ntial increase in lity was attained. Importantly, in most cases the glucagon-XTEN fusion proteins were prepared to achieve target concentrations and were not evaluated to determine the maximum solubility limits for the given construct. However, in the case of glucagon linked to the AF-144 XTEN, the limit of solubility was determined, with the result that a d increase in solubility was achieved, compared to glucagon not linked to XTEN. In addition, the glucagon-AF 144 CFXTEN was evaluated for stability, and was found to be stable in liquid formulation for at least 6 months under refrigerated conditions and for approximately one month at 37°C (data not shown).

] The data support the conclusion that the g of short-length XTEN polypeptides to a ically active protein such as glucagon can markedly enhance the solubility properties of the protein by the resulting fusion protein, as well as confer stability at the higher protein concentrations.

Table 36: Solubility of Glucagon-XTEN constructs Test Article Solubility Glucagon 6O uM Glucagon-Y36 >370 [1V1 Glucagon-Y72 >293 uVI Glucagon-AF108 >145 uVI Glucagon-AF12O >160 uVI Glucagon-Y144 >497 uVI Glucagon-AE144 >467 uVI Glucagon-AF144 >3600 [1M Glucagon-Y288 >163 uVI ] Example 44: is of sequences for secondary structure by prediction algorithms Amino acid sequences can be assessed for secondary structure via certain computer programs or algorithms, such as the well-known Chou-Fasman algorithm (Chou, P. Y., et al. (1974) Biochemistry, 13: 222-45) and the Garnier-Osguthorpe-Robson, or “GOR” method (Garnier J, Gibrat JF, Robson B. (1996). GOR method for predicting protein secondary structure from amino acid sequence. Methods Enzymol 266:540-553). For a given sequence, the algorithms can predict whether there exists some or no secondary structure at all, expressed as total and/or tage of residues of the sequence that form, for example, alpha-helices or beta-sheets or the percentage of residues of the sequence predicted to result in random coil formation.

Several representative sequences from XTEN “families” have been assessed using two algorithm tools for the Chou-Fasman and GOR methods to assess the degree of secondary structure in these sequences. The Chou-Fasman tool was provided by m R. Pearson and the University of Virginia, at the “Biosupport” internet site, URL located on the World Wide Web at .fasta.bioch.virginia.edu/fasta_www2/fasta_www.cgi?rm=misc1 as it existed on June 19, 2009. The GOR tool was provided by Pole lnformatique Lyonnais at the Network Protein Sequence Analysis internet site, URL located on the World Wide Web at .npsa-pbil.ibcp.fr/cgi-bin/secpred_gor4.p1 as it existed on June 19, 2008.

As a first step in the analyses, a single XTEN ce was analyzed by the two algorithms.

The AE864 composition is an XTEN with 864 amino acid es created from multiple copies of four 12 amino acid sequence motifs consisting of the amino acids G, S, T, E, P, and A. The sequence motifs are characterized by the fact that there is limited repetitiveness within the motifs and within the overall sequence in that the ce of any two consecutive amino acids is not repeated more than twice in any one 12 amino acid motif, and that no three uous amino acids of full-length the XTEN are identical. sively longer portions of the AP 864 sequence from the N—terminus were analyzed by the Chou- Fasman and GOR algorithms (the latter requires a minimum length of 17 amino acids). The sequences were analyzed by entering the FASTA format sequences into the prediction tools and running the is. The results from the analyses are presented in Table 37.

The results indicate that, by the Chou-Fasman ations, short XTEN of the AE and AG families, up to at least 288 amino acid residues, have no alpha-helices or beta-sheets, but s of predicted tage of random coil by the GOR algorithm vary from 78-99%. With increasing XTEN lengths of 504 residues to greater than 1300, the XTEN analyzed by the Chou-Fasman algorithm had ted percentages of helices or beta-sheets of 0 to about 2%, while the calculated percentages of random coil increased to from 94-99%. Those XTEN with alpha-helices or beta-sheets were those sequences with one or more instances of three contiguous serine residues, which resulted in predicted beta-sheet formation. However, even these sequences still had approximately 99% random coil formation.

The data provided herein suggests that 1) XTEN created from le sequence motifs of G, S, T, E, P, and A that have d repetitiveness as to contiguous amino acids are predicted to have very low amounts of alpha-helices and beta-sheets; 2) that increasing the length of the XTEN does not appreciably increase the probability of alpha-helix or heet formation; and 3) that progressively sing the length of the XTEN sequence by addition of non-repetitive 12-mers consisting of the amino acids G, S, T, E, P, and A s in increased percentage of random coil formation. Results further indicate that XTEN sequences defined herein (including e. g., XTEN created from sequence motifs of G, S, T, E, P, and A) have limited repetitiveness (including those With no more than two identical contiguous amino acids in any one motif) are expected to have very limited secondary structure.

Any order or combination of sequence motifs from Table 3 can be used to create an XTEN polypeptide that will result in an XTEN sequence that is substantially devoid of secondary structure, though three contiguous serines are not preferred. The unfavorable property of three contiguous series however, can be ameliorated by increasing the length of the XTEN. Such sequences are expected to have the characteristics described in the CFXTEN embodiments of the invention disclosed herein.

Table 37: CHOU-FASMAN and GOR tion calculations of polypeptide ces SEQ ID No. Chou-Fasman GOR SEQ NAME N0: Residues Calculation Calculation AE36-- R'd651 ue ttl:H:0E:00 a S 1489 36 94.44% LCW0402_002 percent: H: 0.0 E: 0.0 AE36-. R'd651 ue ttl:H:0E:00 a S 1490 36 94.44% LCW0402_003 percent: H: 0.0 E: 0.0 AG36-- R'd“1 ue ttl:H:0E:00 a S 1491 36 77.78% LCW0404_001 percent: H: 0.0 E: 0.0 AG36-. Residue totals: H: 0 E: 0 1492 36 83.33 % LCW0404_003 percent: H: 0.0 E: 0.0 e totals: H: 0 E: 0 AE42_1 1493 42 90.48% percent: H: 0.0 E: 0.0 Residue totals: H: 0 E: 0 AE42 1 1494 42 90.48% — percent: H: 0.0 E: 0.0 Residue totals: H: 0 E: 0 AG42 1 1495 42 88.10% — t: H: 0.0 E: 0.0 Residue totals: H: 0 E: 0 AG42_2 1496 42 8810‘Vi 0 percent: H: 0.0 E: 0.0 e totals: H: 0 E: 0 AE144 1497 144 98.61% percent: H: 0.0 E: 0.0 Residue totals: H: 0 E: 0 AG144 1 1498 144 91.67% _ percent: H: 0.0 E: 0.0 Residue : H: 0 E: 0 AE288 1499 288 99.31% percent: H: 0.0 E: 0.0 Residue totals: H: 0 E: 0 AG288_2 1500 288 92 71I percent: H: 0.0 E: 0.0 Residue totals: H: 0 E: 0 AF504 1501 504 94.44% percent: H: 0.0 E: 0.0 e totals: H: 7 E: 0 AD 576 1502 576 99.65%)0 t: H: 1.2 E: 00 AE576 1503 576 Residue totals: H: 2 E: 0 99.65% SEQ I No. Ch0u~Fasman GOR SEQ NAME N0: Residues Calculation Calculation percent: H: 0.4 E: 0.0 Res'd1 ue Gastt1:H: 0 E: 3 AG576 1504 576 99.31% percent: H: 0.4 E: 0.5 Residue totals: H: 2 E: 0 AF540 1505 540 99.65 percent: H: 0.4 E: 0.0 Res1due totals: H: 0 E: 0 AD836 1506 836 98.44% percent: H: 0.0 E: 0.0 R'd651 ue tt1:H:2E:30 a S AE864 1507 864 99.77% percent: H: 0.2 E: 0.4 e totals: H: 2 E: 0 AF864 1508 875 95.20%)0 percent: H: 0.2 E: 00 R'd651 ue tt1:H:0E:00 a S AG864 1509 864 94.91% percent: H: 0.0 E: 0.0 R'd651 ue tt1:H:7E:30 a S AM875 1510 875 98.63% t: H: 0.8 E: 0.3 R”due' t0 a St 1 :H: 7 E: 0 AM1318 1511 1318 99.17% percent: H: 0.7 E: 0.0 Residue totals: H: 4 E: 3 AM923 1512 924 98.70%)0 percent: H: 0.4 E: 03 Residue : H: 8 E: 3 AE912 1513 913 99.45%)0 percent: H: 0.9 E: 0.3 Residue totals: H: 0 E: 0 BC 864 1514 )0 percent: H: 0 E; 0 H: alpha-helix E: beta-sheet Example 45: Analysis of polypeptide sequences for repetitiveness In this Example, different polypeptides, including l XTEN sequences, were assessed for repetitiveness in the amino acid sequence. Polypeptide amino acid sequences can be assessed for repetitiveness by quantifying the number of times a shorter subsequence s within the overall polypeptide. For e, a polypeptide of 200 amino acid residues length has a total of 165 overlapping 36-amino acid s” (or “3 6-mers”) and 198 3-mer “subsequences”, but the number of unique 3-mer subsequences will depend on the amount of repetitiveness within the ce. For the analyses, different polypeptide sequences were assessed for repetitiveness by determining the subsequence score obtained by application of the following equation: @333 > , , . .) Subsequence score = “33"“: {Zalfiffiig l wherein: m = (amino acid length of polypeptide) — (amino acid length of subsequence) + 1; and Countl- = cumulative number of occurrences of each unique subsequence within sequence,- In the analyses of the present e, the subsequence score for the polypeptides of Table 38 were determined using the foregoing equation in a computer program using the algorithm depicted in , wherein the subsequence length was set at 3 amino acids. The resulting subsequence score is a reflection of the degree of repetitiveness within the polypeptide.

] The results, shown in Table 38, indicate that the unstructured polypeptides consisting of 2 or 3 amino acid types have high subsequence scores, while those of consisting of the 12 amino acid motifs of the six amino acids G, S, T, E, P, and A with a low degree of internal repetitiveness, have uence scores of less than 10, and in some cases, less than 5. For example, the L288 sequence has two amino acid types and has short, highly repetitive sequences, resulting in a uence score of 50.0. The polypeptide J288 has three amino acid types but also has short, repetitive sequences, resulting in a subsequence score of 33.3. Y576 also has three amino acid types, but is not made of internal repeats, ed in the subsequence score of 15.7 over the first 200 amino acids. W576 consists of four types of amino acids, but has a higher degree of internal repetitiveness, e. g., “GGSG” (SEQ ID NO: 1694),”, resulting in a subsequence score of 23.4. The AD576 ts of four types of 12 amino acid motifs, each consisting of four types of amino acids. Because of the low degree of internal tiveness of the individual motifs, the overall subsequence score over the first 200 amino acids is 13.6. In contrast, XTEN’s consisting of four motifs contains six types of amino acids, each with a low degree of internal repetitiveness have lower subsequence scores; i.e., AE864 (6.1), AF864 (7.5), and AM875 (4.5), while XTEN consisting of four motifs containing five types of amino acids were intermediate; i.e., AE864, with a score of 7.2.

Conclusions: The results indicate that the combination of 12 amino acid subsequence motifs, each consisting of four to six amino acid types that are non-repetitive, into a longer XTEN polypeptide results in an overall sequence that is substantially non-repetitive, as indicated by overall subsequence scores less than 10 and, in many cases, less than 5. This is despite the fact that each subsequence motif may be used multiple times across the sequence. In contrast, polymers created from smaller numbers of amino acid types resulted in higher subsequence scores, with polypeptides consisting of two amino acid type having higher scores that those consisting of three amino acid types.

Table 38: Subseguence score calculations of polypeptide ces Seq Name SEQ ID NO: Score J288 1515 33.3 K288 1516 46.9 L288 1517 50.0 Y288 1518 26.8 Q576 1519 18.5 U576 1520 18.1 W576 1521 23.4 Y576 1522 15.7 AE288 1523 6.0 AG288_1 1524 6.9 AD576 1525 13.6 AE576 1526 6.1 Seq Name SEQ ID NO: Score AF54O 1527 8.8 AF504 1528 7.0 AE864 1529 6.1 AF864 1530 7.5 AG864 1531 7.2 AG868 1532 7.5 AM875 1533 4.5 AE912 1534 4.5 AM923 1535 45 AM1296 1536 4.5 Example 46: Calculation of TEPITOPE scores ] TEPITOPE scores of 9mer peptide sequence can be calculated by adding pocket potentials as described by Sturniolo [Sturniolo, T., et al. (1999) Nat Biotechnol, 17: 555]. In the present Example, separate Tepitope scores were calculated for individual HLA alleles. Table 39 shows as an e the pocket ials for HLA0101 B, which occurs in high frequency in the Caucasian population. To ate the TEPITOPE score of a peptide with sequence Pl-P2-P3-P4-P5-P6-P7-P8-P9, the corresponding individual pocket potentials in Table 39 were added. The 1B score of a 9mer peptide with the sequence FDKLPRTSG (SEQ ID NO: 1695) is the sum of 0, -l.3, 0, 0.9, 0, -l.8, 0.09, 0, To evaluate the TEPITOPE scores for long peptides one can repeat the process for all 9mer subsequences of the sequences. This process can be ed for the proteins encoded by other HLA alleles. Tables 40-43 give pocket potentials for the protein products of HLA alleles that occur with high frequency in the Caucasian population.

TEPITOPE scores calculated by this method range from approximately -10 to +10. However, 9mer peptides that lack a hydrophobic amino acid (FKLMVWY (SEQ ID NO: 1696)) in P1 position have calculated PE scores in the range of -1009 to -989. This value is biologically gless and reflects the fact that a hobic amino acid serves as an anchor residue for HLA binding and peptides lacking a hydrophobic residue in P1 are considered non binders to HLA. Because most XTEN sequences lack hydrophobic residues, all combinations of 9mer subsequences will have TEPITOPEs in the range in the range of -1009 to -989. This method confirms that XTEN polypeptides may have few or no predicted T-cell epitopes.

Table 39: Pocket otential for HLA0101B allele.

WEE” —mnnninnI-I —mnnnlnnl-I Amino Acid P1 P2 P3 P4 P5 P6 P7 P8 P9 D _ -2 _ Table 40: Pocket potential for 1B allele.

Amino acid P1 P2 P3 P4 P5 P6 P7 P8 P9 A -999 O O O - O O - O C -999 O O O - O O - O .3 - Amino acid Amino acid EQTJD'J o 0 9.0.0.0 OOLAOOI—I‘ .0ch NNOO‘N‘ ._I,'_.<'3._I H'oxkom llll llll I—II—II—IN -I>UIt—t-I> ' I—KIOQHN' llll I III—I I—kI—I- \I I—I I ._I 0 00 I I _o U.) W I—II—I I—II—I oI—ILI] I I—k \l I IIN9 AI—I IO U) I Io U) gr II I—KI—K I—I .I—K )—I )—I .I—K 4; 9.0\OOO‘ II II I—KI—K LILI' $3.0 oo\l' II I OI ; O 00 O U} 0 O )—k U) 0 O\ I )—k .b MPG/Ow N' o .0. 00‘00‘ I I H.?‘- m I .0LII" Io.i—‘.L» o N .0 I I 9.0.— [\J I .0 \I Ifi O O O \l I I—I 0 IO I—I I I I—I [\D 00L. 5N;_‘I—I oOm ll I—AO_ No II ._o_"xo I no H..\I I )—I .5 II O 0 O 00 I I—I O\ I I I—I LI] I I—I [\D I I I—I Table 42: Pocket potential for HLA0701B allele.

Amino acid P1 P2 P3 P4 P5 P6 P7 P8 P9 A O Amino acid p—I "UN "U L») "d.5 *UU} "U OK "U x] "U 00 "UQ l D—I D—I I I L» .5 W p—Ip—I p—Ip—I OD—IL11 I H. L») I II I—‘O I—‘LII ON LII-P I I H. I—I Eb II I—II—I L») ._I .I—I )—I )—I .I—I.5 II .053. #00 II II .053. 000 I—‘N OON II O 00 0 LA I ._I ._I I IO O\ D—I .5 I I0 L11 MPG/Orv NI—A .NN. ll _b—KD—K I—‘Lll III II _b—KD—K b—KD—K OI—I "\I'_." II .99.. cow LII III Io L» 00Mac D—I o O\ o -I> <5 L» ,4 o o D—I -I> I ('3 I—A o O I 0 -'> 0L .N D—I .0 m .0. \o I .0_. 5. o I N. <5 I—A O I b—k I_I I IO 0 I—I -I> I O 00 0 0 O 00 IO Lo I | ._I I—I \l I I—‘ l—‘ Table 43: Pocket potential for 1B allele.

Amino acid Example 46: ment of insertion ofXTEN into permissive loops.

XTEN AE42-4 Insertion The construction and expression of FVIH with XTEN AE42 insertions were described in Example 17 and 24. Thus, Where residue X designates the site of insertion and residue Z designates the next residue in the native FVIII polypeptide sequence, the polypeptide resulting from ion ofXTEN AE42 would contain the sequence: X-GAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPASS-Z (SEQ ID NO: 1697) 16 different sites in the FVIII sequence were selected for XTEN AE42 insertion, and these were designed Batch 1. An additional 21 sites selected for XTEN AE42 insertion were designed Batch 2.

Collectively, the Batch 1 and Batch 2 sites represent 12 sites in the Al domain, 7 sites in the A2 domain, sites in the A3 domain, 4 sites in the Cl domain, and 3 sites in the C2 domain. Locations of Batch 1 and 2 sites in the 3-D structure of FVIII are depicted in .

The location of these Batch 1 and Batch 2 insertion sites results in 37 ucts designated pSDOOOl- pSDOOO4, pSDOOO9- pSDOOl2, pSDOO23- pSDOO32, pSDOO34- pSDOO63 [the foregoing ranges e all intermediate numbers, as well], the sequences of which are set forth in Table 21 and the ions sites of which are set forth in Table 23.

In Vitro assays To assess FVIII tolerability to XTEN AE42-4 insertion, the FVIII actiVity in culture media samples from FVIII-XTEN cell cultures was analyzed using a FVIII chromogenic assay. Antigen expression levels were analyzed by FVIII-HC (FVIII heavy chain) and FVIII-LC (FVIII light chain) ELISA.

FVIII ActiVity Measurement by Chromogenic Assay ] The FVIII actiVity was measured using the COATEST® SP FVIII kit from DiaPharma (lot# N0890l9) and all incubations were performed on a 37°C plate heater with shaking. Cell culture ts from transient transfection media of FVIII-XTEN AE42-4 variants from 6 well plates were diluted to the desired FVIII actiVity range using lx FVIII COATEST® buffer. FVIII rds were ed in 1x FVIII COATEST® buffer containing mock transfection media with matching culture media concentration as the g sample. The range of recombinant Factor VIII (rFVIII) standard was from 100 mIU/mL to 0.78 mIU/mL. The standards, diluted cell culture samples, and a pooled normal human plasma assay control were added to Immulon® 2HB 96-well plates in duplicates (25 uL/well). y ed IXa/FX/Phospholipid mix (50 uL), 25 [LL of 25mM CaCl2, and 50 [LL of FXa substrate were added sequentially into each well, with 5 minutes incubation between each addition. After incubating with the substrate, 25 [LL of 20% acetic acid was added to ate the color reaction, and the absorbance at 405 nm was measured with a SpectraMAX® plus ular DeVices) ment.

Data analysis was performed using SoftMax Pro software (version 5.2). The Lowest Level of Quantification (LLOQ) was 39 mIU/mL. Results are presented in Table 22.

Expression Measurement by FVIII-HC and FVIII-LC ELISA Expression of variants was quantified using ELISA. The FVIII antigen expression levels of DNA constructs corresponding to XTEN insertions in the Al and A2 domains of FVIII were analyzed by FVIII-LC ELISA. The FVIII n expression levels of DNA constructs corresponding to XTEN insertions in the A3, C1 and C2 domains of FVIII were ed by FVIII-HC ELISA. Results are presented in Table 22.

FVIII-XTEN antigens in cell culture media after t were captured by GMA011 antibodies (Green Mountain Antibodies) for FVIII-LC ELISA) or by GMA016 antibodies (Green Mountain Antibodies) for FVIII-HC ELISA. Immulon® 2HB 96-well plates were coated with 100ul/well of anti- FVIII antibody l) by ght incubation at 40C. Plates were then washed four times with Phosphate Buffer saline with 20 (PBST) and blocked with blocking buffer (PBST with 10% heat inactivated horse serum) for 1 hour at room temperature.

Cell culture harvests from ent transfection media of FVIII-XTEN variants from a 6-well plate were diluted to the desired FVIII antigen range using 1x blocking buffer. FVIII standards were prepared in 1x FVIII blocking buffer containing mock transfection media with matching media concentration as the g samples. The range of rFVIII standard was from 50 ng/mL to 0.39 ng/mL.

Standards, diluted cell culture samples, and a pooled normal human plasma assay control were added into Immulon® 2HB 96-well plates in ates (100 uL/well) and incubated at 370C for 2 hours.

Following four times washing with PBST, 100 pl of HRP-sheep anti-hFVIII antibody (Affinity Biologicals, F8C-EIC-D) were added into each well and plates were incubated for 1 hour at 370C. After another four washes with PBST, 100 pl ofTMB Super Sensitive Substrate (BioFX) were added to each well, followed by 5-10 min color development. To ate the color reaction, 50 [LL of H2SO4 were added to each well, and the absorbance of at 450 nm was measured with a SpectraMAX plus (Molecular Devices) instrument.

Data analysis was performed using SoftMax Pro software (version 5.4). The Lowest Level of Quantification (LLOQ) was 0.0039 ug/mL. Results are ted in Table 22.

Permissive sites into which XTEN sequences were inserted without eliminating procoagulant actiVity of the recombinant protein, or the ability of the recombinant proteins to be expressed in the host cell were clustered within loops in each of the three A domains of FVIII. shows the location of insertion sites in the recombinant FVIII proteins that showed FVIII ty on domains A1, A2 and A3. shows a structural representation depicting the location of insertion sites in the recombinant FVIII proteins that showed FVIII actiVity.

The sive sites clustered in solvent exposed, highly flexible surface loops (XTEN permissive loops). The A1 domain loops were located in a region corresponding approximately to amino acid ons 15 to 45, and 201 to 232, respectively, in the sequence of mature human FVIII ().

The A2 domain loops were located in a region corresponding approximately to amino acid positions 395 to 421, and 577 to 635, respectively, in the sequence of mature human FVIII (). The A3 domain loops were located in a region corresponding approximately to amino acid positions 1705 to 1732, and 1884 to 1917, respectively, in the sequence of mature human FVIII (). FIGS. 37A and 37B show the location of the XTEN sive loops relative to secondary structure elements in the tridimensional structure of FVIII.

Example 47: CFXTEN with insertions ofXTEN having 144 amino acids Analysis of the preliminary data presented above (Example 46) suggested the existence of defined regions within the linear polypeptide sequences and 3-D structures of the FVIII A domains that can odate the insertion ofXTEN sequences. To test this hypothesis and further define the boundaries of putative regions that can accommodate the insertion ofXTEN sequences without loss of FVIII activity, 23 additional insertion sites not present in either Batch 1 or 2 were chosen and designated Batch 3.

Batch 3 constructs were generated by the insertion of a 144 residue XTEN AE polypeptide, comprising amino acid residues Gly (G), Ala (A), Pro (P), Ser (S), Thr (T), and Glu (E), or a 144 residue XTEN AG polypeptide, comprising amino acid residues Gly (G), Ala (A), Pro (P), Ser (S), and Thr (T).

Five different version of the 144 residue AE polypeptide were generated and designated XTEN-AEl44- 2A, XTEN-AEl44-3B, XTEN-AEl44-4A, XTEN-AEl44-5A, XTEN-AEl44-6B. The amino acid sequences are as set forth in Table 4. Five ent ns of the 144 residue polypeptide were generated and designated XTEN-AGl44-l, XTEN-AGl44-A, XTEN-AGl44-B, XTEN-AGl44-C, and XTEN-AGl44-F. The amino acid sequences are as set forth in Table 4.

The 144 residue XTEN encoding DNA sequence was introduced by the chemical synthesis of DNA segments (DNA 2.0, Redwood City, CA) spanning the nearest unique restriction sites within the base vector on either side of the site of insertion.

The DNA ces corresponding to the XTEN 144 peptides were inserted such that the resulting DNA construct would encode a FVIII protein in which the XTEN 144 protein sequence is inserted immediatelym the residue indicated in the site selection, and flanked by AscI and X1101 sites.

In addition to these sites, those sites from Batch 1 and 2 at which insertion of the XTEN AE42 ptide did not abolish FVIII procoagulant acitivity were modified by excision of the AE42 polypeptide encoding DNA segment with restriction enzymes AscI and X7101, and introduction ofXTEN AEl44 and XTEN AGl44 coding sequences at the same sites. The location of these Batch 1, Batch 2 and Batch insertion sites is summarized in Table III. presents a structural representation of FVIII showing the location of the XTEN l44 insertion sites.

A total of 48 constructs with 144 XTEN s were created. The constructs are pSDOOOl- pSDOOO4, pSDOOO9-pSDOOl2, pSDOO23-63 [the foregoing ranges include all intermediate numbers, as well], the sequences of which are set forth in Table 21 and the insertion sites of which are ed in Table 22.

Expression of XTEN 144 Variants ] FVIII ts with XTEN l44 insertions were tranfected into HEK293F cells (Invitrogen, Carlsbad, CA) using polyethyleneimine (PEI, Polysciences Inc. Warrington, PA) or Lipofectamine transfection reagent (Invitrogen, Carlsbad, CA). The transiently transfected cells were grown in 293 Free Style medium or a mixture of 293 Free Style and CD Opti CHO media (Invitrogen, ad, CA). The cell culture medium was harvested 3-5 days after transfection and analyzed for FVIII sion by genic FVIII activity assay and FVIII ELISA conducted as described herein.

Cell culture media from ent transfection were trated 10-fold in Centricon® spin columns (lOOkd cut-off). trated material was then flash frozen and stored at -80°C for future in vitro analysis and in vivo PK studies.

In vitro assays To assess FVIII tolerability to insertions, the FVIII activity in e media samples from cell es was analyzed using a FVIII chromogenic assay. Antigen expression levels were analyzed by FVIII-HC (FVIII heavy chain) and FVIII-LC (FVIII light chain) ELISA.

FVIII Activity Measurement by genic Assay and Expression Measurement by FVIII- HC and FVIII-LC ELISA Chromogenic and ELISA assay methods were conducted as described. The results obtained are summarized in Table 23.

Permissive sites into which XTEN ces were inserted without eliminating procoagulant activity of the recombinant protein, or the ability of the recombinant ns to be expressed in the host cell clustered within loops in each of the three A domains of FVIII. The same XTEN permissive loop regions tolerating the shorter XTEN sequences inserted were found to tolerate the insertion of the longer XTEN sequences. shows the location ofXTEN 144 insertion sites in the recombinant FVIII proteins that showed FVIII activity on domains A], A2 and A3. shows a structural representation depicting the location of insertion sites in the inant FVIII proteins that showed FVIII activity.

These observation indicate that two regions within each of the A domains of FVIII are able to accommodate insertion ofXTEN sequences t loss of FVIII cofactor activity. A structural depiction of these so-called XTEN permissive loops (FIGS. 40 and 4]) demonstrate that they occupy structurally analogous positions in each of the A domains and project from one face of the FVIII molecule. The identified XTEN sive loops correspond to highly flexible loops located n beta strands in the three-dimensional structures of the Al, A2, and A3 domains, as shown in FIGS. 37A and 37B.

The in vivo evaluation ofXTEN 144 insertions on FVIII Half-life Extension, as determined by pharmacokinetics, is described in Example 32.

Example 48: Rescue or enhancement of FVIII expression by ion of an XTEN sequence within the a3 acidic peptide region of FVIII.

Adherent HEK293 cells were transfected (as described in Example 24) with FVIII-XTEN DNA constructs in which the coding ce of a B-domain deleted factor VIII contained 2 to 4 XTEN ions of 144 amino acid residues each, of composition and ion location as indicated in Table 44, below. At 5 days post-transfection, cell culture atants were assayed for FVIII activity by the chromogenic assay (as described in e 25). Results are shown in Table 44.

Table 44. Expression levels of FVIII Activity by CFXTEN variants containing an XTEN at position 1720 and one, two, or three additional XTEN insertions.

Construct Domain, Position, and Type ofXTEN Insertion Activity Name (mm/mm 1720 LSD0040.002 26 AGl44 AGl44 175 _—403 AE144_ 1720 __ AG144 1656 1720 LSD0045.002 AG144 AG144 2598 1656 1720 PSD080.002 26 AG144 AG144 AG144 1081 1656 1720 .001 403 AE144 AG144 AG144 789 1720 PSD082.001 26 AG144 AG144 1900 AE144 <LLOQ 1656 1720 PSD090.003 26 AG144 AG144 AG144 1900 AE144 316 For the e of comparison, all FVIII-XTEN constructs had an AG144 XTEN insertion at amino acid position 1720 (numbered relative to ength factor VIII) within the A3 domain.

Expression levels of FVIII-XTEN varians were determined by chromogenic assay and expressed in units of mIU/mL. Constructs with a single additional XTEN insertion at either position 26 in the A1 domain (LSD0040.002) or position 403 in the A2 domain (LSD0041.008) yielded expression levels of 175 and 279 mIU/mL, respectively. In contrast, a construct with a single additional XTEN insertion at position 1656 within the a3 acidic e yielded an expression level of 2598 mIU/mL, demonstrating enhancement of expression level for the a3 XTEN insertion construct ve to the A1 and A2 insertion constructs. In addition, in ison to the FVIII-XTEN construct with XTEN insertions at positions 26 in the A1 domain and 1720 in the A3 domain (LSD0040.002), the construct with an additional XTEN insertion at position 1656 within the a3 acidic peptide region (PSD080.002) yielded significantly higher expression (175 and 1081 mIU/mL, respectively). Consistent with these findings, the construct with XTEN insertions at ons 403 in the A2 domain and 1720 in the A3 domain (LSD0041.008) yielded an expression level of 279 mIU/mL, whereas an additional XTEN insertion at position 1656 within the a3 acidic peptide region 3.001) resulted in an increase in the expression level to 789 mIU/mL.

Lastly, the FVIII-XTEN construct with an XTEN insertion at position 26 within the A1 domain and two XTEN insertions at ons 1720 and 1900 within the A3 domain (PSD082.001) did not yield activity above the lower limit of quantitation. However, the FVIII-XTEN construct with an additional XTEN insertion within the a3 acidic peptide region (PSD090.003) resulted in detectable ty, trating that inclusion of an XTEN sequence within the a3 domain can result in recovery of expression (as measured by activity) in FVIII-XTEN constructs that are otherwise expressed at levels below the lower limit of quantitation. Under the conditions of the experiment, the results support the conclusion that insertion ofXTEN at the 1656 position and, by extension, within the a3 region, results in enhanced expression of gulant FVIII-XTEN compositions.

Example 49: Effect ofXTEN ion 0n FVIII activity measured by aPTT A one stage activated partial prothrombin (aPTT) coagulation assay was employed in on to the chromogenic assay (as described in Example 25) to determine FVIII ty of various FVIII- XTEN fusion proteins.

MLhod: The XTEN aPTT activity was ed using the Sysmex O instrument (Siemens Healthcare Diagnostics Inc.,Tarrytown, NY). To create a standard curve for the assay, WHO factor VIII standard was diluted with 2% mock transfection media to 100 mU/mL and a two-fold serial dilution series was then performed, with the last standard being 0.78 mU/mL. FVIII-XTEN cell culture samples were first d at 1:50 with aPTT assay buffer, further dilutions were made with 2% mock transfection media when needed.

After dilution, the aPTT assay was performed using Sysmex instrument as follow: 50 [ll of diluted standards and samples were mixed with 50 ul human FVIII def1cient plasma and then 50 ul of aPTT reagent. The mixture was incubated at 37°C for 4 min, and following incubation, 50 ul of CaClz was added to the e, and the clotting time was measured immediately.

To determine test samples FVIII ty, the ng time of the standards were plotted using semi-log scale (Clotting time: Linear; Standard concentration: Log) to extrapolates the on between clotting time and FVIII actiVity, and FVIII-XTEN actiVity was then calculated against the standard curve. The assay sensitiVity was 40 mU/mL factor VIII.

RLults: The results are summarized in FIGS. 44-46. When single XTEN of 144 or 288 amino acids were inserted into the FVIII, all of the FVIII-XTEN fusion proteins exhibiting actiVity in the genic assay were also active in aPTT assay. The aPTT actiVity ed the trend of chromogenic assay, for example, those molecules that showed low FVIII actiVity in the chromogenic assay also had low aPTT values. Generally, the aPTT results for the fusion proteins were lower than those obtained by the chromogenic assay, with a chromogenic to aPTT ratio of 1.1 up to 2.2, as rated in , for the single XTEN insertions. The FVIII-XTEN fusion proteins with multiple XTEN insertions, in general, showed further ions in aPTT actiVity in comparison to chromogenic assay. Assays of FVIII-XTEN with two XTEN ions showed actiVity with all constructs, but with chromogenic/aPTT ratios approaching 4, in some instances (). Assays of FVIII-XTEN with some three XTEN insertions also showed actiVity in both assays, with chromogenic/aPTT ratios approaching 5, in some instances (), while the ratios for the BDD-FVIII control were more comparable (right side of FIG 46). Additionally, the site ofXTEN insertion appeared to contribute to the differences seen between aPTT and chromogenic actiVities. For example, while some molecules with 2 XTEN insertions ed in up to 4-fold lower ty than chromogenic values, the aPTT actiVity of other FVIII molecules with 2 XTEN were fairly able to chromogenic activity (). Some molecules with 3 XTEN insertions showed up to 5 —fold lower than chromogenic activities, other FVIII molecules with 3 XTEN have aPTT actiVity less than 2-fold lower than chromogenic actiVity (). Under the conditions of the experiment, the results support the conclusion that FVIII-XTEN fusion protein constructs do retain procoagulant actiVity, but that the chromogenic assay generally provides higher actiVity levels than that in the aPTT assay system employed in the study.

Example 50: Evaluations of the Effect ofXTEN Insertion Site onf FVIII Half-life Extension Methods: Six XTEN fusion proteins with single XTEN AG-144 insertions at defined locations were tested in FVIII/VWF DKO mice (as generally described in Example 32) to evaluate the effect ofXTEN insertion site on FVIII ife. Six representative XTEN variants (listed in table 1) with XTEN insertion in either within A1, A2, a3, A3-region1 (A3-R1), A3-region 2 (A3-R2) or at the C- terminus were selected for this study, and BDD-FVIII generated from the base vector was used as the control. FVIII/VWF DKO mice were treated with a single intravenous administration of transient transfaction cell e media concentrate from the six FVIII-XTEN ucts (or positive control media) at 100-200 IU/kg, and plasma samples were subsequently collected at 5min, 7 hours and 16 hours post-dosing. Plasma FVIII activity was tested using the FVIII chromogenic assay and FVIII-XTEN half- life was estimated using the WinNonlin program. The study data are summarized in Table 45 and FIG RLults: A significantly longer ife was observed for all XTEN variants tested compared to BDD-FVIII l, but the degree of the ife increase varied, with the variant with XTEN at the 403 insertion site conferring the least half-life extension at 10-fold (in comparison to control), while the 1900 insertion t conferred the most ife extension at 18-fold. The differences ofXTEN insertion site on FVIII half-life extension may reflect the roles of different FVIII domains in FVIII clearance in vivo.

Table 45: FVIII-XTEN single AG-144 insertion variants PK in FVIIINWF DKO mice BDD- pSD pSD- pSD- pSD- pSD- pSD- Treatment FVIII -050 0003 0039 0010 063 014 Insertion site None 26 403 1656 1720 1900 CT ry 21.3 33.8 34.8 36.0 33.6 39.6 32.4 th/rz 0.25 3.15 2.4 3.3 4.28 4.54 3.91 t1/2 Increase 13 10 13 17 18 16 (fold) Example 51: tions of the Additive Effect ofXTEN iIsertions 0n FVIII Half-life Extension.

Methods: To evaluate the effects of multiple XTEN insertions on FVIII-XTEN fusion protein half-life, the half-livesof FVIII-XTEN variants with 1-3 XTEN ions were determined in FVIII- XTEN DKO mice using the cell culture concentrate from five constructs (as generally described in e 32). Five FVIII-XTEN variants were tested in the study: pSD-062, with AE144 insertion at position 1900 (numbered relative to full-length factor VIII); pSD-0005 with AE144 in the FVIII B domain (B-domain amino acid position 745); pSD-0019 with AE288 at the FVIII C-terminus (CT); LSD0003.006 with AE144 inserted in the B-domain and AE288 inserted at the C-terminus, and LSD0055.021 with three XTEN ofAE144, AE144, and AE288 inserted at position 1900, with the B domain and at the C-terminus. The FVIII-XTEN half-life values were estimated using the WinNonlin program.

RLults: The study results are summarized in Table 46, and the PK curves are shown in . The study results clearly demonstrated the additive effect of multiple XTEN insertions on FVIII half- life extension. With single XTEN insertions, the half-life of FVIII was extended from 0.25 hr to 3.2-4.0 hr, a 13 tol6-fold increase. When the B and CT XTEN insertions were combined together, the FVIII half-life was further extended to 10.6 hr, a d gation. Finally, in the case of a third XTEN insertion added at position 1900 to the B/CT uct, the half-life d 16 hr in the FVIII-VWF DKO mice, a 64-fold increase.

Table 46: Additive effect ofXTEN insertions 0n FVIII tm in FVIIINWF DKO mice BDD- pSD pSD— pSD— LSD- LSD- FVIII -062 0005 0019 0003.006 0055.021 Insertion s1te 0.25 3.8 3.2 4.0 10.6 16.0 t1/2Increase 13 (fold) - Example 52: Evaluation of FVIII-XTEN Interference with the Binding of Anti-FVIII Antibodies using the Bethesday Assay The ability ofXTEN insertions in the FVIII molecule to er with g by pre-existing anti-FVIII antibodies to the XTEN fusion protein was evaluated in order to determine their utility in treating patients with anti-FVIII inhibitory antibodies.

Methods: To assess the binding of anti-FVIII antibodies, two FVIII-XTEN variants (PSD088, with 144 XTEN inserted at the locations of 26/403/1656/1900; and PSD-090, with 144 XTEN inserted at the locations of 26/1656/1720/1900) were tested in comparison with Refacto (a marketed rFVIII) t plasma samples from three ilia A patients with factor VIII inhibitors (designated 04-483, 05-505, and GK1838-2079), as well as a sheep anti-FVIII poly-clonal antibody from Affinity Biologicals Inc (F8C-EIA—C). The Bethesda titer of the four anti-FVIII ab against the two FVIII-XTEN variants (pSD- 088 and pSD-090) and the Refacto control were determined using modified Bethesda assay methods, detailed as follows. Heat inactivated anti-FVIII antibody samples at various dilutions were incubated with 1 IU/mL of each FVIII variant (diluted in 1X in FVIII chromogenic assay buffer) at a 1:1 ratio. The FVIII/antibody mixtures were then incubated for 2 hours in a 37°C tor. After the incubation, the samples were diluted for 10-fold with 1 x FVIII chromogenic assay buffer, and 25 [AL of diluted mixture were then used for a FVIII chromogenic assay, The percentage of remaining FVIII activity was calculated t the post-incubation ty of a known non-neutralizing . Bethesda units were calculated using the following formula: BU=dilution factor X (LN(percent of remaining activity) + 6. 643 8).

RLults: The results are listed in Table 47. Decreased Bethesda unit (BU) titers were observed for all four antibodies when tested against the two XTEN variants, in comparison with Refacto. A to 8-fold fold se t PSD-088 and a 3 to 5-fold decrease against pSD-090, respectively, were obtained. The inhibition curves t FVIII variants for each antibody were plotted () and compared to Refacto, and demonstrates a clear left-shift of the inhibition curve for the two FVIII-XTEN molecules,with the pSD-O88 FVIII-XTEN variant resulting in a further left-shift compared to pSD-O90.

These s clearly demonstrate that: 1) both FVIII-XTEN variant fusion proteins are more resistant to isting VIII inhibitory dies than Refacto; and 2) PSD-O88 is more resistant to anti-FVIII antibodies than PSD-O90, which may provide information useful in determining the differences on the XTEN insertion sites in interferring with the binding of anti-FVIII antibodies. Under the conditions of the experiment, the s provide some support for the potential use of FVIII-XTEN compositions for treating hemophilia A patients with factor VIII inhibitors.

Table 47: Anti-FVIII antibod Bethesda titer a ainst FVIII-XTEN variants Anti-FVIII ab. 04-483 05-505 GKl838-2079 F8C-EIA—C ———“ Example 53: Half-life Evaluations of FVIII XTEN fusion les containing four XTEN insertions in Hemophilia A mice Methods: Eight FVIII-XTEN fusion proteins with four XTEN insertions each at defined locations were tested in FVIII/VWF DKO mice to evaluate the effect of the XTEN insertions on FVIII half-life extension: LSDOO71.00l, contains 403-AG144, l900-AE144, 745(B)-AE144, 2332(CT)-AE288 XTEN insertions (designated as the FVIII amino acid number and the XTEN inserted); LSDOO71.002, containing 403-AE144, l900-AE144, 745(B)-AE144, 2332(CT)-AE288 XTEN insertions; LSDOO72.001, containing 403-AG144, l900-AG144, 745(B)-AE144, 2332(CT)-AE288 XTEN insertions; LSDOO72.002, containing 403-AE144, G144, -AE144, 2332(CT)-AE288 XTEN insertions; pBCO247.004, ning l8-AG144, 403-AE144, l656-AG144, 2332(CT)-AE288 XTEN ions; pBCO251.002, containing l8-AG144, l656-AG144, E144, 2332(CT)-AE288 XTEN insertions; pSDO88, containing 26-AG144, 403-AE144, AG144, l900-AE144 XTEN insertions and pSDO90, containing 26-AG144, l656-Agl44, l720-AG144, l900-AE144 XTEN insertions. VWF DKO mice were treated, as generally described in Example 32, with a single intravenous administration of FVIII-XTEN transfection cell media concentrate of the eight constructs at 100-200 IU/kg, and plasma samples were subsequently collected at 5 min, 8 hrs, 24 hrs, 48 hrs, 72 hrs and 96 hrs post-dosing. Plasma FVIII actiVity was tested using the FVIII genic assay and FVIII- XTEN half-life was estimated using the WinNonlin m.

RLults: All of the eight FVIII XTEN fusion molecules containing four XTEN ions exhibited longer half-life than unmodified FVIII (results in Table 48). Three molecules with XTEN insertions at positions 403, 1900, B domain, and C-terminal achieved half-life up to 16.3 hrs, which is a 65-fold improvement in comparison to unmodified BDD FVIII. However, the molecules tested with XTEN insertions at 26/403/1656/1900 (pSD088), or at 26/1656/1720/1900 (pSD090) showed half-life of 9.1 hrs and 9.5 hrs, respectively, Which, in comparison to BDD FVHI, represents an increase of 36-fold and 38-fold, tively. pBC247.004 (XTEN ions at 18/403/1656/CT) and pBC251.002 (XTEN insertions at 18/1900/1656/CT) achieved half-life values of 14.1 hrs and 13 hrs, respectively. The results demonstrate that multiple XTEN insertions (in this case, four XTEN insertions for each FVHI molecule) can significantly improve FVHI half-life. It further shows that the effect ofXTEN on FVHI ife is ion site ent, even in the event of multiple XTEN ions.

Table 48: PK of FVIII-XTEN variants with four XTEN insertions in FVIIINWF DKO mice Treatment XTEN Insertions t1/2 (hr) t1/2 Increase (fold) BDD-FVIH None 0.25 NA LSD0071.001 403AG/1900AE/B/CT 16.2 64.8 LSD0071.002 403AE/1900AE/B/CT 16.3 65.2 LSD0072.001 1900AG/B/CT 11.8 47.2 LSD0072.002 403AE/1900AG/B/CT 16.1 64.4 ch247.004 /1656/CT 14.1 56.4 pBC251.002 18/1900/1656/CT 13.0 52 pSD088 /1656/1900 9.1 36.4 pSD090 26/1656/1720/1900 9.5 38 Table 49: Exem la Biolo icalActivi Exem la Assa s and Preferred Indications Biologically Active Exemplary Activity Protein Biological Activity Assays Preferred Indication: Factor VIII Coagulation factor VIII is a Chromogenix assay Hemophilia A; (Factor VIII; factor essential for (Rosen 8, Scand J bleeding; Octocog alfa; hemostasis. This gene Haematol (1984) 33 Factor VIII Moroctocog encodes coagulation (Suppl 40):139—45); deficiency; alfa; factor VIII, which participates Chromogenix bleeding episodes Recombinant in the intrinsic pathway of Coamatic® Factor VIII in patients with Antihemophilic blood coagulation; factor VIII assay; one-stage factor VIII inhibitor; factor; is a cofactor for factor lXa clotting assay Surgeay~related Nordiate; which, in the presence of Ca (Lethagen, 8., et al., hemorrhagic ReFacto; + 2 and olipids, Scandinavian J episodes Kogenate; converts factor X to the Haematology (1986) Kogenate activated form Xa. This gene 37:448—453.

SF; Helixate; produces two alternatively One-stage clotting Recombinate) spliced transcripts. assay and two-stage Transcript variant l encodes clotting assay a large glycoprotein, isoform (Barrowcliffe TW, a, which circulates in plasma Semin Thromb and associates with von . (2002) Willebrand 28(3):247-256); factor in a noncovalent Development of a complex. This protein simple undergoes multiple chromogenic factor VIII cleavage . ript assay for variant 2 encodes a putative clinical use.

Biologically Active Exemplary Activity n Biological Activity Assays Preferred tion: small protein, isoform b, (Wagenvoord RJ, which consists primarily of Hendrix HH, the phospholipid binding Hemker HC. domain of factor Vlllc. This Haemostasis binding domain is essential 1989; 19(4): 196-204) for coagulant activity. Bethesda assay Defects in this gene results (Verbruggen B, et al. in hemophilia A, a common Improvements in factor recessive X-linked VIII inhibitor detection: coagulation disorder. From Bethesda to Nijmegen. Semin Thromb Hemost. 2009 Nov;35(8):752-759) Table 50: Exemplary CFXTEN comprising FVIII and internal/external XTEN seguences (SEQ ID NOS 1537-1554 res ectivel in order of a e CFXTEN Amino Acid Sequence Name FVIII BDD2 ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI (Al-K127 — AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ AE144- REKEDDKGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE V128—N745- GSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPG AE288- SEPATSGSETPGTSTEPSEGSAPGVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDL P1640- VKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASA Y2332) RAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQAS LEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYD DDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSY KSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQA YPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRY YSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRF LPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGY TFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTG DYYEDSYEDISAYLLSKNNAIEPRSFSQNGGTSESATPESGPGSEPATSGSETPGTSESATPES GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSE PATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP SESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEG TSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGPPVLK RHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERL WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEV EDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTK DEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKS MERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMG SIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAG MSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSW IKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDS SGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYF TNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTS MYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVH QIALRMEVLGCEAQDLY FVIII BDD2 ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI (Al-A375- AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ AE576- KVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR K376-N745- EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN AE144- RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD P1640- LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD CFXTEN Amino Acid ce Name Y2332) DNSPSFIQIRSVAGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTS TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT SESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG TSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEP SEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESG PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTST EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE SGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGT SESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPS EGSAPGKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRF MAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRL PKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLI CYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIM HSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGE ENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNA IEPRSFSQNGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPS EGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGP GSEPATSGSETPGTSTEPSEGSAPGPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDF DIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEF TDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGA EPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCH TNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHA INGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPG VFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQ YGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMY SLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELM GCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPK EWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGN QDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY FVIII BDD2 LGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI (A1-Y1792- AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ AF144- REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR E1793- EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN Y2332- RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD AE864) LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS PRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPR SFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRG ELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYGGTSTPESGSASPGTSPSGESS TAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGTSPSGESSTAPGT STPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGEEDQRQ GAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLV CHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRF HAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNL YPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITA WAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFI IMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRME LMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNN PKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQ GNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGGSPAGSPTSTEE GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSES ATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS APGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP ESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG CFXTEN Amino Acid Sequence Name EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESG PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPA GSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGS ETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGT SESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPS EGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETP GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTE PSEGSAP FVIII BDD2 ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI (A1-Y2043- AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ AG144- REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR G2044- EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN Q2222- RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD AG864- LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD V2223- DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY Y2332) KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS PRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPR SFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRG ELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNET KTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQV TVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLV MAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKA GIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGPGSSPSASTGT GPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGT SSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSG TASSSGGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYIS QFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLR MELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQG SSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPS PGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSST GSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPG SSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPG TSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTG SPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGA SPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSG ATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGS PGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTP GSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSG ATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSS PGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSST PSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGA TGSPGSSTPSGATGSPGASPGTSSTGSPGVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTS MYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVH QIALRMEVLGCEAQDLY FVIII BDD2 ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFN (A1-G1799- IAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTS AE144- QREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLV A1800- CREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGY F2093- VNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTL AE42- LMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVV $2094- SPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQR V2223- IGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGIT AE42- DVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERD N2224- LASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLE AE42- DPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYE CFXTEN Amino Acid Sequence Name N2225- DTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYED G2278— ISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDE AE42- DENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGS K2279- FTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGGGSEP Y2332) TPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSG SETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP GTSTEPSEGSAPGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEK DVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQ MEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVR KKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPL GMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKT FGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPGSSLYISQFIIMYS QTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMG CDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVGPAGS GTSTEPSEGSAPGTSESATPESGPGSEPATSGGNNPKEWLQVDFQKTMKVTGVTT QGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGGTEPSEGSAPGSPAGSPTSTEEGTSESAT PESGPGSEPATSGSKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLG CEAQDLY FVIII BDD2 ATRRYYLGAVELSWDYMQSDLGELPVDARGPGSSPSASTGTGPGSSPSASTGTGPGTPGSG (Al-R28- TASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGS AG144-F29- PGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSGFPPRVPKSFPFNTSV G244- VYKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSY AG288- AEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDL L245- VKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASA R2090- RAWPKMHTVNGYVNRSLPGGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTP AG576- SGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGAT Q2091- GSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG Y2332- SSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSA AG864) STGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGLIGCHRKSVY WHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQH DGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKK HPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETF KTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLK DFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQR GNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDS LQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENP GLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNP PVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAA VERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYI RAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHM APTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFD ETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAGINGYIMDTLPGLVMAQDQRIRWY LLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGE HLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWST KEPFSWIKVDLLAPMIIHGIKTQGARGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGS PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATS GSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPA TSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTST EEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTS TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE GSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPG TSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESA TPESGPGTSTEPSEGSAPGQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSS GIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFT NMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSM YVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQ IALRMEVLGCEAQDLYGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST EEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSP AGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE CFXTEN Amino Acid Sequence Name GSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG EGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESA TPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSE SATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEG SAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGT STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESAT PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP GSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSES ATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGS APGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNI (Al-T1651- AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ AG576- REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR R1652- EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN K1808- RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD AG144- LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD P1809- DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY F2093- KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL AG288- YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI S2094- GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA Y2332) SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS KNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLR QSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQL GTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDT TLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGP ALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTP LIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPE SARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLK EMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKN LFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQI VEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTP STLTQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSS HLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKK VENTVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWN EANRPGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDT ILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITGPGTPGSGT ASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSP GASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASP GTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSST GSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPG SSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPG TSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTG SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSS TPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGT ASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSSGRTTLQSDQEEI DYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRA QSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYS FYSSLISYEEDQRQGAEPRKNFVKGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSG ATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSS PGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGPNETKTYFWKVQHHMAPTKD EFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSW YFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGS NENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGM STLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWI PMIIHGIKTQGARQKFGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSST SPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGA TGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSP GSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPS CFXTEN Amino Acid Sequence Name PGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGSSLYISQFII MYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRME LMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNN PKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQ GNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY FVIII BDD2 ATRRYYLGAVELSWDYMQSDLGELPVDAGGAPSPSASTGTGPGTPGSGTASSSPGSSTPSG (Al-A28- ATGSPGPSGPGRFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEV AG42-F29- TLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEGGPGTPGSGTASSSPG E124- SSTPSGATGSPGSSPSASTGTGPGASPGDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYS AG42- YLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGGSPSASTGTGPGAS D125-E124- PGTSSTGSPGTPGSGTASSSPGSSTPSGAGKSWHSETKNSLMQDRDAASARAWPKMHTVN AG42- LPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQT D125-P333- LLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPGSASTGTGPGASPGTSSTGSPGTPGSG AG42- TASSSPGSSTPSGATGGQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKK Q334- HPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETF Y2332) KTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLK DFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQR GNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDS LQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENP GLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNP PVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAA VERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYI RAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHM APTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFD ETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYL LSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEH LHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTK EPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFF GNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQIT ASSYFTNMFATWTPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVK SLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQ SWVHQIALRMEVLGCEAQDLY FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNI (Al-D345- AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ AE144- REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR Y346- EKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN D403- IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD AE144- LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDGGSEPATSGSETPGTSES R405- ATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSE R1797- TPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGYD AE288- DDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDGGT Q1798- STPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAE Y2322) SPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPG TSPSGESSTAPGRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLY GEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTV EDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVF DENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILS IGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGM TALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPEN DIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDS NNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTI PSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESG SSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSATNRK THIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKN MEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGP EKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKI QEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLN DSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALK QFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSI PQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNL CFXTEN Amino Acid Sequence Name SLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKD LFPTETSNGSPG WDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWA KQGRTERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPR SFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRG ELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRGGTSESATPESGPGSE PATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPT STEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG TSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPAT SGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESG PGTSTEPSEGSAPGQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDL EKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNI QMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVR KKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLG MASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQG ARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRL HPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHL QGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQW TLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY FVIII (A1- ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI N745)- AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ AE864- KVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR (P1640- EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN Y2332) RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS KNNAIEPRSFSQNGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGT GSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSP TSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP GTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPES GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP ESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG TSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESG PSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPA GSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPE SGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGS SETPGTSESATPESGPGTSTEPSEGSAPGPPVLKRHQREITRTTLQSDQEEIDYDDTIS VEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQ FKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISY EEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSG LIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTF KENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYK MALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHI RDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFS SLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSI RSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNA WRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQN QGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY FVIII BDD9 ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTS V V Y KKTLFVEFTVHLFNI (Al- N745)- AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ AE288- REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR CFXTEN Amino Acid Sequence Name (P1640- EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN Y2332) RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA INGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP FMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS KNNAIEPRSFSQNGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGT SESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESAT PESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTE PSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGPPVLKRHQREITRTTLQSDQ EEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRN RAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRP YSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVD LEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPC NIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFT VRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTP LGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKT QGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYI RLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARL HLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGH QWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQ FVIII BDD9 LGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI (Al- S743)- AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ AE288- REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR (Q1638- EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN Y2332) RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA INGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS KNNAIEPRSFSGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTS ESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE GSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGQNPPVLKRHQREITRTTLQSDQE EIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNR AQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRP LISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVD LEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPC NIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFT VRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTP LGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKT QGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYI RLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARL HLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGH QWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQ FVIII BDD9 ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI (Al- N745)- AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ AG288_2- REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR (P1640- EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN Y2332)- RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD CFXTEN Amino Acid Sequence Name AG288_2 LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI YKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS KNNAIEPRSFSQNGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG TPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSA STGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGT GPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGA SPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGPPVLKRHQREITRTTLQS DQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVL RNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQA SRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFS DVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCR MEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGH KEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKC QTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIH GIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPII ARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSK GRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQ DGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGC EAQDLYGAGSPGAETAPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG SPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSS PSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSAST GTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGP GASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGAETAEQKLISEEDLSP FVIII BDD9 ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNI (Al- S743)- WMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ AG288_2- REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR (Q1638- EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN Y2332)- RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD AG288_2 LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS KNNAIEPRSFSGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTP GSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSAST GTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGP GSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASP GTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGQNPPVLKRHQREITRTTLQS DQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVL RNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQA SRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFS DVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCR APCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGH VFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKC ASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIH GIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPII ARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSK ARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQ DGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGC EAQDLYGAGSPGAETAPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG SPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSS PSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSAST CFXTEN Amino Acid Sequence Name GTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGP GASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGAETAEQKLISEEDLSP FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNI BDD10 AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ (Al- N745)- REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR AE288- EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN (P1640- RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD Y2332)- LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD AE288 DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS KNNAIEPRSFSQNGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGT SESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESAT SPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTE PSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGPPVLKRHQAEITRTTLQSDQ EEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRN RAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRP YSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVD LEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPC NIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFT VRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTP LGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKT QGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYI RLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARL HLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGH QWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQ DLYGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAG SPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPES GPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSE ETPGTSESATPESGPGTSTEPSEGSAP FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNI BDD10 AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ (Al- S743)- REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR AE288- EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN (Q1638- RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD Y2332)- LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD AE288 DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI YKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP FMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS KNNAIEPRSFSGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTS SGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE GSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGQNPPVLKRHQAEITRTTLQSDQE TISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNR AQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRP YSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVD LEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPC NIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFT VRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTP LGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKT CFXTEN Amino Acid Sequence Name QGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYI RLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARL HLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGH QWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQ DLYGGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAG SPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPES ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSE PATSGSETPGTSESATPESGPGTSTEPSEGSAP FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNI BDD10 AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ (Al- N745)- REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR AG288_2- EKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN - RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD Y2332)- LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD AG288_2 DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS KNNAIEPRSFSQNGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG TPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSA STGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGT GPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGA SPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPPVLKRHQAEITRTTLQSD QEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLR NRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQAS RPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSD VDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRA PCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVF TVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQT PLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIK TQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARY IRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARL NAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGH QWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQ DLYGAGSPGAETAPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG TPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSA STGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGT SASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGA SPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGAETAEQKLISEEDLSPAT FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNI BDD10 WMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ (Al- S743)- REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCR AG288_2- EGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVN (Q1638- RSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMD Y2332)- LGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD AG288_2 DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLI GPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQA SNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFP FSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLS KNNAIEPRSFSGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTP GSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSAST GTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGP GSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASP GTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSQNPPVLKRHQAEITRTTLQSD CFXTEN Amino Acid Sequence Name QEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLR NRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQAS RPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSD VDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRA PCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVF TVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQT PLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIK TQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARY IRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARL HLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGH QWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQ DLYGAGSPGAETAPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPG TPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSA STGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGT GPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGA SPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGAETAEQKLISEEDLSPAT Sequence name reflects N— to C-terminus configuration of the FVIH segments (amino acid spanning numbers relative to mature sequence) and XTEN ents Table 51: ary CFXTEN comprising FVIII, cleavage seguences and XTEN ces (SEQ ID NOS 590 res ectivel in order of a earance CFXTEN Amino Acid Sequence Name SP-AE288- MQIELSTCFFLCLLRFCFSGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP CS-L- GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESA _1- TPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP 745)— GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS AE288- EGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPQSPRSFQGPEGPSATRRYYLGAVE (FVIII_168 LSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIAKPRPPWMGLLG 6-2332)-L- PTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSH CS-AE288 TYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFI LLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWH VIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGM EAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKT WVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAI QHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEI VTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDK RNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSlNGYVFDSLQLSVCLHEV AYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDF RNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST EEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTST EPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSE TPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTST EPSEGSAPQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTD GSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRK NFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNP AHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMD TLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEML PSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLA RLHYSGSlNAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTY TLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLG MESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKV CFXTEN Amino Acid Sequence Name TGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLL TRYLRIHPQSWVHQIALRMEVLGCEAQDLYGPEGPSQSPRSFQGTSESATPESGPGSEPATSGS ETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTS SGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGS APGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPA GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS SP-AE576- MQIELSTCFFLCLLRFCFSGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE CS-L- GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS (FVIII_1- PTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP 745)— GTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPS AE576- EGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPG (FVIII_168 SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE 6-2332)-L- GSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGS CS-AE288 EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATP ESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPQS PRSFQGPSGPATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFV EFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEY REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGA LLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNG YVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLL MDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFD DDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKY KKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLY SRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGP LLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNI MHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGE TVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIE PRSFSQNGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSA PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSES ATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA ATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEP SEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP GTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG TSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSP TSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPQSPRSFQKKTRHY FIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLG PYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHH MAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFD ETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLS MGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHA GMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSW IKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSG NPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNM FATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKE FLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRME VLGCEAQDLYGPEGPSQSPRSFQGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPAT SGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESA GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAP GTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP SP- MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSV (FVIII_1- VYKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYW 745)— KASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVK AE576- IGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAW (FVIII_168 PKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPI 6-2332)-L- TFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTD CS-AE576 SEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNN GPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPH CFXTEN Amino Acid Sequence Name GITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMER IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLED PEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTL TLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYL LSKNNAIEPRSFSQNGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTS TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTS TEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS TEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGS EPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS APGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPES GPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPQSPR SFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGE LNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKT HHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQ EFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQD QRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVE CLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINA WSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLM VFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQ ITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVK SLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQS WVHQIALRMEVLGCEAQDLYGPEGPSQSPRSFQGSPAGSPTSTEEGTSESATPESGPGTSTEPS SPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG GSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSP TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG SEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSP TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPG TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE GSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT STEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPE SGPGTSTEPSEGSAP SP-AE576- MQIELSTCFFLCLLRFCFSGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE CS-L- GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS (FVIII_1- PTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP 745)— SEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPS AE576- EGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPG (FVIII_168 SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE 6-2332) GSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGS EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATP ESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPQS PRSFQGPEGPSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLF VEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAE YDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIG ALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVN GYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTL LMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRF DDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRK YKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIG PLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNI MHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGE TVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIE PRSFSQNGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSA PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSES ATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA PGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEP SEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP GTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS CFXTEN Amino Acid Sequence Name EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSP TSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPQSPRSFQKKTRHY FIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLG PYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHH MAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFD ETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLS MGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHA LVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSW APMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSG IKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNM FATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKE FLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRME VLGCEAQDLY SP-AE576- MQIELSTCFFLCLLRFCFSGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE CS-L- GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS (FVIII_1- PTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP 743)— SEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPS AE288- EGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPG (FVIII_168 SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE 6-2332)-L- GSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGS CS-AE576 EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATP PAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPIEP RSPSGSPGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFT VHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDD QTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALL VCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGY VNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLM DLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYK KVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSR RLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLI CYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHS INGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVF MSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRS FSGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTE PSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTE EGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPA TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETP GTSESATPESGPGTSTEPSEGSAPQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQS GSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYS SLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDV HSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDP TFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYK MALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRD FQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYI SQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLR MELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVN NPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQG NQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGSPGIEPRSPSGSPAGS PTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPS EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP SESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT STEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEG SAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSE PATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTS TEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP SP-AG288- MQIELSTCFFLCLLRFCFSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG CFXTEN Amino Acid Sequence Name CS-L- SPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSP (FVIII_1- SASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGT 743)— GPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASP AG576- SPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSIEPRSPSGSPGATRRYYLGAVEL (FVIII_168 SWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIAKPRPPWMGLLGP 6-2332)-L- TIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHT CS-AG288 YVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFIL EGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHV IGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGME AYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTW VHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQ HESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIF KYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKR NVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVA YWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFR NRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSPGTPGSGTASSSPGSSTPS GATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP GASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGT SSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSP GSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPS GATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSP GASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPS GTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSP GSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSA STGTGPGSSPSASTGTGPGASPGTSSTGSQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLR NRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRP LISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDL EKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQ MEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKK EEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMAS GHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQK FSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSI RSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAW RPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKV KVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGSPGQSPRSFQ PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTP SGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTG PGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPG TSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTG PGTPGSGTASSSPGSSTPSGATGS SP-AG576- MQIELSTCFFLCLLRFCFSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGT CS-L- GPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPG (FVIII_1- SGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASS 745)— SPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASP AG288- SPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGT (FVIII_168 GPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASP 6-2332)-L- GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATG CS-AE576 SPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASP GTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTG SQSPRSFQGSPGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLF VEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAE YDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIG ALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVN GYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTL LMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRF DDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRK MAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPL YSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIG PLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNI MHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGE CFXTEN Amino Acid Sequence Name ENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIE PRSFSQNPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASS SPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSP SASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGT GPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSP SASTGTGPGTPGSGTASSSPGSSTPSGATGSQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVL RNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASR PYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVD LEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNI QMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRK KEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMA SGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQ KFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHY SIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNA WRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNG GNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGSPGQSPR SFQGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPES GPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSE SATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS APGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS APGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTST GSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP SP- MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSV (FVIII_1- FVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYW 743)— KASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVK AG576- DLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAW (FVIII_168 PKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPI 6-2332)-L- TFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTD CS-AG576 SEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNN GPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPH GITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMER DLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLED PEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTL TLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYL LSKNNAIEPRSFSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSST PSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASS SPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASP GTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTG SPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASP GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTG SPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPG SGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTG SPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSQSPRS FQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGEL LGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTY FWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQE FALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQD QRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVE CLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINA WSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLM VFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQ ITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVK SLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQS WVHQIALRMEVLGCEAQDLYGSPGQSPRSFQPGTPGSGTASSSPGSSTPSGATGSPGSSPSAST GTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPG STGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSAS TGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPG CFXTEN Amino Acid ce Name SSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSG ATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPG ASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGT ASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPG TGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAS TGTGPGASPGTSSTGS SP-AG288- MQIELSTCFFLCLLRFCFSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG CS-L- SPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSP (FVIII_1- SASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGT 743)— SASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASP AG288- GTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSQSPRSFQGPSGPATRRYYLGAV (FVIII_168 ELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIAKPRPPWMGLL 6-2332)-L- GPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGS 88 HTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHK FILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYW HVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDG MEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPK TWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREA IQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGE TVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDK RNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEV AYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDF RNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSPGASPGTSSTGSPGASPG TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSS PGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGS GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGS PGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTP SGATGSQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDG SFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSL1SYEEDQRQGAEPRKN FVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPA HGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTL PGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPS KAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARL HYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRG NSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGME SKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTG VTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTR YLRIHPQSWVHQIALRMEVLGCEAQDLYGPSGPQSPRSFQGTSESATPESGPGSEPATSGSETP GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESA TPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAP GTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP SP-AE576- MQIELSTCFFLCLLRFCFSGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE CS-L- GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS (FVIII_1- PTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP 743)— GTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPS AG576- EGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPG (FVIII_168 SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE ) GSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGS EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATP ESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPQS PRSFQGSPGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVE FTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYD DQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGAL LVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGY VNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLM DLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDD DNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYK KVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSR RLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLI CFXTEN Amino Acid Sequence Name CYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHS INGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVF MSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRS FSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSS TPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSST PGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGAS PGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTAS SSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGAS PGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST GSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSS TPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTAS SSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSQSPRSFQKKTRHYFIA AVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYI RAEVEDNIMVTFRNQASRPYSFYSSL1SYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAP TKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKS WYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGS NENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMS TLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKV DLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKH NIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFAT RLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLI SSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVL GCEAQDLY FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIA BDDZ KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK S3 67- EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL FXIa- AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNSSLPGL AE42- IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF F3 68- CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSKLT Y23 32- RAETGEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSGFIQIRSVAKKHPKTWVHY FXIa- IAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESG AE864 YGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYK WTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVIL FSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWY QTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRG MTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSD QEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNR AQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYS FYSSL1SYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEK IGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQME DPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEY KMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIR DFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSL YISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTL RMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWTPSKARLHLQGRSNAWRPQV NNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQ GNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYKLTRAETGGSPAGSP TSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPG TSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSE GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGT STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPE SGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS APGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTST EEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES GPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSE SATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTST EEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP CFXTEN Amino Acid ce Name FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIA BDDZ KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK N745 - EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL FIXa- AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL AGZ88- IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF FIXa- CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI P 1 640- RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY Y23 32- TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK FIXa- HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD AG864 QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPLGR IVGGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPG SSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAS TGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPG ASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSAS TGTGPGTPGSGTASSSPGSSTPSGATGSGPLGRIVGGPPVLKRHQREITRTTLQSDQEEIDYDDT ISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQ FKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYE EDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIG PLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKEN YRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYN LYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITAS GQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIM YSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMG SMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEW LQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSF TPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYPLGRIVGGGASPGTSSTGSPG SSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTS STGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPG TPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS TGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPG ASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSAS TGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPG SSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSG ATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPG ASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGT ASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPG SSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAS TGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPG SSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP FVIII LGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIA BDDZ KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK V1 2 8- EDDKVLQVRIVGGGAPSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGPSGPGLQVRIVGG FVIIa- FPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQ AG42- TLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRK FVIIa- SVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSH G2044- QHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAK FVIIa- VHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETF AG144- QHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDF Y23 32- IFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQ FVIIa- IMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSV AG576 CLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGC HNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQRE ITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMS SSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTF RNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWA YFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNC RAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHV FTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTP CFXTEN Amino Acid Sequence Name LGMASGHIRDFQITASGQYGLQVRIVGGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTP SGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSS PGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGLQVRIVGGQWAPKLARLHYSGS INAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGT NVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISD AQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQG VKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHP QSWVHQIALRMEVLGCEAQDLYLQVRIVGGPGTPGSGTASSSPGSSTPSGATGSPGSSPSAST GTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPG ASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSAS TPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPG SSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSG ATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPG ASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGT ASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPG SSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAS TGTGPGASPGTSSTGS AE864- GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS FVIII- EGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPG in- TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP AE144 ESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGT SESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPE SGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPA GSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE TPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSA PGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAG SPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP GATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNI AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQRE KEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGS LAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPG LIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLL FCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQ IRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHP STRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKY ETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDF KVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEE NNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKT SNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSN KTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQ LVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQ EKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRS LNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRAL LEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSI PQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLS LAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFP TETSNGSPG HYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRT ERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTR HYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGL AEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQH HMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIF CFXTEN Amino Acid Sequence Name DETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYL LSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLH AGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFS WIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDS SGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTN MFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYV KEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALR MEVLGCEAQDLYGLTPRSLLVGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGS PTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAP GTSESATPESGPGSEPATSGSETPGTSTEPSEGSAP FVIII LGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIA BDD3 - KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK FXIIa- EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL AE144 AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG HNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL ITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERL WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEF DCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFT ENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIH SIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLV YSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP MIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNP PIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPS QGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQD GHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEA QDLYGTMTRIVGGGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTS TEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPES GPGSEPATSGSETPGTSTEPSEGSAP FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIA BDD3 - KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK Elastase- EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL AE144 AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY TREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL KRHQGEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERL WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEF DCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFT ENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIH SIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLV YSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP MIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNP PIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPS KARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQD FFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEA QDLYGGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAP CFXTEN Amino Acid Sequence Name GSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPAT SGSETPGTSTEPSEGSAP FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIA BDD3 - MGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK FXIa- EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL AE144 AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY TREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL ITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERL WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEF DCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFT ENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIH HVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLV YSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP MIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNP PIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPS KARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQD GHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEA QDLYGKLTRAETGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTST EPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPES GPGSEPATSGSETPGTSTEPSEGSAP FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTVHLFNIA BDD3 - KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK Thrombin- EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL AE144 AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY TREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL KRHQGEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERL WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEF DCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFT ENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIH SIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLV YSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP MIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNP PIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPS KARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQD GHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEA TPRSLLVGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTS TEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPES GPGSEPATSGSETPGTSTEPSEGSAP AE144- GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPAT FVIII SGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP BDDZ- GTSTEPSEGSAPGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKT MMP- 1 7- LFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEG AE864 AEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGL IGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHT CFXTEN Amino Acid Sequence Name VNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQ TLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVV RFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIG RKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVR PLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQAS NIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFS GETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNN AIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKK TRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHL GLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKV TKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFF TIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRW YLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEH TLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEP DLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNV HNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYF TNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSM YVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIA LRMEVLGCEAQDLYGAPLGLRLRGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPA GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE TPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE TPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSES PGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGS PTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGP GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPAT SGSETPGTSESATPESGPGTSTEPSEGSAP AE144- GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPAT FVIII SGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP BDDZ- GTSTEPSEGSAPGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKT FXIIa- LFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEG AE864 AEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGL IGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHT VNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQ TLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVV RFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIG RKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVR PLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQAS NIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFS GETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNN AIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKK TRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHL GLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKV QHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFF TIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRW YLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEH LHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEP FSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNV DSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYF TNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSM YVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIA GCEAQDLYGTMTRIVGGGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPA GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE CFXTEN Amino Acid Sequence Name TPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST APGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSES PGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGS PTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGP GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPAT SGSETPGTSESATPESGPGTSTEPSEGSAP AG144- SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGT FVIII GPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSP BDDZ- SASTGTGPGASPGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKT FXIa- LFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEG AG576 AEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGL IGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHT VNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQ TLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVV RFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIG RKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVR PLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL IGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQAS NIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFS GETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNN AIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKK TRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHL GLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKV QHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFF TIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRW YLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEH LHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEP FSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNV DSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYF TNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSM YVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIA LRMEVLGCEAQDLYGKLTRAETGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSP SASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTG SPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPG SGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATG SPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSP SASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTG SPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSST PSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGT GPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASP GTSSTGS AE144- GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPAT FXIa-FVIII GSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP BDDZ- GTSTEPSEGSAPGKLTRAETGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNT AE864 SVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVS GAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDL VKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASAR AWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEI SPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDL VVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYL NNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIY PHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNM ERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYED CFXTEN Amino Acid Sequence Name TLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISA YLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQ SPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLY RGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNE TKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQV TVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVM RWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIW GEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGS INAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGT LMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISD AQITASSYFTNMFATWSPSKARLHQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQG VKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHP QSWVHQIALRMEVLGCEAQDLYGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPA GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE TPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE TPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGS PTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGP GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPAT SGSETPGTSESATPESGPGTSTEPSEGSAP AE144- GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPAT FVIII SGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP BDDZ- GTSTEPSEGSAPGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKT Y23 32- LFVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEG Thrombin- AEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGL AE864 IGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHT VNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQ TLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVV RFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIG RKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVR PLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGL CYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQAS NIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFS SMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNN AIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKK TRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHL GLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKV QHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFF TIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRW SNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEH LHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEP FSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNV DSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYF WSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSM YVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIA LRMEVLGCEAQDLYGLTPRSLLVGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPA GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE TPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE TPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA CFXTEN Amino Acid Sequence Name ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGS PTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGP GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPAT SGSETPGTSESATPESGPGTSTEPSEGSAP AE864- GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS FVIII- EGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPG MMP TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP AE144 ESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGT SESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPE SGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPA GSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSA ATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAG SPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP GATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNI AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQRE KEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGS LAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPG LIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLL FCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQ IRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHP STRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKY ETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDF KVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEE NNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKT SNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSN KTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQ LVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQ EKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRS LNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRAL KQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSI PQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLS LAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFP TETSNGSPG HYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRT ERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTR HYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGL LGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQH HMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIF DETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYL ENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLH AGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFS WIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDS IFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTN MFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYV KEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALR EAQDLYGAPLGLRLRGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGS PTSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAP GTSESATPESGPGSEPATSGSETPGTSTEPSEGSAP AF144- GTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSGTAPGSTSSTA CFXTEN Amino Acid Sequence Name FXIIa- ESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESSTAPGT FVIII- SPSGESSTAPGTMTRIVGGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSV FXIIa- VYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYW AF864 KASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVK IGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAW PKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPI TFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTD SEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNN GPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPH PLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMER DLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLED PEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTL TLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYL LSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLL RQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLR LNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLF GKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLT KDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDR MLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQ RTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSR NLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQN VEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRI QNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKE KGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSG VQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTS GKVELLPKV SSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINE GQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFD IYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTD GSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRK ETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNP AHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMD TLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEML PSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLA RLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTY RGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLG MESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKV TGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLL TRYLRIHPQSWVHQIALRMEVLGCEAQDLYGTMTRIVGGGSTSESPSGTAPGTSPSGESSTAP GSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESP SGTAPGTSPSGESSTAPGSTSESPSGTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGT STAPGTSPSGESSTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSG TAPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSP SGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGSTSSTAESPG PGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPE SGPXXXGASASGAPSTXXXXSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAP GSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTA ESPGPGTSPSGESSTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGS TSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAES PGPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTST PESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSSTAESPG GESSTAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSP AE864- GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS FVIII- EGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPG FXIa- TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP AE144 ESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGT SESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPE SGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPA CFXTEN Amino Acid Sequence Name EEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE TPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSA PGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAG SPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP GATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNI AKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQRE KEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGS LAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPG LIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLL FCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQ IRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHP STRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKY ETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDF KVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEE NNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKT SNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSN KTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQ LVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQ EKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRS LNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRAL KQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSI PLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLS EMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFP TETSNGSPG HYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRT ERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTR HYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGL LGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQH HMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIF YFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYL LSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLH AGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFS WIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDS SGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTN PSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYV KEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALR MEVLGCEAQDLYGKLTRAETGGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSP TSTEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPG TSESATPESGPGSEPATSGSETPGTSTEPSEGSAP AE144- GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGSEPAT VIII SGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP BDD9- GTSTEPSEGSAPGKLTRAETGATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNT AE864 SVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVS YWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDL VKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASAR AWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEI SPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDL TDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYL NNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIY PHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNM ERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQL EDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYED TLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISA YLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQ CFXTEN Amino Acid Sequence Name SPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLY RGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNE TKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQV TVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVM AQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIW RVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGS INAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGT LMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISD AQITASSYFTNMFATWSPSKARLPEQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQG SMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHP QSWVHQIALRMEVLGCEAQDLYGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPA GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE GSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE TPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGS PTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGP GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPAT SGSETPGTSESATPESGPGTSTEPSEGSAP AE48— MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGKLTRAETGATRRYY FXIa-FVIII LGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPW BDD9- MGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVF AE864 PGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQ TLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRK IGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSH QHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAK KHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETF KTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDF PILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQ IMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSV YWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGC HNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQRE ITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMS SSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTF RNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWA YFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNC RAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHV FTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTP LGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQ GARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLH PTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGR SNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQ NGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYGGSPAG SPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP TPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPS EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATP ESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT STEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEG AGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSE PATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTS TEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTS ESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES GPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSE SATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTST CFXTEN Amino Acid Sequence Name EPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP FVIII LGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIA BDD9- KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK FXIa- EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL AG288_2 AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL KRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERL WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEF DCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFT ENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIH SIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLV YSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP MIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNP PIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPS KARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQD GHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEA QDLYKLTRAETGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPG SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGT GPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSP SASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTG SPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGS FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIA BDD9- KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK FXIa- EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL AG864 AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY TREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL KRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERL SSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEF DCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFT ENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIH SIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLV YSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP MIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNP PIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPS KARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQD FFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEA TRAETGGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTP SGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSS PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGS GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGS PGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPG TSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGS PGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTP SGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGS CFXTEN Amino Acid Sequence Name PGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGS GTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGS PGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTP SGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGS ASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTP SGATGSPGASPGTSSTGSP FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIA BDD9 (1— KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK 745) EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL AG288_2- AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL (1640- IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF Y2332)- CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI FXIa- HPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY AG864 TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNGPG ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSG ATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPG ASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTS STGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPG TPGSGTASSSPGSSTPSGATGSPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDE DENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFT QPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFV KPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAH GRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLP DQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPS KAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARL HYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRG NSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGME SKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTG VTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTR YLRIHPQSWVHQIALRMEVLGCEAQDLYKLTRAETGGASPGTSSTGSPGSSPSASTGTGPGSS PSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTAS SSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSS TPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG TGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTP GSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTAS SSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGAS PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTG TGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGAS PGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGAT GSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGAS PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSST GSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTP GSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIA BDD9 (1— KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK 743) EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL AG288_2- AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL (163 8- SVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF Y2332)- CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI FXIa- RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY AG864 TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK ILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD CLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSGPGASP GTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATG CFXTEN Amino Acid Sequence Name SPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASP SPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTG SPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPG SGTASSSPGSSTPSGATGSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDE DENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFT QPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFV KPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAH GRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLP GLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPS KAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARL NAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRG NSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGME SKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTG VTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTR QSWVHQIALRMEVLGCEAQDLYKLTRAETGGASPGTSSTGSPGSSPSASTGTGPGSS PSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTAS TPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSS TPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG TGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTP GSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTAS SSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGAS PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTG TGPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGAS PGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGAT GSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGAS PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSST GSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTP GSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP BDD10 (1— ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIA 745) KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK AG288_2- EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL (1640- AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL Y2332)- IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF FXIa- CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI AG864 RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNGPG ASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSG ATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPG ASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTS STGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPG TPGSGTASSSPGSSTPSGATGSPPVLKRHQAEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDE DENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFT ELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFV TYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAH GRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLP GLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPS KAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARL HYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRG NSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGME SKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTG VTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTR YLRIHPQSWVHQIALRMEVLGCEAQDLYKLTRAETGGASPGTSSTGSPGSSPSASTGTGPGSS PSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTAS SSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSS TPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTG TGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTP CFXTEN Amino Acid Sequence Name GSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTAS SSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGAS PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTG PGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGAS PGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGAT GSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGAS PGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSST GSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTP GSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIA BDD10- MGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK FXIa- EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL AG288_2 AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL ITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERL WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEF DCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFT ENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIH SIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLV TPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP MIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNP PIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPS KARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQD GHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEA QDLYKLTRAETGAGSPGAETAPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSG ATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPG SSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSAS TGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPG ASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSGAETAEQKLISEEDLSPATG FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIA BDD 1 0- KPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK FXIa- EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL AG864 AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF QHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL ITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERL WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEF DCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFT ENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIH SIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLV YSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP MIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNP PIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPS KARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQD GHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEA CFXTEN Amino Acid Sequence Name QDLYKLTRAETGGASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTP SGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSS PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGS GTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGS TSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGASPGTSSTGSPGASPG TSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGS PGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTP SGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGS PGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPGS GTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGS PGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTP SGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGS PGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTP SGATGSPGASPGTSSTGSP FVIII ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIA BDD l 0- MGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREK FXIa- EDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL AE864 AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGL IGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLF CHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQI RSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAY TDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVK HLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVD QRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFD SLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPG LWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVL KRHQAEITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERL WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEF DCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFT ENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIH SIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLV YSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAP MIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNP PIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPS KARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQD GHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEA QDLYKLTRAETGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTE EGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTE PSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSA PGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPA TSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP GTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPAT SGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP PTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESA TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGS PTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETP GSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESA TPESGPGTSTEPSEGSAP Sequence name reflects N— to inus configuration of the FVIII variant and XTEN components: signal peptide (SP); linker (L); cleavage sequence (CS) may be d by protease name active on the sequence, and XTEN components by family name and length, With insertion points for components denoted by FVIII amino acid and numbered ons adjacent to the inserted sequence or Al being the N—terminus and Y2332 being the C-terminus of the FVIII.

Definitions of the specific embodiments of the invention as claimed herein follow.

According to a first embodiment of the ion, there is provided a recombinant factor VIII fusion protein sing a factor VIII polypeptide fused to an extended recombinant polypeptide (XTEN), n the factor VIII polypeptide comprises A1 domain, A2 domain, A3 domain, a3 domain, C1 domain, C2 domain, and optionally a B domain or a portion f, wherein the XTEN is inserted within the factor VIII polypeptide between two adjacent domains.

According to a second embodiment of the invention, there is provided an isolated nucleic acid encoding the recombinant factor VIII fusion n of the first embodiment.

According to a third embodiment of the ion, there is provided a vector comprising the isolated nucleic acid of the second embodiment.

According to a fourth embodiment of the invention, there is provided an isolated host cell comprising the vector of the third embodiment. ing to a fifth embodiment of the invention, there is ed a pharmaceutical composition sing the recombinant factor VIII fusion protein of the first embodiment and a pharmaceutically acceptable carrier.

According to a sixth embodiment of the invention, there is provided a pharmaceutical composition comprising the isolated nucleic acid of the second embodiment, the vector of the third embodiment, or the isolated host cell of the fourth embodiment, and a pharmaceutically acceptable carrier.

According to a seventh embodiment of the invention, there is provided a method of making a recombinant factor VIII fusion n comprising culturing the isolated host cell of the fourth embodiment in media under suitable conditions.

According to an eighth embodiment of the invention, there is provided use of the pharmaceutical ition of the fifth embodiment in the manufacture of a ment for the ent of a coagulopathy in a subject in need thereof.

According to a ninth embodiment of the invention, there is provided use of the recombinant factor VIII fusion protein of the first embodiment in the manufacture of a medicament for the treatment of a coagulopathy in a subject in need thereof.

Claims (30)

WHAT WE CLAIMED IS:

1. A recombinant factor VIII fusion protein comprising a factor VIII polypeptide fused to an extended recombinant polypeptide (XTEN), wherein the factor VIII polypeptide comprises A1 domain, A2 domain, A3 domain, a3 domain, C1 domain, C2 domain, and optionally a B domain or a portion thereof, n the XTEN is inserted within the factor VIII polypeptide between two adjacent domains.

2. The recombinant factor VIII fusion protein of claim 1, wherein the XTEN is inserted within the factor VIII polypeptide between the A1 domain and the A2 domain.

3. The recombinant factor VIII fusion n of claim 1, n the XTEN is inserted within the factor VIII polypeptide between the A2 domain and the B domain.

4. The recombinant factor VIII fusion protein of claim 1, wherein the XTEN is inserted within the factor VIII polypeptide between the B domain and the a3 domain.

5. The recombinant factor VIII fusion protein of claim 1, wherein the XTEN is inserted within the factor VIII polypeptide between the a3 domain and the A3 domain.

6. The recombinant factor VIII fusion n of claim 1, wherein the XTEN is inserted within the factor VIII polypeptide between the A3 domain and the C1 domain.

7. The recombinant factor VIII fusion protein of claim 1, wherein the XTEN is inserted within the factor VIII ptide between the C1 domain and the C2 domain.

8. The recombinant factor VIII fusion n of any one of claims 1 to 7, comprising two XTENs.

9. The recombinant factor VIII fusion protein of any one of claims 1 to 8, comprising three XTENs, four XTENs, five XTENs, or six XTENs.

10. The recombinant factor VIII fusion protein of any one of claims 1 to 9, wherein the XTEN comprises 36 amino acids, 42 amino acids, 72 amino acids, 96 amino acids, 144 amino acids, or 288 amino acids.

11. The recombinant factor VIII fusion n of claim 10, wherein the XTEN comprises 72 amino acids, 144 amino acids, or 288 amino acids.

12. The inant factor VIII fusion protein of any one of claims 1 to 11, wherein the XTEN ses one or more XTEN sequence motifs, which are selected from the group consisting of SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, and 48.

13. The recombinant factor VIII fusion protein of claim 12, wherein the one or more XTEN sequence motifs are selected from the group consisting of SEQ ID NOs: 23, 24, 25, and 26.

14. The recombinant factor VIII fusion protein of any one of claims 1 to 13, wherein the XTEN comprises an amino acid ce having 90%, 96%, 97%, 98%, 99%, 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 49, 50, 51, 52, 57, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 78, and 79.

15. The inant factor VIII fusion protein of any one of claims 1 to 14, wherein the XTEN comprises an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99%, or 100% ce ty to an amino acid sequence selected from the group consisting of SEQ ID NOs: 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, and 351.

16. The recombinant factor VIII fusion protein of any one of claims 1 to 15, wherein the factor VIII polypeptide comprises a native factor VIII polypeptide.

17. The inant factor VIII fusion protein of any one of claims 1 to 16, wherein the factor VIII polypeptide comprises a partially deleted B domain or a fully deleted B domain.

18. The recombinant factor VIII fusion protein of any one of claims 1 to 17, wherein the factor VIII polypeptide comprises a single chain factor VIII polypeptide.

19. The recombinant factor VIII fusion protein of any one of claims 1 to 18, wherein a heterologous ptide is fused to the factor VIII polypeptide.

20. The recombinant factor VIII fusion protein of claim 19, wherein the heterologous ptide is an Fc fragment of immunoglobulin or an FcRn binding domain.

21. The recombinant factor VIII fusion protein of claim 19, wherein the heterologous polypeptide is selected from the group consisting of n or a fragment thereof, a β subunit of C-terminal e of human chorionic gonadotropin, a HAP sequence, a transferrin, a PAS polypeptide, a polyglycine linker, a polyserine linker, and a combination thereof.

22. An isolated nucleic acid encoding the recombinant factor VIII fusion protein of any one of claims 1 to 21.

23. A vector comprising the isolated nucleic acid of claim 22.

24. The vector of claim 23, wherein the vector is a plasmid, a cosmid, a viral particle, or phage.

25. An isolated host cell comprising the vector of claim 23 or 24.

26. A pharmaceutical composition comprising the recombinant factor VIII fusion protein of any one of claims 1 to 21 and a ceutically acceptable carrier.

27. A pharmaceutical composition comprising the isolated nucleic acid of claim 22, the vector of claim 23 or 24, or the isolated host cell of claim 25, and a ceutically acceptable carrier.

28. A method of making a recombinant factor VIII fusion protein comprising culturing the ed host cell of claim 25 in media under suitable conditions.

29. Use of the pharmaceutical composition of claim 26 or 27 in the manufacture of a ment for the treatment of a opathy in a subject in need thereof.

30. Use of the recombinant factor VIII fusion protein of any one of claims 1 to 21 in the manufacture of a medicament for the treatment of a coagulopathy in a subject in need thereof. Date: 15 February 2018