AU2016244273B2

AU2016244273B2 - Immunoglobulin Chimeric Monomer-Dimer Hybrids

Info

Publication number: AU2016244273B2
Application number: AU2016244273A
Authority: AU
Inventors: Alan J. Bitonti; Susan C. Low; Adam R. Mezo; Robert T. Peters; Daniel S. Rivera; James M. Stattel
Original assignee: Bioverativ Therapeutics Inc
Current assignee: Bioverativ Therapeutics Inc
Priority date: 2003-05-06
Filing date: 2016-10-13
Publication date: 2018-10-18
Anticipated expiration: 2024-05-06
Also published as: AU2016244273A1; AU2019200306A1; AU2019200306B2

Abstract

The invention relates to a chimeric monomer-dimer hybrid protein wherein said protein comprises a first and a second polypeptide chain, said first polypeptide chain comprising at least a portion of an immunoglobulin constant region and a biologically active molecule, and said second polypeptide chain comprising at least a portion of an immunoglobulin constant region without the biologically active molecule of the first chain. The invention also relates to methods of using and methods of making the chimeric monomer-dimer hybrid protein of the invention.

Description

IMMUNOGLOBULIN CHIMERIC MONOMER-DIMER HYBRIDS

2016244273 13 Oct 2016 [001] This application is a divisional of Australian patent application no. 2013245463, filed on 15 October 2013, which is a divisional of Australian patent application no. 2013204787, filed on 12 April 2013 and is related to International patent application no. PCT/US2004/014064, filed on 6 May 2004 and claiming priority from United States patent application no. 60/469,600, filed on 6 May 2003, United States patent application no. 60/487,964, filed on 17 July 2003 and United States patent application no. 60/539,207, filed on 26 January 2004; each of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION [002] The invention relates generally to therapeutic chimeric proteins, comprised of two polypeptide chains, wherein the first chain is comprised of a therapeutic biologically active molecule and the second chain is not comprised of the therapeutic biologically active molecule of the first chain. More specifically, the invention relates to chimeric proteins, comprised of two polypeptide chains, wherein both chains are comprised of at least a portion of an immunoglobulin constant region wherein the first chain is modified to further comprise a biologically active molecule, and the second chain is not so modified. The invention, thus relates to a chimeric protein that is a monomer-dimer hybrid, i.e., a chimeric protein having a dimeric aspect and a monomeric aspect, wherein the dimeric aspect relates to the fact that it is comprised of two polypeptide chains each comprised of a portion of an immunoglobulin constant region, and wherein the monomeric aspect relates to the fact that only one of the two chains is comprised of a therapeutic biologically active molecule. Figure 1 illustrates one example of a monomer-dimer hybrid wherein the

2016244273 13 Oct 2016 biologically active molecule is erythropoietin (EPO) and the portion of an immunoglobulin constant region is an IgG Fc region.

BACKGROUND OF THE INVENTION [003] Immunoglobulins are comprised of four polypeptide chains, two heavy chains and two light chains, which associate via disulfide bonds to form tetramers. Each chain is further comprised of one variable region and one constant region. The variable regions mediate antigen recognition and binding, while the constant regions, particularly the heavy chain constant regions, mediate a variety of effector functions, e.g., complement binding and Fc receptor binding (see, e.g., U.S. Patent Nos.: 6,086,875; 5,624,821; 5,116,964).

[004] The constant region is further comprised of domains denoted CH (constant heavy) domains (CH1, CH2, etc.). Depending on the isotype, (i.e. IgG, IgM, IgA IgD, IgE) the constant region can be comprised of three or four CH domains. Some isotypes (e.g. IgG) constant regions also contain a hinge region Janeway et al. 2001, Immunobiology, Garland Publishing, N.Y., N.Y.

[005] The creation of chimeric proteins comprised of immunoglobulin constant regions linked to a protein of interest, or fragment thereof, has been described (see, e.g., U.S. Patent Nos. 5,480,981 and 5,808,029; Gascoigne et al. 1987, Proc. Natl. Acad. Sci. USA 84:2936; Capon et ai. 1989, Nature 337:525; Traunecker et al. 1989, Nature 339:68; Zettmeissl et al. 1990, DNA Cell Biol. USA 9:347; Byrn et al. 1990, Nature 344:667; Watson et al. 1990, J. Cell. Biol. 110:2221; Watson et al. 1991, Nature 349:164; Aruffo et al. 1990, Cell 61:1303; Linsley et al. 1991, J. Exp. Med. 173:721; Linsley et al. 1991, J. Exp. Med. 174:561; Stamenkovic et al., 1991, Cell 66:1133; Ashkenazi et al. 1991, Proc. Natl. Acad. Sci. USA

2016244273 13 Oct 2016

88:10535; Lesslauer et al. 1991, Eur. J. Immunol. 27:2883; Peppel et al. 1991, J.

Exp. Med. 174:1483; Bennett et al. 1991, J. Biol. Chem. 266:23060; Kurschner et al. 1992, J. Biol. Chem. 267:9354; Chalupny et al. 1992, Proc. Natl. Acad. Sci. USA 89:10360; Ridgway and Gorman, 1991, J. Cell. Biol. 115, Abstract No. 1448; Zheng et al. 1995, J. Immun. 154:5590). These molecules usually possess both the biological activity associated with the linked molecule of interest as well as the effector function, or some other desired characteristic associated with the immunoglobulin constant region (e.g. biological stability, cellular secretion).

[006] The Fc portion of an immunoglobulin constant region, depending on the immunoglobulin isotype can include the CH2, CH3, and CH4 domains, as well as the hinge region. Chimeric proteins comprising an Fc portion of an immunoglobulin bestow several desirable properties on a chimeric protein including increased stability, increased serum half life (see Capon et al. 1989, Nature 337:525) as well as binding to Fc receptors such as the neonatal Fc receptor (FcRn) (U.S. Patent ) Nos. 6,086,875, 6,485,726, 6,030,613; WO 03/077834; US2003-0235536A1).

[007] FcRn is active in adult epithelial tissue and expressed in the lumen of the intestines, pulmonary airways, nasal surfaces, vaginal surfaces, colon and rectal surfaces (U.S. Patent No. 6,485,726). Chimeric proteins comprised of FcRn binding partners (e.g. IgG, Fc fragments) can be effectively shuttled across epithelial barriers by FcRn, thus providing a non-invasive means to systemically administer a desired therapeutic molecule. Additionally, chimeric proteins comprising an FcRn binding partner are endocytosed by cells expressing the FcRn. But instead of being marked for degradation, these chimeric proteins are recycled out into circulation again, thus increasing the in vivo half life of these proteins.

2016244273 13 Oct 2016 [008] Portions of immunoglobulin constant regions, e.g., FcRn binding partners typically associate, via disulfide bonds and other non-specific interactions, with one another to form dimers and higher order multimers. The instant invention is based in part upon the surprising discovery that transcytosis of chimeric proteins comprised of FcRn binding partners appears to be limited by the molecular weight of the chimeric protein, with higher molecular weight species being transported less efficiently.

[009] Chimeric proteins comprised of biologically active molecules, once administered, typically will interact with a target molecule or cell. The instant invention is further based in part upon the surprising discovery that monomer-dimer hybrids, with one biologically active molecule, but two portions of an immunoglobulin constant region, e.g., two FcRn binding partners, function and can be transported more effectively than homodimers, also referred to herein simply as “dimers” or higher order multimers with two or more copies of the biologically active molecule. This is due in part to the fact that chimeric proteins, comprised of two or more biologically active molecules, which exist as dimers and higher order multimers, can be sterically hindered from interacting with their target molecule or cell, due to the presence of the two or more biologically active molecules in close proximity to one another and that the biologically active molecule can have a high affinity for itself.

[010] Accordingly one aspect of the invention provides chimeric proteins comprised of a biologically active molecule that is transported across the epithelium barrier. An additional aspect of the invention provides chimeric proteins comprised of at least one biologically active molecule that is able to interact with its target molecule or cell with little or no steric hindrance or self aggregation.

2016244273 13 Oct 2016 [011] The aspects of the invention provide for chimeric proteins comprising a first and second polypeptide chain, the first chain comprising at least a portion of immunoglobulin constant region, wherein the portion of an immunoglobulin constant region has been modified to include a biologically active molecule and the second chain comprising at least a portion of immunoglobulin constant region, wherein the portion of an immunoglobulin constant region has not been so modified to include the biologically active molecule of the first chain.

SUMMARY OF THE INVENTION [012] The invention relates to a chimeric protein comprising one biologically active molecule and two molecules of at least a portion of an immunoglobulin constant region. The chimeric protein is capable of interacting with a target molecule or cell with less steric hindrance compared to a chimeric protein comprised of at least two biologically active molecules and at least a portion of two immunoglobulin constant regions. The invention also relates to a chimeric protein comprising at least one biologically active molecule and two molecules of at least a portion of an immunoglobulin constant region that is transported across an epithelium barrier more efficiently than a corresponding homodimer, i.e., wherein both chains are linked to the same biologically active molecule. The invention, thus relates to a chimeric protein comprising a first and a second polypeptide chain linked together, wherein said first chain comprises a biologically active molecule and at least a portion of an immunoglobulin constant region, and said second chain comprises at least a portion of an immunoglobulin constant region, but no immunoglobulin variable region and without any biologically active molecule attached.

2016244273 13 Oct 2016 [013] The invention relates to a chimeric protein comprising a first and a second polypeptide chain linked together, wherein said first chain comprises a biologically active molecule and at least a portion of an immunoglobulin constant region, and said second chain comprises at least a portion of an immunoglobulin constant region without an immunoglobulin variable region or any biologically active molecule and wherein said second chain is not covalently bonded to any molecule having a molecular weight greater than 1 kD, 2 kD, 5 kD, 10 kD, or 20 kD. In one embodiment, the second chain is not covalently bonded to any molecule having a molecular weight greater than 0-2 kD. In one embodiment, the second chain is not covalently bonded to any molecule having a molecular weight greater than 5-10 kD. In one embodiment, the second chain is not covalently bonded to any molecule having a molecular weight greater than 15-20 kD.

[014] The invention relates to a chimeric protein comprising a first and a second polypeptide chain linked together, wherein said first chain comprises a biologically active molecule and at least a portion of an immunoglobulin constant region, and said second chain comprises at least a portion of an immunoglobulin constant region not covalently linked to any other molecule except the portion of an immunoglobulin of said first polypeptide chain.

[015] The invention relates to a chimeric protein comprising a first and a second polypeptide chain linked together, wherein said first chain comprises a biologically active molecule and at least a portion of an immunoglobulin constant region, and said second chain consists of at least a portion of an immunoglobulin constant region and optionally an affinity tag.

2016244273 13 Oct 2016 [016] The invention relates to a chimeric protein comprising a first and a second polypeptide chain linked together, wherein said first chain comprises a biologically active molecule and at least a portion of an immunoglobulin constant region, and said second chain consists essentially of at least a portion of an immunoglobulin constant region and optionally an affinity tag.

[017] The invention relates to a chimeric protein comprising a first and a second polypeptide chain linked together, wherein said first chain comprises a biologically active molecule and at least a portion of an immunoglobulin constant region, and said second chain comprises at least a portion of an immunoglobulin constant region without an immunoglobulin variable region or any biologically active molecule and optionally a molecule with a molecular weight less than 10 kD, 5 kD, 2 kD or 1 kD. In one embodiment, the second chain comprises a molecule less than 15-20 kD. In one embodiment, the second chain comprises a molecule less than 510 kD. In one embodiment, the second chain comprises a molecule less than 1-2 kD.

[018] The invention relates to a chimeric protein comprising a first and second polypeptide chain, wherein said first chain comprises a biologically active molecule, at least a portion of an immunoglobulin constant region, and at least a first domain, said first domain having at least one specific binding partner, and wherein said second chain comprises at least a portion of an immunoglobulin constant region, and at least a second domain, wherein said second domain is a specific binding partner of said first domain, without any immunoglobulin variable region or a biologically active molecule.

2016244273 13 Oct 2016 [019] The invention relates to a method of making a chimeric protein comprising a first and second polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are not the same, said method comprising transfecting a ceil with a first DNA construct comprising a DNA molecule encoding a first polypeptide chain comprising a biologically active molecule and at least a portion of an immunoglobulin constant region and optionally a linker, and a second DNA construct comprising a DNA molecule encoding a second polypeptide chain comprising at least a portion of an immunoglobulin constant region without any biologically active molecule or an immunoglobulin variable region, and optionally a linker, culturing the cells under conditions such that the polypeptide chain encoded by the first DNA construct is expressed and the polypeptide chain encoded by the second DNA construct is expressed and isolating monomer-dimer hybrids comprised of the polypeptide chain encoded by the first DNA construct and the polypeptide chain encoded by the second DNA construct.

[020] The invention relates to a method of making a chimeric protein comprising a first and second polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are not the same, and wherein said first polypeptide chain comprises a biologically active molecule, at least a portion of an immunoglobulin constant region, and at least a first domain, said first domain, having at least one specific binding partner, and wherein said second polypeptide chain comprises at least a portion of an immunoglobulin constant region and a second domain, wherein said second domain, is a specific binding partner of said first domain, without any biologically active molecule or an immunoglobulin variable region, said method comprising transfecting a cell with a first DNA construct

2016244273 13 Oct 2016 comprising a DNA molecule encoding said first polypeptide chain and a second DNA construct comprising a DNA molecule encoding, said second polypeptide chain, culturing the cells under conditions such that the polypeptide chain encoded by the first DNA construct is expressed and the polypeptide chain encoded by the second DNA construct is expressed and isolating monomer-dimer hybrids comprised of the polypeptide chain encoded by the first DNA construct and polypeptide chain encoded by the second DNA construct, [021] The invention relates to a method of making a chimeric protein of the invention said method comprising transfecting a cell with a first DNA construct comprising a DNA molecule encoding a first polypeptide chain comprising a biologically active molecule and at least a portion of an immunoglobulin constant region and optionally a linker, culturing the cell under conditions such that the polypeptide chain encoded by the first DNA construct is expressed, isolating the polypeptide chain encoded by the first DNA construct and transfecting a cell with a second DNA construct comprising a DNA molecule encoding a second polypeptide chain comprising at least a portion of an immunoglobulin constant region without any biologically active molecule or immunoglobulin variable region, culturing the cell under conditions such that the polypeptide chain encoded by the second DNA construct is expressed, isolating the polypeptide chain, encoded by the second DNA construct, combining the polypeptide chain, encoded by the first DNA construct and the polypeptide chain encoded by the second DNA construct under conditions such that monomer-dimer hybrids comprising the polypeptide chain encoded by the first DNA construct and the polypeptide chain encoded by the second DNA construct form, and isolating said monomer-dimer hybrids.

2016244273 13 Oct 2016 [022] The invention relates to a method of making a chimeric protein comprising a first and second polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are not the same, said method comprising transfecting a cell with a DNA construct comprising a DNA molecule encoding a polypeptide chain comprising at least a portion of an immunoglobulin constant region, culturing the cells under conditions such that the polypeptide chain encoded by the DNA construct is expressed with an N terminal cysteine such that dimers of the polypeptide chain form and isolating dimers comprised of two copies of the polypeptide chain encoded by the DNA construct and chemically reacting the isolated dimers with a biologically active molecule, wherein said biologically active molecule has a C terminus thioester, under conditions such that the biologically active molecule reacts predominantly with oniy one polypeptide chain of the dimer thereby forming a monomer-dimer hybrid.

[023] The invention relates to a method of making a chimeric protein comprising a first and second polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are not the same, said method comprising transfecting a cell with a DNA construct comprising a DNA molecule encoding a polypeptide chain comprising at least a portion of an immunoglobulin constant region, culturing the cells under conditions such that the polypeptide chain encoded by the DNA construct is expressed with an N terminal cysteine such that dimers of the polypeptide chains form, and isolating dimers comprised of two copies of the polypeptide chain encoded by the DNA construct, and chemically reacting the isolated dimers with a biologically active molecule, wherein said biologically active molecule has a C terminus thioester, such that the biologically active molecule is io

2016244273 13 Oct 2016 linked to each chain of the dimer, denaturing the dimer comprised of the portion of the immunoglobulin linked to the biologically active molecule such that monomeric chains form, combining the monomeric chains with a polypeptide chain comprising at least a portion of an immunoglobulin constant region without a biologically active molecule linked to it, such that monomer-dimer hybrids form, and isolating the monomer-dimer hybrids.

[024] The invention relates to a method of making a chimeric protein comprising a first and second polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are not the same, said method comprising transfecting a cell with a DNA construct comprising a DNA molecule encoding a polypeptide chain comprising at least a portion of an immunoglobulin constant region, culturing the cells under conditions such that the polypeptide chain encoded by the DNA construct is expressed as a mixture of two polypeptide chains, wherein the mixture comprises a polypeptide with an N terminal cysteine, and a polypeptide with a cysteine in close proximity to the N terminus, isolating dimers comprised of the mixture of polypeptide chains encoded by the DNA construct and chemically reacting the isolated dimers with a biologically active molecule, wherein said biologically active molecule has an active thioester, such that at least some monomer-dimer hybrid forms and isolating the monomer-dimer hybrid from said mixture.

[025] The invention relates to a method of treating a disease or condition comprising administering a chimeric protein of the invention thereby treating the disease or condition.

2016244273 29 Sep 2017 [026] Additional objects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[027] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

[027a] Definitions of the specific embodiments of the invention as claimed herein follow.

[027b] According to a first embodiment of the invention, there is provided a chimeric protein comprising one biologically active molecule and two immunoglobulin constant regions or portions thereof, wherein the chimeric protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the biologically active molecule and an immunoglobulin constant region, or a portion thereof, that is an Fc neonatal receptor (FcRn) binding partner and wherein the biologically active molecule is a protein selected from the group consisting of a cytokine and a hormone; and wherein the second polypeptide chain comprises an immunoglobulin constant region, or a portion thereof, that is an FcRn binding partner, but does not comprise a biologically active molecule.

[027c] According to a second embodiment of the invention, there is provided a pharmaceutical composition comprising the chimeric protein according to the first embodiment and a pharmaceutically acceptable excipient.

[027d] According to a third embodiment of the invention, there is provided a first polynucleotide encoding the first polypeptide chain of the chimeric protein according to the first embodiment, and a second polynucleotide encoding the second polypeptide chain of said chimeric protein.

2016244273 29 Sep 2017 [027e] According to a fourth embodiment of the invention, there is provided a method of treating a disease or condition in a subject in need thereof comprising administering the chimeric protein according to the first embodiment or the pharmaceutical composition of the second embodiment.

[027f] According to a fifth embodiment of the invention, there is provided a method of making the chimeric protein according to the first embodiment wherein the method comprises:

transfecting a host cell with a first polynucleotide encoding the first polypeptide chain and a second polynucleotide encoding the second polypeptide chain;

culturing the cell under conditions such that the first and second polypeptide chains are expressed; and isolating the chimeric protein from said host cell.

[Text continues on page 13.]

12a

2016244273 13 Oct 2016

BRIEF DESCRIPTION OF THE DRAWINGS [028] Figure 1 is a schematic diagram comparing the structure of an EPOFc homodimer, or dimer, and the structure of an Epo-FC monomer-dimer hybrid.

[029] Figure 2a is the amino acid sequence of the chimeric protein Factor Vll-Fc. Included in the sequence is the signal peptide (underlined), which is cleaved by the cell and the propeptide (bold), which is recognized by the vitamin independent γ carboxylase which modifies the Factor VII to achieve full activity. The sequence is subsequently cleaved by PACE to yield Factor Vll-Fc.

[030] Figure 2b is the amino acid sequence of the chimeric protein Factor IX-Fc. Included in the sequence is the signal peptide (underlined) which is cleaved by the cell and the propeptide (bold) which is recognized by the vitamin K-dependent γ carboxylase which modifies the Factor IX to achieve full activity. The sequence is subsequently cleaved by PACE to yield Factor IX-Fc.

[031] Figure 2c is the amino acid sequence of the chimeric protein IFNa-Fc. Included in the sequence is the signal peptide (underlined), which is cleaved by the cell resulting in the mature IFNa-Fc.

[032] Figure 2d is the amino acid sequence of the chimeric protein IFNa-Fc Δ linker. Included in the sequence is the signal peptide (underlined) which is cleaved by the cell resulting in the mature IFNa- Fc Δ linker.

[033] Figure 2e is the amino acid sequence of the chimeric protein Flag-Fc. Included in the sequence is the signal peptide (underlined), which is cleaved by the cell resulting in the mature Flag-Fc.

[034] Figure 2f is the amino acid sequence of the chimeric protein EpoCCA-Fc. Included in the sequence is the signal peptide (underlined), which is

2016244273 13 Oct 2016 cleaved by the cell resulting in the mature Epo-CCA-Fc. Also shown in bold is the acidic coiled coil domain.

[035] Figure 2g is the amino acid sequence of the chimeric protein CCB-Fc. Included in the sequence is the signal peptide (underlined), which is cleaved by the cell resulting in the mature CCB-Fc. Also shown in bold is the basic coiled coil domain.

[036] Figure 2h is the amino acid sequence of the chimeric protein Cys-Fc. Included in the sequence is the signal peptide (underlined), which is cleaved by the cell resulting in the mature Cys-Fc. When this sequence is produced in CHO cells a percentage of the molecules are incorrectly cleaved by the signal peptidase such that two extra amino acids are left on the N terminus, thus preventing the linkage of a biologically active molecule with a C terminal thioester (e.g., via native ligation). When these improperly cleaved species dimerize with the properly cleaved Cys-Fc and are subsequently reacted with biologically active molecules with C terminal thioesters, monomer-dimer hybrids form.

[037] Figure 2i is the amino acid sequence of the chimeric protein IFNaGS15-Fc. Included in the sequence is the signal peptide (underlined) which is cleaved by the cell resulting in the mature IFNa- GS15-Fc.

[038] Figure 2j is the amino acid sequence of the chimeric protein Epo-Fc. Included in the sequence is the signal peptide (underlined), which is cleaved by the cell resulting in the mature Epo-Fc. Also shown in bold is the 8 amino acid linker.

[039] Figure 3a is the nucleic acid sequence of the chimeric protein Factor Vll-Fc. Included in the sequence is the signal peptide (underlined) and the propeptide (bold) which is recognized by the vitamin K-dependent γ carboxylase

2016244273 13 Oct 2016 which modifies the Factor VII to achieve full activity. The translated sequence is subsequently cleaved by PACE to yield mature Factor VI l-Fc.

[040] Figure 3b is the nucleic acid sequence of the chimeric protein Factor IX-Fc. Included in the sequence is the signal peptide (underlined) and the propeptide (bold) which is recognized by the vitamin K-dependent γ carboxylase which modifies the Factor IX to achieve full activity. The translated sequence is subsequently cleaved by PACE to yield mature Factor IX-Fc.

[041] Figure 3c is the nucleic acid sequence of the chimeric protein IFNaFc. Included in the sequence is the signal peptide (underlined), which is cleaved by the cell after translation resulting in the mature IFNa-Fc.

[042] Figure 3d is the nucleic acid sequence of the chimeric protein IFNaFc Δ linker. Included in the sequence is the signal peptide (underlined) which is cleaved by the cell after translation resulting in the mature IFNa- FcA linker.

[043] Figure 3e is the amino acid sequence of the chimeric protein Flag-Fc. Included in the sequence is the signal peptide (underlined), which is cleaved by the cell after translation resulting in the mature Flag-Fc.

[044] Figure 3f is the nucleic acid sequence of the chimeric protein EpoCCA-Fc. Included in the sequence is the signal peptide (underlined), which is cleaved by the cell after translation resulting in the mature Epo-CCA-Fc. Also shown in bold is the acidic coiled coil domain.

[045] Figure 3g is the nucleic acid sequence of the chimeric protein CCBFc. Included in the sequence is the signal peptide (underlined), which is cleaved by the cell after translation resulting in the mature CCB-Fc. Also shown in bold is the basic coiled coil domain.

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2016244273 13 Oct [046] Figure 3h is the nucleic acid sequence of the chimeric protein Cys-Fc. included in the sequence is the signal peptide (underlined), which is cleaved by the cell after translation resulting in the mature Cys-Fc.

[047] Figure 3i is the nucleic acid sequence of the chimeric protein IFNaGS15-FC. Included in the sequence is the signal peptide (underlined) which is cleaved by the cell after translation resulting in the mature IFNa-GS15-Fc.

[048] Figure 3j is the nucleic acid sequence of the chimeric protein Epo-Fc. Included in the sequence is the signal peptide (underlined), which is cleaved by the cell after translation resulting in the mature Epo-Fc. Also shown in bold is a nucleic acid sequence encoding the 8 amino acid linker.

[049] Figure 4 demonstrates ways to form monomer-dimer hybrids through native ligation.

[050] Figure 5a shows the amino acid sequence of Fc MESNA (SEQ ID

NO:4).

[051] Figure 5b shows the DNA sequence of Fc MESNA (SEQ ID NO:5).

[052] Figure 6 compares antiviral activity of IFNa homo-dimer (i.e. comprised of 2 IFNa molecules) with an IFNa monomer-dimer hybrid (i.e. comprised of1 IFNa molecule).

[053] Figure 7 is a comparison of clotting activity of a chimeric monomerdimer hybrid Factor VIla-Fc (one Factor VII molecule) and a chimeric homodimer Factor VIIa-Fc (two Factor VII molecules).

[054] Figure 8 compares oral dosing in neonatal rats of a chimeric monomer-dimer hybrid Factor VIIa-Fc (one Factor VII molecule) and a chimeric homodimer Factor VIIa-Fc (two Factor VII molecules).

2016244273 13 Oct 2016 [055] Figure 9 compares oral dosing in neonatal rats of a chimeric monomer-dimer hybrid Factor IX-Fc (one Factor IX molecule) with a chimeric homodimer.

[056] Figure 10 is a time course study comparing a chimeric monomerdimer hybrid Factor IX-Fc (one Factor IX molecule) administered orally to neonatal rats with an orally administered chimeric homodimer.

[057] Figure 11 demonstrates pharmokinetics of Epo-Fc dimer compared to Epo-Fc monomer-dimer hybrid in cynomolgus monkeys after a single pulmonary dose.

[058] Figure 12 compares serum concentration in monkeys of subcutaneously administered Epo-Fc monomer-dimer hybrid with subcutaneously administered Aranesp® (darbepoetin alfa).

[059] Figure 13 compares serum concentration in monkeys of intravenously administered Epo-Fc monomer-dimer hybrid with intravenously administered Aranesp® (darbepoetin alfa) and Epogen® (epoetin alfa).

[060] Figure 14 shows a trace from a Mimetic Red 2™ column (ProMetic LifeSciences, inc., Wayne, NJ) and an SDS-PAGE of fractions from the column containing EpoFc monomer-dimer hybrid, EpoFc dimer, and Fc. EpoFc monomerdimer hybrid is found in fractions 11, 12,13, and 14. EpoFc dimer is found in fraction 18. Fc is found in fractions 1/2.

[061] Figure 15 shows the pharmacokinetics of IFNpFc with an 8 amino acid linker in cynomolgus monkeys after a single pulmonary dose.

[062] Figure 16 shows neopterin stimulation in response to the IFNp-Fc homodimer and the IFNp-Fc N297A monomer-dimer hybrid in cynomolgus monkeys.

2016244273 13 Oct 2016 [063] Figure 17a shows the nucleotide sequence of interferon β-Fc; Figure 17b shows the amino acid sequence of interferon β-Fc.

[064] Figure 18 shows the amino acid sequence of T20(a); T21 (b) and T1249(c).

DESCRIPTION OF THE EMBODIMENTS

A. Definitions [065] Affinity tag, as used herein, means a molecule attached to a second molecule of interest, capable of interacting with a specific binding partner for the purpose of isolating or identifying said second molecule of interest.

[066] Analogs of chimeric proteins of the invention, or proteins or peptides substantially identical to the chimeric proteins of the invention, as used herein, means that a relevant amino acid sequence of a protein or a peptide is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to a given sequence. By way of example, such sequences may be variants derived from various species, or they may be derived from the given sequence by truncation, deletion, amino acid substitution or addition. Percent identity between two amino acid sequences is determined by standard alignment algorithms such as, for example, Basic Local Alignment Tool (BLAST) described in Altschul et al. 1990, J. Mol. Biol., 215:403-410, the algorithm of Needleman et al. 1970, J. Mol. Biol., 48:444-453; the algorithm of Meyers et al. 1988, Comput. Appl. Biosci., 4:11-17; or Tatusova et al. 1999, FEMS Microbiol. Lett., 174:247-250, etc. Such algorithms are incorporated into the BLASTN, BLASTP and “BLAST 2 Sequences” programs (see www.ncbi.nlm.nih.gov/BLAST). When utilizing such programs, the default parameters can be used. For example, for nucleotide sequences the following

2016244273 13 Oct 2016 settings can be used for “BLAST 2 Sequences”: program BLASTN, reward for match 2, penalty for mismatch -2, open gap and extension gap penalties 5 and 2 respectively, gap x_dropoff 50, expect 10, word size 11, filter ON. For amino acid sequences the following settings can be used for “BLAST 2 Sequences”: program BLASTP, matrix BLOSUM62, open gap and extension gap penalties 11 and 1 respectively, gap x_dropoff 50, expect 10, word size 3, filter ON.

[067] Bioavailability, as used herein, means the extent and rate at which a substance is absorbed into a living system or is made available at the site of physiological activity.

[068] Biologically active molecule, as used herein, means a nonimmunoglobulin molecule or fragment thereof, capable of treating a disease or condition or localizing or targeting a molecule to a site of a disease or condition in the body by performing a function or an action, or stimulating or responding to a function, an action or a reaction, in a biological context (e.g. in an organism, a ceil, or an in vitro model thereof). Biologically active molecules may comprise at least one of polypeptides, nucleic acids, small molecules such as small organic or inorganic molecules.

[069] A chimeric protein, as used herein, refers to any protein comprised of a first amino acid sequence derived from a first source, bonded, covalently or noncovaiently, to a second amino acid sequence derived from a second source, wherein the first and second source are not the same. A first source and a second source that are not the same can include two different biological entities, or two different proteins from the same biological entity, or a biological entity and a non-bioiogical entity. A chimeric protein can include for example, a protein derived from at least 2

2016244273 13 Oct 2016 different biological sources. A biological source can include any non-synthetically produced nucleic acid or amino acid sequence (e.g. a genomic or cDNA sequence, a plasmid or viral vector, a native virion or a mutant or analog, as further described herein, of any of the above). A synthetic source can include a protein or nucleic acid sequence produced chemically and not by a biological system (e.g. solid phase synthesis of amino acid sequences). A chimeric protein can also include a protein derived from at least 2 different synthetic sources or a protein derived from at least one biological source and at least one synthetic source. A chimeric protein may also comprise a first amino acid sequence derived from a first source, covalently or noncovalently linked to a nucleic acid, derived from any source or a small organic or inorganic molecule derived from any source. The chimeric protein may comprise a linker molecule between the first and second amino acid sequence or between the first amino acid sequence and the nucieic acid, or between the first amino acid sequence and the small organic or inorganic molecule.

[070] Clotting factor, as used herein, means any molecule, or analog thereof, naturally occurring or recombinantly produced which prevents or decreases the duration of a bleeding episode in a subject with a hemostatic disorder, in other words, it means any molecule having clotting activity.

[071] Clotting activity, as used herein, means the ability to participate in a cascade of biochemical reactions that culminates in the formation of a fibrin clot and/or reduces the severity, duration or frequency of hemorrhage or bleeding episode.

[072] Dimer as used herein refers to a chimeric protein comprising a first and second polypeptide chain, wherein the first and second chains both comprise a

2016244273 13 Oct 2016 biologically active molecule, and at least a portion of an immunoglobulin constant region. A homodimer refers to a dimer where both biologically active molecules are the same.

[073] Dimerically linked monomer-dimer hybrid refers to a chimeric protein comprised of at least a portion of an immunloglobulin constant region, e.g. an Fc fragment of an immunoglobulin, a biologically active molecule and a linker which links the two together such that one biologically active molecule is bound to 2 polypeptide chains, each comprising a portion of an immunoglobulin constant region. Figure 4 shows an example of a dimerically linked monomer-dimer hybrid.

[074] DNA construct, as used herein, means a DNA molecule, or a clone of such a molecule, either single- or double-stranded that has been modified through human intervention to contain segments of DNA combined in a manner that as a whole would not otherwise exist in nature. DNA constructs contain the information necessary to direct the expression of polypeptides of interest. DNA constructs can include promoters, enhancers and transcription terminators. DNA constructs containing the information necessary to direct the secretion of a polypeptide will also contain at least one secretory signal sequence.

[075] Domain, as used herein, means a region of a polypeptide (including proteins as that term is defined) having some distinctive physical feature or role including for example an independently folded structure composed of one section of a polypeptide chain. A domain may contain the sequence of the distinctive physical feature of the polypeptide or it may contain a fragment of the physical feature which retains its binding characteristics (i.e., it can bind to a second domain). A domain

2016244273 13 Oct 2016 may be associated with another domain. In other words, a first domain may naturally bind to a second domain.

[076] A fragment, as used herein, refers to a peptide or polypeptide comprising an amino acid sequence of at least 2 contiguous amino acid residues, of at least 5 contiguous amino acid residues, of at least 10 contiguous amino acid residues, of at least 15 contiguous amino acid residues, of at least 20 contiguous amino acid residues, of at least 25 contiguous amino acid residues, of at least 40 contiguous amino acid residues, of at least 50 contiguous amino acid residues, of at least 100 contiguous amino acid residues, or of at least 200 contiguous amino acid residues or any deletion or truncation of a protein, peptide, or polypeptide.

[077] Hemostasis, as used herein, means the stoppage of bleeding or hemorrhage; or the stoppage of blood flow through a blood vessel or body part.

[078] Hemostatic disorder, as used herein, means a genetically inherited or acquired condition characterized by a tendency to hemorrhage, either spontaneously or as a result of trauma, due to an impaired ability or inability to form a fibrin clot.

[079] Linked, as used herein, refers to a first nucleic acid sequence covalently joined to a second nucleic acid sequence. The first nucleic acid sequence can be directly joined or juxtaposed to the second nucleic acid sequence or alternatively an intervening sequence can covalently join the first sequence to the second sequence. Linked as used herein can also refer to a first amino acid sequence covalently, or non-covalently, joined to a second amino acid sequence. The first amino acid sequence can be directly joined or juxtaposed to the second amino acid sequence or alternatively an intervening sequence can covalently join the first amino acid sequence to the second amino acid sequence.

2016244273 13 Oct 2016 [080] Operatively linked, as used herein, means a first nucleic acid sequence linked to a second nucleic acid sequence such that both sequences are capable of being expressed as a biologically active protein or peptide.

[081] Polypeptide, as used herein, refers to a polymer of amino acids and does not refer to a specific length ofthe product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term does not exclude post-expression modifications ofthe polypeptide, for example, glycosyiation, acetylation, phosphorylation, pegylation, addition of a lipid moiety, or the addition of any organic or inorganic molecule. Included within the definition, are for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids) and polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.

[082] High stringency, as used herein, includes conditions readily determined by the skilled artisan based on, for example, the length ofthe DNA. Generally, such conditions are defined in Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1, pp. 1.101-104, Cold Spring Harbor Laboratory Press (1989), and include use of a prewashing solution for the nitrocellulose filters 5X SSC, 0.5% SDS, 1.0 mM EDTA (PH 8.0), hybridization conditions of 50% formamide, 6X SSC at 42°C (or other similar hybridization solution, such as Stark's solution, in 50% formamide at 42°C, and with washing at approximately 68°C, 0.2X SSC, 0.1% SDS. The skilled artisan will recognize that the temperature and wash solution salt concentration can be adjusted as necessary according to factors such as the length of the probe.

Ο

CM

2016244273 13 Oct [083] Moderate stringency, as used herein, include conditions that can be readily determined by those having ordinary skill in the art based on, for example, the length of the DNA. The basic conditions are set forth by Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed. Vol. 1, pp. 1.101-104, Cold Spring Harbor Laboratory Press (1989), and include use of a prewashing solution for the nitrocellulose filters 5X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of 50% formamide, 6X SSC at 42°C (or other similar hybridization solution, such as Stark's solution, in 50% formamide at 42°C), and washing conditions of 60°C, 0.5X SSC, 0.1% SDS.

[084] A small inorganic molecule, as used herein means a molecule containing no carbon atoms and being no larger than 50 kD.

[085] A small organic molecule, as used herein means a molecule containing at least one carbon atom and being no larger than 50 kD.

[086] Treat, treatment, treating, as used herein means, any of the following: the reduction in severity of a disease or condition; the reduction in the duration of a disease course; the amelioration of one or more symptoms associated with a disease or condition; the provision of beneficial effects to a subject with a disease or condition, without necessarily curing the disease or condition, the prophylaxis of one or more symptoms associated with a disease or condition.

B. Improvements Offered by Certain Embodiments of the Invention [087] The invention provides for chimeric proteins (monomer-dimer hybrids) comprising a first and a second polypeptide chain, wherein said first chain comprises a biologically active molecule and at least a portion of an immunoglobulin constant region, and said second chain comprises at least a portion of an immunoglobulin

2016244273 13 Oct 2016 constant region without any biologically active molecule or variable region of an immunoglobulin. Figure 1 contrasts traditional fusion protein dimers with one example of the monomer-dimer hybrid of the invention. In this example, the biologically active molecule is EPO and the portion of an immunoglobulin is IgG Fc region.

[088] Like other chimeric proteins comprised of at least a portion of an immunoglobulin constant region, the invention provides for chimeric proteins which afford enhanced stability and increased bioavailability of the chimeric protein compared to the biologically active molecule alone. Additionally, however, because only one of the two chains comprises the biologically active molecule, the chimeric protein has a lower molecular weight than a chimeric protein wherein all chains comprise a biologically active molecule and while not wishing to be bound by any theory, this may result in the chimeric protein being more readily transcytosed across the epithelium barrier, e.g., by binding to the FcRn receptor thereby increasing the half-life of the chimeric protein. In one embodiment, the invention thus provides for an improved non-invasive method (e.g. via any mucosal surface, such as, orally, buccally, sublingually, nasally, rectally, vaginally, or via pulmonary or occular route) of administering a therapeutic chimeric protein of the invention. The invention thus provides methods of attaining therapeutic levels of the chimeric proteins of the invention using less frequent and lower doses compared to previously described chimeric proteins (e.g. chimeric proteins comprised of at least a portion of an immunoglobulin constant region and a biologically active molecule, wherein all chains of the chimeric protein comprise a biologically active molecule).

2016244273 13 Oct 2016 [089] In another embodiment, the invention provides an invasive method, e.g., subcutaneously, intravenously, of administering a therapeutic chimeric protein of the invention. Invasive administration of the therapeutic chimeric protein of the invention provides for an increased half life of the therapeutic chimeric protein which results in using less frequent and lower doses compared to previously described chimeric proteins (e.g. chimeric proteins comprised of at least a portion of an immunoglobulin constant region and a biologically active molecule, wherein all chains of the chimeric protein comprise a biologically active molecule).

[090] Yet another advantage of a chimeric protein wherein only one of the chains comprises a biologically active molecule is the enhanced accessibility of the biologically active molecule for its target cell or molecule resulting from decreased steric hindrance, decreased hydrophobic interactions, decreased ionic interactions, or decreased molecular weight compared to a chimeric protein wherein all chains are comprised of a biologically active molecule.

C. Chimeric Proteins [091] The invention relates to chimeric proteins comprising one biologically active molecule, at least a portion of an immunoglobulin constant region, and optionally at least one linker. The portion of an immunoglobulin will have both an N, or an amino terminus, and a C, or carboxy terminus. The chimeric protein may have the biologically active molecule linked to the N terminus of the portion of an immunoglobulin. Alternatively, the biologically active molecule may be linked to the C terminus of the portion of an immunoglobulin. In one embodiment, the linkage is a covalent bond. In another embodiment, the linkage is a non-covalent bond.

2016244273 13 Oct 2016 [092] The chimeric protein can optionally comprise at least one linker; thus, the biologically active molecule does not have to be directly linked to the portion of an immunoglobulin constant region. The linker can intervene in between the biologically active molecule and the portion of an immunoglobulin constant region. The linker can be linked to the N terminus of the portion of an immunoglobulin constant region, or the C terminus of the portion of an immunoglobulin constant region. If the biologically active molecule is comprised of at least one amino acid the biologically active molecule will have an N terminus and a C terminus and the linker can be linked to the N terminus of the biologically active molecule, or the C terminus the biologically active molecule.

[093] The invention relates to a chimeric protein of the formula X-L_a-F:F or F:F-L_a-X , wherein X is a biologically active molecule, L is an optional linker, F is at least a portion of an immunoglobulin constant region and, a is any integer or zero. The invention also relates to a chimeric protein of the formula T_a-X-L_a-F:F or T_a-F:FL_a-X, wherein X is a biologically active molecule, L is an optional linker, F is at least a portion of an immunoglobulin constant region, a is any integer or zero, T is a second linker or alternatively a tag that can be used to facilitate purification of the chimeric protein, e.g., a FLAG tag, a histidine tag, a GST tag, a maltose binding protein tag and (:) represents a chemical association, e.g. at least one non-peptide bond. In certain embodiments, the chemical association, i.e., (:) is a covalent bond. In other embodiments, the chemical association, i.e., (:) is a non-covalent interaction, e.g., an ionic interaction, a hydrophobic interaction, a hydrophilic interaction, a Van der Waals interaction, a hydrogen bond. It will be understood by

2016244273 13 Oct 2016 the skilled artisan that when a equals zero X will be directly linked to F. Thus, for example, a may be 0,1,2, 3,4, 5, or more than 5.

[094] in one embodiment, the chimeric protein of the invention comprises the amino acid sequence of figure 2a (SEQ ID NO:6). In one embodiment, the chimeric protein of the invention comprises the amino acid sequence of figure 2b (SEQ ID NO:8). In one embodiment, the chimeric protein of the invention comprises the amino acid sequence of figure 2c (SEQ ID NO: 10). In one embodiment, the chimeric protein of the invention comprises the amino acid sequence of figure 2d (SEQ ID NO:12). In one embodiment, the chimeric protein of the invention comprises the amino acid sequence of figure 2e (SEQ ID NO:14). In one embodiment, the chimeric protein of the invention comprises the amino acid sequence of figure 2f (SEQ ID NO: 16). In one embodiment, the chimeric protein of the invention comprises the amino acid sequence of figure 2g (SEQ ID NO:18). In one embodiment, the chimeric protein of the invention comprises the amino acid sequence of figure 2h (SEQ ID NO:20). In one embodiment, the chimeric protein of the invention comprises the amino acid sequence of figure 2i (SEQ ID NO:22). In one embodiment, the chimeric protein of the invention comprises the amino acid sequence of figure 2j (SEQ ID NO:24). In one embodiment, the chimeric protein of the invention comprises the amino acid sequence of figure 17b (SEQ ID NO:27).

1. Chimeric Protein Variants [095] Derivatives of the chimeric proteins of the invention, antibodies against the chimeric proteins of the invention and antibodies against binding partners of the chimeric proteins of the invention are all contemplated, and can be made by altering their amino acids sequences by substitutions, additions, and/or deletions/truncations

2016244273 13 Oct 2016 or by introducing chemical modification that result in functionally equivalent molecules. It will be understood by one of ordinary skill in the art that certain amino acids in a sequence of any protein may be substituted for other amino acids without adversely affecting the activity of the protein.

[096] Various changes may be made in the amino acid sequences of the chimeric proteins of the invention or DNA sequences encoding therefore without appreciable loss of their biological activity, function, or utility. Derivatives, analogs, or mutants resulting from such changes and the use of such derivatives is within the scope of the present invention. In a specific embodiment, the derivative is functionally active, i.e., capable of exhibiting one or more activities associated with the chimeric proteins of the invention, e.g., FcRn binding, viral inhibition, hemostasis, production of red blood cells. Many assays capable of testing the activity of a chimeric protein comprising a biologically active molecule are known in the art. Where the biologically active molecule is an HIV inhibitor, activity can be tested by measuring reverse transcriptase activity using known methods (see, e.g., BarreSinoussi et al. 1983, Science 220:868; Gallo et al. 1984, Science 224:500). Alternatively, activity can be measured by measuring fusogenic activity (see, e.g., Nussbaum etal. 1994, J. Virol. 68(9):5411). Where the biological activity is hemostasis, a StaCLot FVIIa-rTF assay can be performed to assess activity of Factor Vila derivatives (Johannessen et al. 2000, Blood Coagulation and Fibrinolysis 11:S159).

[097] Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs (see Table 1). Furthermore, various amino acids are commonly substituted with neutral amino

2016244273 13 Oct 2016 acids, e.g., alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine (see, e.g., MacLennan et al. 1998, Acta Physiol. Scand. Suppl. 643:55-67; Sasaki et ai. 1998, Adv. Biophys. 35:1-24).

TABLE 1

Original Residues	Exemplary Substitutions	Typical Substitutions
Ala (A)	Val, Leu, Ile	Val
Arg (R)	Lys, Gin, Asn	Lys
Asn (N)	Gin	Gin
Asp (D)	Glu	Glu
Cys (C)	Ser, Ala	Ser
Gin (Q)	Asn	Asn
Gly (G)	Pro, Ala	Ala
His (H)	Asn, Gin, Lys, Arg	Arg
He (I)	Leu, Val, Met, Ala, Phe, Norleucine	Leu
Leu (L)	Norleucine, Ile, Val, Met, Ala, Phe	lie
Lys (K)	Arg, 1,4-Diamino-butyric Acid, Gin, Asn	Arg
Met (M)	Leu, Phe, lie	Leu
Phe (F)	Leu, Val, lie, Ala, Tyr	Leu
Pro (P)	Aia	Giy
Ser (S)	Thr, Ala, Cys	Thr
Thr (T)	Ser	Ser
Trp (W)	Tyr, Phe	Tyr
Tyr (Y)	Trp, Phe, Thr, Ser	Phe
Val (V)	lie, Met, Leu, Phe, Ala, Norleucine	Leu

2016244273 13 Oct 2016

2. Biologically Active Molecules [098] The invention contemplates the use of any biologically active molecule as the therapeutic molecule of the invention. The biologically active molecule can be a polypeptide. The biologically active molecule can be a single amino acid. The biologically active molecule can include a modified polypeptide.

[099] The biologically active molecule can include a lipid molecule (e.g. a steroid or cholesterol, a fatty acid, a triacylgiycerol, glycerophospholipid, or sphingolipid). The biologically active molecule can include a sugar molecule (e.g. glucose, sucrose, mannose). The biologically active molecule can include a nucleic acid molecule (e.g. DNA, RNA). The biologically active molecule can include a small organic molecule or a small inorganic molecule.

a. Cytokines and Growth Factors [0100] In one embodiment, the biologically active molecule is a growth factor, hormone or cytokine or analog or fragment thereof. The biologically active molecule can be any agent capable of inducing cell growth and proliferation. In a specific embodiment, the biologically active molecule is any agent which can induce erythrocytes to proliferate. Thus, one example of a biologically active molecule contemplated by the invention is EPO. The biologically active molecule can also include, but is not limited to, RANTES, ΜΙΡ1α, ΜΙΡ1β, IL-2, IL-3, GM-CSF, growth hormone, tumor necrosis factor (e.g. TNFa or β).

[0101] The biologically active molecule can include interferon a, whether synthetically or recombinantly produced, including but not limited to, any one of the about twenty-five structurally related subtypes, as for example interferon-a2a, now commercially available for clinical use (ROFERON®, Roche) and interferon-a2b also

2016244273 13 Oct 2016 approved for clinical use (INTRON®, Schering) as well as genetically engineered versions of various subtypes, including, but not limited to, commercially available consensus interferon a (INFERGEN®, Intermune, developed by Amgen) and consensus human leukocyte interferon see, e.g., U.S. Patent Nos.: 4,695,623; 4,897,471, interferon β, epidermal growth factor, gonadotropin releasing hormone (GnRH), leuprolide, follicle stimulating hormone, progesterone, estrogen, or testosterone.

[0102] A list of cytokines and growth factors which may be used in the chimeric protein of the invention has been previously described (see, e.g., U.S. Patent Nos. 6,086,875, 6,485,726, 6,030,613; WO 03/077834; US2003-0235536A1).

b. Antiviral Agents [0103] in one embodiment, the biologically active molecule is an antiviral agent, including fragments and analogs thereof. An antiviral agent can include any molecule that inhibits or prevents viral replication, or inhibits or prevents viral entry into a cell, or inhibits or prevents viral egress from a cell. In one embodiment, the antiviral agent is a fusion inhibitor. In one embodiment, the antiviral agent is a cytokine which inhibits viral replication. In another embodiment, the antiviral agent is interferon a.

[0104] The viral fusion inhibitor for use in the chimeric protein can be any molecule which decreases or prevents viral penetration of a cellular membrane of a target cell. The viral fusion inhibitor can be any molecule that decreases or prevents the formation of syncytia between at least two susceptible ceils. The viral fusion inhibitor can be any molecule that decreases or prevents the joining of a lipid bilayer membrane of a eukaryotic cell and a lipid bilayer of an enveloped virus. Examples

2016244273 13 Oct 2016 of enveloped virus include, but are not limited to HIV-1, HIV-2, SIV, influenza, parainfluenza, Epstein-Barr virus, CMV, herpes simplex 1, herpes simplex 2 and respiratory syncytia virus.

[0105] The viral fusion inhibitor can be any molecule that decreases or prevents viral fusion including, but not limited to, a polypeptide, a small organic molecule or a small inorganic molecule. In one embodiment, the fusion inhibitor is a polypeptide. In one embodiment, the viral fusion inhibitor is a polypeptide of 3-36 amino acids. In another embodiment, the viral fusion inhibitor is a polypeptide of 350 amino acids, 10-65 amino acids, 10-75 amino acids. The polypeptide can be comprised of a naturally occurring amino acid sequence (e.g. a fragment of gp41) including analogs and mutants thereof or the polypeptide can be comprised of an amino acid sequence not found in nature, so long as the polypeptide exhibits viral fusion inhibitory activity.

[0106] In one embodiment, the viral fusion inhibitor is a polypeptide, identified as being a viral fusion inhibitor using at least one computer algorithm, e.g., ALLMOTI5, 107x178x4 and PLZIP (see, e.g., U.S. Patent Nos.: 6,013,263; 6,015,881; 6,017,536; 6,020,459; 6,060,065; 6,068,973; 6,093,799; and 6,228,983).

[0107] In one embodiment, the viral fusion inhibitor is an HIV fusion inhibitor. In one embodiment, HIV is HIV-1. In another embodiment, HIV is HIV-2. In one embodiment, the HIV fusion inhibitor is a polypeptide comprised of a fragment of the gp41 envelope protein of HIV-1. The HIV fusion inhibitor can comprise, e.g., T20 (SEQ ID NO:1) or an analog thereof, T21 (SEQ ID NO:2) or an analog thereof, T1249 (SEQ ID NO:3) or an analog thereof, Nccg9P41 (Louis et al. 2001, J. Biol.

2016244273 13 Oct 2016

Chem. 276:(31)29485) or an analog thereof, or 5 helix (Root et al. 2001, Science 291:884) or an analog thereof. .

[0108] Assays known in the art can be used to test for viral fusion inhibiting activity of a polypeptide, a small organic molecule, or a small inorganic molecule. These assays include a reverse transcriptase assay, a p24 assay, or syncytia formation assay (see, e.g., U.S. Patent No. 5,464,933).

[0109] A list of antiviral agents which may be used in the chimeric protein of the invention has been previously described (see, e.g., U.S. Patent Nos. 6,086,875, 6,485,726, 6,030,613; WO 03/077834; US2003-0235536A1).

c. Hemostatic Agents [0110] In one embodiment, the biologically active molecule is a clotting factor or other agent that promotes hemostasis, including fragments and analogs thereof. The clotting factor can include any molecule that has clotting activity or activates a molecule with clotting activity. The clotting factor can be comprised of a polypeptide. The clotting factor can be, as an example, but not limited to Factor VIII, Factor IX, Factor Xi, Factor XII, fibrinogen, prothrombin, Factor V, Factor VII, Factor X, Factor XIII or von Willebrand Factor. In one embodiment, the clotting factor is Factor VII or Factor Vila. The clotting factor can be a factor that participates in the extrinsic pathway. The clotting factor can be a factor that participates in the intrinsic pathway. Alternatively, the clotting factor can be a factor that participates in both the extrinsic and intrinsic pathway.

[0111] The clotting factor can be a human clotting factor or a non-human clotting factor, e.g., derived from a non-human primate, a pig or any mammal. The clotting factor can be chimeric clotting factor, e.g., the clotting factor can comprise a

2016244273 13 Oct 2016 portion of a human clotting factor and a portion of a porcine clotting factor or a portion of a first non-human clotting factor and a portion of a second non-human clotting factor.

[0112] The clotting factor can be an activated clotting factor. Alternatively, the clotting factor can be an inactive form of a clotting factor, e.g., a zymogen. The inactive clotting factor can undergo activation subsequent to being linked to at least a portion of an immunoglobulin constant region. The inactive clotting factor can be activated subsequent to administration to a subject. Alternatively, the inactive clotting factor can be activated prior to administration.

[0113] in certain embodiments an endopeptidase, e.g., paired basic amino acid cleaving enzyme (PACE), or any PACE family member, such as PCSK1-9, including truncated versions thereof, or its yeast equivalent Kex2 from S. cerevisiae and truncated versions of Kex2 (Kex2 1-675) (see, e.g., U.S. Patent Nos. 5,077,204; 5,162,220; 5,234,830; 5,885,821; 6,329,176) may be used to cleave a propetide to form the mature chimeric protein of the invention (e.g. factor VII, factor IX).

d. Other Proteinaceous Biologically Active Molecules [0114] In one embodiment, the biologically active molecule is a receptor or a fragment or analog thereof. The receptor can be expressed on a cell surface, or alternatively the receptor can be expressed on the interior of the cell. The receptor can be a viral receptor, e.g., CD4, CCR5, CXCR4, CD21, CD46. The biologically active molecule can be a bacterial receptor. The biologically active molecule can be an extra-cellular matrix protein or fragment or analog thereof, important in bacterial colonization and infection (see, e.g., U.S. Patent Nos.: 5,648,240; 5,189,015; 5,175,096) ora bacterial surface protein important in adhesion and infection (see,

2016244273 13 Oct 2016

e.g., U.S. Patent No. 5,648,240). The biologically active molecule can be a growth factor, hormone or cytokine receptor, or a fragment or analog thereof, e.g., TNFa receptor, the erythropoietin receptor, CD25, CD122, or CD132.

[0115] A list of other proteinaceous molecules which may be used in the chimeric protein of the invention has been previously described (see, e.g., U.S. Patent Nos. 6,086,875; 6,485,726; 6,030,613; WO 03/077834; US2003-0235536A1).

e. Nucleic Acids [0116] In one embodiment, the biologically active molecule is a nucleic acid, e.g., DNA, RNA. in one specific embodiment, the biologically active molecule is a nucleic acid that can be used in RNA interference (RNAi). The nucleic acid molecule can be as an example, but not as a limitation, an anti-sense molecule or a ribozyme or an aptamer.

[0117] Antisense RNA and DNA molecules act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation. Antisense approaches involve the design of oligonucleotides that are complementary to a target gene mRNA. The antisense oligonucleotides will bind to the complementary target gene mRNA transcripts and prevent translation. Absolute compiementarily, is not required.

[0118] A sequence complementary to a portion of an RNA, as referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the

2016244273 13 Oct 2016 longer the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.

[0119] Antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects, the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.

[0120] The oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucleotide may include other appended groups such as polypeptides (e.g. for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. 1989, Proc. Natl. Acad. Sci. USA 86:6553; Lemaitre et al. 1987, Proc. Natl. Acad. Sci. USA 84:648; WO 88/09810,) or the blood-brain barrier (see, e.g., WO 89/10134), hybridization-triggered cleavage agents (see, e.g., Krol et ai. 1988, BioTechniques 6:958) or intercalating agents (see, e.g., Zon 1988, Pharm. Res. 5:539). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a polypeptide, hybridization triggered crosslinking agent, transport agent, or hybridization-triggered cleavage agent.

[0121] Ribozyme molecules designed to catalytically cleave target gene mRNA transcripts can also be used to prevent translation of target gene mRNA and,

2016244273 13 Oct 2016 therefore, expression of target gene product. (See, e.g., WO 90/11364; Sarver et al 1990, Science 247, 1222-1225).

[0122] Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. (See Rossi 1994, Current Biology 4:469). The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage event. The composition of ribozyme molecules must include one or more sequences complementary to the target gene mRNA, and must include the well known catalytic sequence responsible for mRNA cleavage. For this sequence, see, e.g., U.S. Pat No. 5,093,246.

[0123] In one embodiment, ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy target gene mRNAs. In another embodiment, the use of hammerhead ribozymes is contemplated. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases; 5'-UG-3'. The construction and production of hammerhead ribozymes is well known in the art and is described more fully in Myers 1995, Molecular Biology and Biotechnology: A Comprehensive Desk Reference, VCH Publishers, New York, and in Haseioffand Gerlach 1988, Nature, 334:585.

f. Small Molecules [0124] The invention also contemplates the use of any therapeutic small molecule or drug as the biologically active molecule in the chimeric protein of the invention. A list of small molecules and drugs which may be used in the chimeric

2016244273 13 Oct 2016 protein ofthe invention has been previously described (see, e.g., U.S. Patent Nos. 6,086,875; 6,485,726; 6,030,613; WO 03/077834; US2003-0235536A1).

2. Immunoglobulins [0125] The chimeric proteins ofthe invention comprise at least a portion of an immunoglobulin constant region. Immunoglobulins are comprised of four protein chains that associate covalently—two heavy chains and two light chains. Each chain is further comprised of one variable region and one constant region. Depending upon the immunoglobulin isotype, the heavy chain constant region is comprised of 3 or 4 constant region domains (e.g. CH1, CH2, CH3, CH4). Some isotypes are further comprised of a hinge region.

[0126] The portion of an immunoglobulin constant region can be obtained from any mammal. The portion of an immunoglobulin constant region can include a portion of a human immunoglobulin constant region, a non-human primate immunoglobulin constant region, a bovine immunoglobulin constant region, a porcine immunoglobulin constant region, a murine immunoglobulin constant region, an ovine immunoglobulin constant region or a rat immunoglobulin constant region.

[0127] The portion of an immunoglobulin constant region can be produced recombinantly or synthetically. The immunoglobulin can be isolated from a cDNA library. The portion of an immunoglobulin constant region can be isolated from a phage library (See, e.g., McCafferty et al. 1990, Nature 348:552, Kang et al. 1991, Proc. Natl. Acad. Sci. USA 88:4363; EP 0 589 877 B1). The portion of an immunoglobulin constant region can be obtained by gene shuffling of known sequences (Mark et al. 1992, Bio/Technol. 10:779). The portion of an immunoglobulin constant region can be isolated by in vivo recombination

2016244273 13 Oct 2016 (Waterhouse etal. 1993, Nucl. Acid Res. 21:2265). The immunoglobulin can be a humanized immunoglobulin (U.S. Patent No. 5,585,089, Jones etal. 1986, Nature 332:323).

[0128] The portion of an immunoglobulin constant region can include a portion of an IgG, an IgA, an IgM, an IgD, or an IgE. In one embodiment, the immunoglobulin is an IgG. in another embodiment, the immunoglobulin is lgG1. In another embodiment, the immunoglobulin is lgG2.

[0129] The portion of an immunoglobulin constant region can include the entire heavy chain constant region, or a fragment or analog thereof. In one embodiment, a heavy chain constant region can comprise a CH1 domain, a CH2 domain, a CH3 domain, and/or a hinge region. In another embodiment, a heavy chain constant region can comprise a CH1 domain, a CH2 domain, a CH3 domain, and/or a CH4 domain.

[0130] The portion of an immunoglobulin constant region can include an Fc fragment. An Fc fragment can be comprised of the CH2 and CH3 domains of an immunoglobulin and the hinge region of the immunoglobulin. The Fc fragment can be the Fc fragment of an IgG 1, an lgG2, an lgG3 or an lgG4. In one specific embodiment, the portion of an immunoglobulin constant region is an Fc fragment of an lgG1. In another embodiment, the portion of an immunoglobulin constant region is an Fc fragment of an IgG2.

[0131] In another embodiment, the portion of an immunoglobulin constant region is an Fc neonatal receptor (FcRn) binding partner. An FcRn binding partner is any molecule that can be specifically bound by the FcRn receptor with consequent active transport by the FcRn receptor of the FcRn binding partner. Specifically

2016244273 13 Oct 2016 bound refers to two molecules forming a complex that is relatively stable under physiologic conditions. Specific binding is characterized by a high affinity and a low to moderate capacity as distinguished from nonspecific binding which usually has a low affinity with a moderate to high capacity. Typically, binding is considered specific when the affinity constant Ka is higher than 10⁶M'¹, or more preferably higher than 10⁸ M'¹. If necessary, non-specific binding can be reduced without substantially affecting specific binding by varying the binding conditions. The appropriate binding conditions such as concentration of the molecules, ionic strength of the solution, temperature, time allowed for binding, concentration of a blocking agent (e.g. serum albumin, milk casein), etc., may be optimized by a skilled artisan using routine techniques.

[0132] The FcRn receptor has been isolated from several mammalian species including humans. The sequences of the human FcRn, monkey FcRn rat FcRn, and mouse FcRn are known (Story et al. 1994, J. Exp. Med. 180:2377). The FcRn receptor binds IgG (but not other immunoglobulin classes such as IgA, IgM, IgD, and IgE) at relatively low pH, actively transports the IgG transcellularly in a luminal to serosal direction, and then releases the IgG at relatively higher pH found in the interstitial fluids. It is expressed in adult epithelial tissue (U.S. Patent Nos.

6,485,726, 6,030,613, 6,086,875; WO 03/077834; US2003-0235536A1) including lung and intestinal epithelium (Israel et al. 1997, Immunology 92:69) renal proximal tubular epithelium (Kobayashi et al. 2002, Am. J. Physiol. Renal Physiol. 282:F358) as well as nasal epithelium, vaginal surfaces, and biliary tree surfaces.

[0133] FcRn binding partners of the present invention encompass any molecule that can be specifically bound by the FcRn receptor including whole IgG,

2016244273 13 Oct 2016 the Fc fragment of IgG, and other fragments that include the complete binding region of the FcRn receptor. The region of the Fc portion of IgG that binds to the FcRn receptor has been described based on X-ray crystallography (Burmeister et al. 1994, Nature 372:379). The major contact area of the Fc with the FcRn is near the junction of the CH2 and CH3 domains. Fc-FcRn contacts are all within a single Ig heavy chain. The FcRn binding partners include whole IgG, the Fc fragment of IgG, and other fragments of IgG that include the complete binding region of FcRn. The major contact sites include amino acid residues 248, 250-257, 272, 285, 288, 290291,308-311, and 314 of the CH2 domain and amino acid residues 385-387, 428, and 433-436 of the CH3 domain. References made to amino acid numbering of immunoglobulins or immunoglobulin fragments, or regions, are all based on Kabat et al. 1991, Sequences of Proteins of Immunological Interest, U.S. Department of Public Health, Bethesda, MD.

[0134] The Fc region of IgG can be modified according to well recognized procedures such as site directed mutagenesis and the like to yield modified IgG or Fc fragments or portions thereof that will be bound by FcRn. Such modifications include modifications remote from the FcRn contact sites as well as modifications within the contact sites that preserve or even enhance binding to the FcRn. For example, the following single amino acid residues in human lgG1 Fc (Fcy1) can be substituted without significant loss of Fc binding affinity for FcRn; P238A, S239A, K246A, K248A, D249A, M252A, T256A, E258A, T260A, D265A, S267A, H268A, E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A, E283A, H285A, N286A, T289A, K290A, R292A, E293A, E294A, Q295A, Y296F, N297A, S298A, Y300F, R301A, V303A, V305A, T307A, L309A, Q311A, D312A, N315A, K317A,

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E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A330Q, P331A, E333A, K334A, T335A, S337A, K338A, K340A, Q342A, R344A, E345A, Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A, Y373A, S375A, D376A, A378Q, E380A, E382A, S383A ,N384A, Q386A, E388A, N389A, N390A, Y391F, K392A, L398A, S400A, D401A, D413A, K414A, R416A, Q418A, Q419A, N421A, V422A, S424A, E430A, N434A, T437A, Q438A, K439A, S440A, S444A, and K447A, where for example P238A represents wildtype proline substituted by alanine at position number 238. As an example, one specifc embodiment, incorporates the N297A mutation, removing a highly conserved N-glycosylation site. In addition to alanine other amino acids may be substituted for the wildtype amino acids at the positions specified above. Mutations may be introduced singly into Fc giving rise to more than one hundred FcRn binding partners distinct from native Fc. Additionally, combinations of two, three, or more of these individual mutations may be introduced together, giving rise to hundreds more FcRn binding partners. Moreover, one of the FcRn binding partners of the monomer-dimer hybrid may be mutated and the other FcRn binding partner not mutated at all, or they both may be mutated but with different mutations. Any of the mutations described herein, including N297A, may be used to modify Fc, regardless of the biologically active molecule (e.g., EPO, IFN, Factor IX, T20).

[0135] Certain of the above mutations may confer new functionality upon the FcRn binding partner. For example, one embodiment incorporates N297A, removing a highly conserved N-glycosylation site. The effect of this mutation is to reduce immunogenicity, thereby enhancing circulating half life of the FcRn binding partner, and to render the FcRn binding partner incapable of binding to FcyRI,

2016244273 13 Oct 2016

FcyRIlA, FcyRHB, and FcyRIIIA, without compromising affinity for FcRn (Routledge et al. 1995, Transplantation 60:847; Friend et al. 1999, Transplantation 68:1632; Shields et ai. 1995, J. Biol. Chem. 276:6591). As a further example of new functionality arising from mutations described above affinity for FcRn may be increased beyond that of wild type in some instances. This increased affinity may reflect an increased “on” rate, a decreased “off” rate or both an increased “on” rate and a decreased “off” rate. Mutations believed to impart an increased affinity for FcRn include T256A, T307A, E380A, and N434A (Shields et al. 2001, J. Biol. Chem. 276:6591).

[0136] Additionally, at least three human Fc gamma receptors appear to recognize a binding site on IgG within the lower hinge region, generally amino acids 234-237. Therefore, another example of new functionality and potential decreased immunogenicity may arise from mutations of this region, as for example by replacing amino acids 233-236 of human IgG 1 “ELLG” to the corresponding sequence from lgG2 “PVA” (with one amino acid deletion). It has been shown that FcyRI, FcyRIl, and FcyRIII, which mediate various effector functions will not bind to lgG1 when such mutations have been introduced. Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al.1999, Eur. J. Immunol. 29:2613.

[0137] in one embodiment, the FcRn binding partner is a polypeptide including the sequence PKNSSMISNTP (SEQ ID NO:26) and optionally further including a sequence selected from HQSLGTQ (SEQ ID NO:27), HQNLSDGK (SEQ ID NO:28), HQNISDGK (SEQ ID NO:29), or VISSHLGQ (SEQ ID NO:30) (U.S. Patent No. 5,739,277).

2016244273 13 Oct 2016 [0138] Two FcRn receptors can bind a single Fc molecule. Crystallographic data suggest that each FcRn molecule binds a single polypeptide of the Fc homodimer. In one embodiment, linking the FcRn binding partner, e.g., an Fc fragment of an IgG, to a biologically active molecule provides a means of delivering the biologically active molecule orally, buccally, sublingually, rectally, vaginaliy, as an aerosol administered nasally or via a pulmonary route, or via an ocular route. In another embodiment, the chimeric protein can be administered invasively, e.g., subcutaneously, intravenously.

[0139] The skilled artisan will understand that portions of an immunoglobulin constant region for use in the chimeric protein of the invention can include mutants or analogs thereof, or can include chemically modified immunoglobulin constant regions (e.g. pegylated), or fragments thereof (see, e.g., Aslam and Dent 1998, Bioconjugation: Protein Coupling Techniques For the Biomedical Sciences Macmifan Reference, London). In one instance, a mutant can provide for enhanced binding of an FcRn binding partner for the FcRn. Also contemplated for use in the chimeric protein of the invention are peptide mimetics of at least a portion of an immunoglobulin constant region, e.g., a peptide mimetic of an Fc fragment or a peptide mimetic of an FcRn binding partner. In one embodiment, the peptide mimetic is identified using phage display or via chemical library screening (see, e.g., McCafferty etal. 1990, Nature 348:552, Kang et al. 1991, Proc. Natl. Acad. Sci. USA 88:4363; EP 0 589 877 B1).

3. Optional Linkers [0140] The chimeric protein of the invention can optionally comprise at least one linker molecule. The linker can be comprised of any organic molecule. In one

2016244273 13 Oct 2016 embodiment, the linker is polyethylene glycol (PEG). In another embodiment, the linker is comprised of amino acids. The linker can comprise 1-5 amino acids, 1-10 amino acids, 1-20 amino acids, 10-50 amino acids, 50-100 amino acids, 100-200 amino acids. In one embodiment, the linker is the eight amino acid linker EFAGAAAV (SEQ ID NO:31). Any of the linkers described herein may be used in the chimeric protein of the invention, e.g., a monomer-dimer hybrid, including EFAGAAAV, regardless of the biologically active molecule (e.g. EPO, IFN, Factor IX).

[0141] The linker can comprise the sequence G_n. The linker can comprise the sequence (GA)_n(SEQ ID NO:32). The linker can comprise the sequence (GGS)_n(SEQ ID NO:33). The linker can comprise the sequence (GGS)_n(GGGGS)_n (SEQ ID NO:34). In these instances, n may be an integer from 1-10, i.e., ),2,3, 4, 5, 6, 7, 8, 9, 10. Examples of linkers include, but are not limited to, GGG (SEQ ID NO:35), SGGSGGS (SEQ ID NO:36), GGSGGSGGSGGSGGG (SEQ ID NO:37), GGSGGSGGGGSGGGGS (SEQ ID NO:38), GGSGGSGGSGGSGGSGGS (SEQ ID NO:39). The linker does not eliminate or diminish the biological activity of the chimeric protein. Optionally, the linker enhances the biological activity of the chimeric protein, e.g., by further diminishing the effects of steric hindrance and making the biologically active molecule more accessible to its target binding site.

[0142] In one specific embodiment, the linker for interferon a is 15-25 amino acids long. In another specific embodiment, the linker for interferon a is 15-20 amino acids long. In another specific embodiment, the linker for interferon a is 10-25 amino acids long, in another specific embodiment, the linker for interferon a is 15 amino acids long. In one embodiment, the linker for interferon a is (GGGGS)_n (SEQ ID

2016244273 13 Oct 2016

NO:40) where G represents glycine, S represents serine and n is an integer from ΙΙΟ. Ina specific embodiment, n is 3.

[0143] The linker may also incorporate a moiety capable of being cleaved either chemically (e.g. hydrolysis of an ester bond), enzymatically (i.e. incorporation of a protease cleavage sequence) or photolytically (e.g.,a chromophore such as 3amino-3-(2-nitrophenyl) proprionic acid (ANP)) in order to release the biologically active molecule from the Fc protein.

4. Chimeric Protein Dimerization Using Specific Binding Partners [0144] In one embodiment, the chimeric protein of the invention comprises a first polypeptide chain comprising at least a first domain, said first domain having at feast one specific binding partner, and a second polypeptide chain comprising at least a second domain, wherein said second domain, is a specific binding partner of said first domain. The chimeric protein thus comprises a polypeptide capable of dimerizing with another polypeptide due to the interaction of the first domain and the second domain. Methods of dimerizing antibodies using heterologous domains are known in the art (U.S. Patent Nos.: 5,807,706 and 5,910,573; Kostelny et al. 1992, J. Immunol. 148(5):1547).

[0145] Dimerization can occur by formation of a covalent bond, or alternatively a non-covalent bond, e.g., hydrophobic interaction, Van der Waal’s forces, interdigitation of amphiphilic peptides such as, but not limited to, alpha helices, charge-charge interactions of amino acids bearing opposite charges, such as, but not limited to, lysine and aspartic acid, arginine and glutamic acid. In one embodiment, the domain is a helix bundle comprising a helix, a turn and another helix. In another embodiment, the domain is a leucine zipper comprising a peptide

2016244273 13 Oct 2016 having several repeating amino acids in which every seventh amino acid is a leucine residue. In one embodiment, the specific binding partners are fos/jun. (see Branden et al. 1991, Introduction To Protein Structure, Garland Publishing, New York).

[0146] In another embodiment, binding is mediated by a chemical linkage (see, e.g., Brennan et al. 1985, Science 229:81). in this embodiment, intact immunoglobulins, or chimeric proteins comprised of at least a portion of an immunoglobulin constant region are cleaved to generate heavy chain fragments. These fragments are reduced in the presence ofthe dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The fragments generated are then converted to thio nitrobenzoate (TNB) derivatives. One ofthe TNB derivatives is then reconverted to the heavy chain fragment thiol by reduction with mercaptoethylamine and is then mixed with an equimolar amount of the other TNB derivative to form a chimeric dimer.

D. Nucleic Acids [0147] The invention relates to a first nucleic acid construct and a second nucleic acid construct each comprising a nucleic acid sequence encoding at least a portion of the chimeric protein of the invention. In one embodiment, the first nucleic acid construct comprises a nucleic acid sequence encoding a portion of an immunoglobulin constant region operatively linked to a second DNA sequence encoding a biologically active molecule, and said second DNA construct comprises a DNA sequence encoding an immunoglobulin constant region without the second DNA sequence encoding a biologically active molecule.

[0148] The biologically active molecule can include, for example, but not as a limitation, a viral fusion inhibitor, a clotting factor, a growth factor or hormone, or a

2016244273 13 Oct 2016 receptor, or analog, or fragment of any of the preceding. The nucleic acid sequences can also include additional sequences or elements known in the art (e.g., promoters, enhancers, poly A sequences, affinity tags), in one embodiment, the nucleic acid sequence of the second construct can optionally include a nucleic acid sequence encoding a linker placed between the nucleic acid sequence encoding the biologically active molecule and the portion of the immunoglobulin constant region. The nucleic acid sequence of the second DNA construct can optionally include a linker sequence placed before or after the nucleic acid sequence encoding the biologically active molecule and/or the portion of the immunoglobulin constant region.

[0149] In one embodiment, the nucleic acid construct is comprised of DNA. In another embodiment, the nucleic acid construct is comprised of RNA. The nucleic acid construct can be a vector, e.g., a viral vector or a plasmid. Examples of viral vectors include, but are not limited to adeno virus vector, an adeno associated virus vector or a murine leukemia virus vector. Examples of plasmids include but are not limited to pUC, pGEM and pGEX.

[0150] In one embodiment, the nucleic acid construct comprises the nucleic acid sequence of figure 3a (SEQ ID NO;7). In one embodiment, the nucleic acid construct comprises the nucleic acid sequence of figure 3b (SEQ ID NO:9 ). In one embodiment, the nucleic acid construct comprises the nucleic acid sequence of figure 3c (SEQ ID NO:11). In one embodiment, the nucleic acid construct comprises the nucleic acid sequence of figure 3d (SEQ ID NO:13). In one embodiment, the nucleic acid construct comprises the nucleic acid sequence of figure 3e (SEQ ID ΝΟ.Ί5). In one embodiment, the nucleic acid construct comprises the nucleic acid

2016244273 13 Oct 2016 sequence of figure 3f (SEQ ID NO:17). In one embodiment, the nucleic acid construct comprises the nucleic acid sequence of figure 3g (SEQ ID NO:19). In one embodiment, the nucleic acid construct comprises the nucleic acid sequence of figure 3h (SEQ ID NO:21). in one embodiment, the nucleic acid construct comprises the nucleic acid sequence of figure 3i (SEQ ID NO:23). In one embodiment, the nucleic acid construct comprises the nucleic acid sequence of figure 3j (SEQ ID NO:25). In one embodiment, the nucleic acid construct comprises the nucleic acid sequence of figure 17a (SEQ ID NO:27).

[0151] Due to the known degeneracy of the genetic code, wherein more than one codon can encode the same amino acid, a DNA sequence can vary from that shown in SEQ ID NOS:7, 9, 11, 13, 15,17,19, 21,23, 25 or 27 and still encode a polypeptide having the corresponding amino acid sequence of SEQ ID NOS:6, 8,10, 12, 14, 16,18, 20, 22, 24 or 26 respectively. Such variant DNA sequences can result from silent mutations (e.g. occurring during PCR amplification), or can be the product of deliberate mutagenesis of a native sequence. The invention thus provides isolated DNA sequences encoding polypeptides of the invention, chosen from: (a) DNA comprising the nucleotide sequence of SEQ ID NOS:7, 9, 11, 13, 15, 17, 19, 21,23, 25 or 27; (b) DNA encoding the polypeptides of SEQ ID NOS:6, 8,

10, 12,14, 16,18, 20, 22, 24 or 26; (c) DNA capable of hybridization to a DNA of (a) or (b) under conditions of moderate stringency and which encodes polypeptides of the invention; (d) DNA capable of hybridization to a DNA of (a) or (b) under conditions of high stringency and which encodes polypeptides of the invention, and (e) DNA which is degenerate as a result of the genetic code to a DNA defined in (a),

2016244273 13 Oct 2016 (b), (c), or (d) and which encode polypeptides of the invention. Of course, polypeptides encoded by such DNA sequences are encompassed by the invention.

[0152] In another embodiment, the nucleic acid molecules comprising a sequence encoding the chimeric protein of the invention can also comprise nucleotide sequences that are at least 80% identical to a native sequence. Also contemplated are embodiments in which a nucieic acid molecules comprising a sequence encoding the chimeric protein of the invention comprises a sequence that is at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least 99.9% identical to a native sequence. A native sequence can include any DNA sequence not altered by the human hand. The percent identity may be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two nucleic acid sequences can be determined by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et at. 1984, Nucl. Acids Res. 12:387, and available from the University of Wisconsin Genetics Computer Group (UWGCG). The preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess 1986, Nucl. Acids Res. 14:6745, as described by Schwartz and Dayhoff, eds. 1979, Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps. Other programs used by one skilled in the art of sequence comparison may also be used.

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E. Synthesis of Chimeric Proteins [0153] Chimeric proteins comprising at least a portion of an immunoglobulin constant region and a biologically active molecule can be synthesized using techniques well known in the art. For example, the chimeric proteins of the invention can be synthesized recombinantly in cells (see, e.g., Sambrook et al. 1989, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. and Ausubel et al. 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y.). Alternatively, the chimeric proteins of the invention can be synthesized using known synthetic methods such as solid phase synthesis. Synthetic techniques are well known in the art (see, e.g., Merrifield, 1973, Chemical Polypeptides, (Katsoyannis and Panayotis eds.) pp. 335-61; Merrifield 1963, J. Am. Chem. Soc. 85:2149; Davis et al. 1985, Biochem. Inti. 10:394; Finn et al. 1976, The Proteins (3d ed.) 2:105; Erikson et al. 1976, The Proteins (3d ed.) 2:257; U.S. Patent No. 3,941,763. Alternatively, the chimeric proteins of the invention can be synthesized using a combination of recombinant and synthetic methods. In certain applications, it may be beneficial to use either a recombinant method or a combination of recombinant and synthetic methods.

[0154] Nucleic acids encoding a biologically active molecule can be readily synthesized using recombinant techniques well known in the art. Alternatively, the peptides themselves can be chemically synthesized. Nucleic acids of the invention may be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. 1988, Nucl. Acids Res. 16:3209,

2016244273 13 Oct 2016 methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports as described in Sarin et al. 1988, Proc. Natl. Acad. Sci. USA 85:7448. Additional methods of nucleic acid synthesis are known in the art. (see, e.g., U.S. Patent Nos. 6,015,881; 6,281,331; 6,469,136).

[0155] DNA sequences encoding immunoglobulin constant regions, or fragments thereof, may be cloned from a variety of genomic or cDNA libraries known in the art. The techniques for isolating such DNA sequences using probe-based methods are conventional techniques and are well known to those skilled in the art. Probes for isolating such DNA sequences may be based on published DNA sequences (see, for example, Hieter et at. 1980, Cell 22:197-207). The polymerase chain reaction (PCR) method disclosed by Mullis et al. (U.S. Patent No. 4,683,195) and Mullis (U.S. Patent No. 4,683,202) may be used. The choice of library and selection of probes for the isolation of such DNA sequences is within the level of ordinary skill in the art. Alternatively, DNA sequences encoding immunoglobulins or fragments thereof can be obtained from vectors known in the art to contain immunoglobulins or fragments thereof.

[0156] For recombinant production, a first polynucleotide sequence encoding a portion of the chimeric protein of the invention (e.g. a portion of an immunoglobulin constant region) and a second polynucleotide sequence encoding a portion of the chimeric protein of the invention (e.g. a portion of an immunoglobulin constant region and a biologically active molecule) are inserted into appropriate expression vehicles, i.e. vectors which contains the necessary elements for the transcription and translation of the inserted coding sequence, or in the case of an RNA viral vector,

2016244273 13 Oct 2016 the necessary elements for replication and translation. The nucleic acids encoding the chimeric protein are inserted into the vector in proper reading frame.

[0157] The expression vehicles are then transfected or co-transfected into a suitable target cell, which wiil express the polypeptides. Transfection techniques known in the art include, but are not limited to, calcium phosphate precipitation (Wigler et al. 1978, Cell 14:725) and electroporation (Neumann et al. 1982, EMBO,

J. 1:841), and liposome based reagents. A variety of host-expression vector systems may be utilized to express the chimeric proteins described herein including both prokaryotic or eukaryotic cells. These include, but are not limited to, microorganisms such as bacteria (e.g. E. coli) transformed with recombinant bacteriophage DNA or plasmid DNA expression vectors containing an appropriate coding sequence; yeast or filamentous fungi transformed with recombinant yeast or fungi expression vectors containing an appropriate coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g. baculovirus) containing an appropriate coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g. cauliflower mosaic virus or tobacco mosaic virus) or transformed with recombinant plasmid expression vectors (e.g. Ti plasmid) containing an appropriate coding sequence; or animal cell systems, including mammalian cells (e.g. CHO, Cos, HeLa cells).

[0158] When the chimeric protein of the invention is recombinantly synthesized in a prokaryotic cell it may be desirable to refold the chimeric protein. The chimeric protein produced by this method can be refolded to a biologically active conformation using conditions known in the art, e.g., denaturing under reducing conditions and then dialyzed slowly into PBS.

2016244273 13 Oct 2016 [0159] Depending on the expression system used, the expressed chimeric protein is then isolated by procedures well-established in the art (e.g. affinity chromatography, size exclusion chromatography, ion exchange chromatography).

[0160] The expression vectors can encode for tags that permit for easy purification of the recombinantly produced chimeric protein. Examples include, but are not limited to vector pUR278 (Ruther et al. 1983, EMBO J. 2:1791) in which the chimeric protein described herein coding sequences may be ligated into the vector in frame with the lac z coding region so that a hybrid protein is produced; pGEX vectors may be used to express chimeric proteins of the invention with a glutathione

S-transferase (GST) tag. These proteins are usually soluble and can easily be purified from cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The vectors include cleavage sites (thrombin or Factor Xa protease or PreScission Protease™ (Pharmacia, Peapack, N.J.)) for easy removal of the tag after purification.

[0161] To increase efficiency of production, the polynucleotides can be designed to encode multiple units of the chimeric protein of the invention separated by enzymatic cleavage sites. The resulting polypeptide can be cleaved (e.g. by treatment with the appropriate enzyme) in order to recover the polypeptide units. This can increase the yield of polypeptides driven by a single promoter. When used in appropriate viral expression systems, the translation of each polypeptide encoded by the mRNA is directed internally in the transcript; e.g., by an internal ribosome entry site, IRES. Thus, the polycistronic construct directs the transcription of a single, large polycistronic mRNA which, in turn, directs the translation of multiple, individual polypeptides. This approach eliminates the production and enzymatic

2016244273 13 Oct 2016 processing of polyproteins and may significantly increase yield of polypeptide driven by a single promoter.

[0162] Vectors used in transformation will usually contain a selectable marker used to identify transformants. In bacterial systems, this can include an antibiotic resistance gene such as ampiciliin or kanamycin. Selectable markers for use in cultured mammalian cells include genes that confer resistance to drugs, such as neomycin, hygromycin, and methotrexate. The selectable marker may be an amplifiable selectable marker. One amplifiable selectable marker is the DHFR gene. Another amplifiable marker is the DHFR cDNA (Simonsen and Levinson 1983, Proc. Natl. Acad. Sci. USA 80:2495). Selectable markers are reviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers, Stoneham, MA) and the choice of selectable markers is well within the level of ordinary skill in the art.

[0163] Selectable markers may be introduced into the cell on a separate plasmid 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. Constructs of this type are known in the art (for example, U.S. Pat. No. 4,713,339).

[0164] The expression elements of the expression systems vary in their strength and specificities. Depending on the host/vector system utilized, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used in the expression vector. For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used; when cloning in

2016244273 13 Oct 2016 insect cell systems, promoters such as the baculovirus polyhedron promoter may be used; when cloning in plant cell systems, promoters derived from the genome of plant cells (e.g. heat shock promoters; the promoter for'the small subunit of RUBISCO; the promoter for the chlorophyll a/b binding protein) or from plant viruses (e.g. the 35S RNA promoter of CaMV; the coat protein promoter of TMV) may be used; when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g. metallothionein promoter) or from mammalian viruses (e.g. the adenovirus late promoter; the vaccinia virus 7.5 K promoter) may be used; when generating cell lines that contain multiple copies of expression product, SV40-, BPVand EBV-based vectors may be used with an appropriate selectable marker.

[0165] In cases where plant expression vectors are used, the expression of sequences encoding linear or non-cyciized forms of the chimeric proteins of the invention may be driven by any of a number of promoters. For example, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al. 1984, Nature 310:511-514), or the coat protein promoter of TMV (Takamatsu et al. 1987, EMBO J. 6:307-311) may be used; alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi et al. 1984, EMBO J. 3:1671-1680; Broglie et al. 1984, Science 224:838-843) or heat shock promoters, e.g., soybean hsp17.5-E or hsp17.3-B (Gurley et al. 1986, Mol. Cell. Biol. 6:559-565) may be used. These constructs can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, etc. For reviews of such techniques see, e.g., Weissbach & Weissbach 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp. 421-463; and Grierson & Corey 1988, Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9.

2016244273 13 Oct 2016 [0166] In one insect expression system that may be used to produce the chimeric proteins ofthe invention, Autographa californica nuclear polyhidrosis virus (AcNPV) is used as a vector to express the foreign genes. The virus grows in Spodoptera frugiperda cells. A coding sequence may be cloned into non-essential regions (for example, the polyhedron gene) ofthe virus and placed under control of an AcNPV promoter (for example, the polyhedron promoter). Successful insertion of a coding sequence will result in inactivation ofthe polyhedron gene and production of non-occluded recombinant virus (i.e. virus lacking the proteinaceous coat coded for by the polyhedron gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed, (see, e.g., Smith et al. 1983, J. Virol. 46:584; U.S. Patent No. 4,215,051). Further examples of this expression system may be found in Ausubel et al., eds. 1989, Current Protocols in Molecular Biology, Voi. 2, Greene Publish. Assoc. & Wiley Interscience.

[0167] Another system which can be used to express the chimeric proteins of the invention is the glutamine synthetase gene expression system, also referred to as the “GS expression system” (Lonza Biologies PLC, Berkshire UK). This expression system is described in detail in U.S. Patent No. 5,981,216.

[0168] In mammalian host cells, a number of viral based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, a coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region ofthe viral genome (e.g. region E1 or E3) will result in a recombinant virus that is viable and capable of expressing peptide in

2016244273 13 Oct 2016 infected hosts (see, e.g., Logan & Shenk 1984, Proc. Natl. Acad. Sci. USA 81:3655). Alternatively, the vaccinia 7.5 K promoter may be used (see, e.g., Mackett et al. 1982, Proc. Natl. Acad. Sci. USA 79:7415; Mackett et al. 1984, J. Virol. 49:857; Panicali et al. 1982, Proc. Natl. Acad. Sci. USA 79:4927).

[0169] In cases where an adenovirus is used as an expression vector, a coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g. region E1 or E3) will result in a recombinant virus that is viable and capable of expressing peptide in infected hosts (see, e.g., Logan & Shenk 1984, Proc. Natl. Acad. Sci. USA 81:3655). Alternatively, the vaccinia 7.5 K promoter may be used (see, e.g., Mackett et al. 1982, Proc. Natl. Acad. Sci. USA 79:7415; Mackett et al. 1984, J. Virol. 49:857; Panicali et al. 1982, Proc. Natl. Acad. Sci. USA 79:4927).

[0170] Host cells containing DNA constructs of the chimeric protein are grown in an appropriate growth medium. As used herein, the term appropriate growth medium means a medium containing nutrients required for the growth of cells. Nutrients required for cell growth may include a carbon source, a nitrogen source, essential amino acids, vitamins, minerals and growth factors. Optionally the media can contain bovine calf serum or fetal calf serum. In one embodiment, the media contains substantially no IgG. The growth medium will generally select for cells containing the DNA construct by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker on the DNA construct or co-transfected with the DNA construct. Cultured mammalian cells are

2016244273 13 Oct 2016 generally grown in commercially available serum-containing or serum-free media (e.g. MEM, DMEM). Selection of a medium appropriate for the particular cell line used is within the level of ordinary skill in the art.

[0171] The recombinantly produced chimeric protein of the invention can be isolated from the culture media. The culture medium from appropriately grown transformed or transfected host cells is separated from the cell material, and the presence of chimeric proteins is demonstrated. One method of detecting the chimeric proteins, for example, is by the binding of the chimeric proteins or portions of the chimeric proteins to a specific antibody recognizing the chimeric protein of the invention. An anti-chimeric protein antibody may be a monoclonal or polyclonal antibody raised against the chimeric protein in question. For example, the chimeric protein contains at least a portion of an immunoglobulin constant region. Antibodies recognizing the constant region of many immunoglobulins are known in the art and are commercially available. An antibody can be used to perform an ELISA or a western blot to detect the presence of the chimeric protein of the invention.

[0172] The chimeric protein of the invention can be synthesized in a transgenic animal, such as a rodent, cow, pig, sheep, or goat. The term transgenic animals refers to non-human animals that have incorporated a foreign gene into their genome. Because this gene is present in germline tissues, it is passed from parent to offspring. Exogenous genes are introduced into single-celled embryos (Brinster et al. 1985, Proc. Natl. Acad. Sci. USA 82:4438). Methods of producing transgenic animals are known in the art, including transgenics that produce immunoglobulin molecules (Wagner et al. 1981, Proc. Natl. Acad. Sci. USA 78:6376; McKnight et al. 1983, Cell 34:335; Brinster et al. 1983, Nature 306:332; Ritchie et al.

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1984, Nature 312:517; Baldassarre et al. 2003, Theriogenology 59:831; Robl et al. 2003, Theriogenology 59:107; Malassagne et al. 2003, Xenotransplantation 10(3):267).

[0173] The chimeric protein of the invention can also be produced by a combination of synthetic chemistry and recombinant techniques. For example, the portion of an immunoglobulin constant region can be expressed recombinantly as described above. The biologically active molecule, can be produced using known chemical synthesis techniques (e.g. solid phase synthesis).

[0174] The portion of an immunoglobulin constant region can be ligated to the biologically active molecule using appropriate ligation chemistry and then combined with a portion of an immunoglobulin constant region that has not been ligated to a biologically active molecule to form the chimeric protein of the invention. In one embodiment, the portion of an immunoglobulin constant region is an Fc fragment. The Fc fragment can be recombinantly produced to form Cys-Fc and reacted with a biologically active molecule expressing a thioester to make a monomer-dimer hybrid. In another embodiment, an Fc-thioester is made and reacted with a biologically active molecule expressing an N terminus Cysteine (Figure 4).

[0175] In one embodiment, the portion of an immunoglobulin constant region ligated to the biologically active molecule will form homodimers. The homodimers can be disrupted by exposing the homodimers to denaturing and reducing conditions (e.g. beta-mercaptoethanol and 8M urea) and then subsequently combined with a portion of an immunoglobulin constant region not linked to a biologically active molecule to form monomer-dimer hybrids. The monomer-dimer hybrids are then

2016244273 13 Oct 2016 renatured and refolded by dialyzing into PBS and isolated, e.g., by size exclusion or affinity chromatography.

[0176] In another embodiment, the portion of an immunoglobulin constant region will form homodimers before being linked to a biologically active molecule. In this embodiment, reaction conditions for linking the biologically active molecule to the homodimer can be adjusted such that linkage of the biologically active molecule to only one chain of the homodimer is favored (e.g. by adjusting the molar equivalents of each reactant).

[0177] The biologically active molecule can be chemically synthesized with an N terminal cysteine. The sequence encoding a portion of an immunoglobulin constant region can be sub-cloned into a vector encoding intein linked to a chitin binding domain (New England Biolabs, Beverly, MA). The intein can be linked to the G terminus of the portion of an immunoglobulin constant region. In one embodiment, the portion of the immunoglobulin with the intein linked to its C terminus can be expressed in a prokaryotic cell. In another embodiment, the portion of the immunoglobulin with the intein linked to its C terminus can be expressed in a eukaryotic cell. The portion of immunoglobulin constant region linked to intein can be reacted with MESNA. In one embodiment, the portion of an immunoglobulin constant region linked to intein is bound to a column, e.g., a chitin column and then eluted with MESNA. The biologically active molecule and portion of an immunoglobulin can be reacted together such that nucleophilic rearrangement occurs and the biologically active molecule is covalently linked to the portion of an immunoglobulin via an amide bond. (Dawsen et al. 2000, Annu. Rev. Biochem. 69:923). The chimeric protein synthesized this way can optionally include a linker

2016244273 13 Oct 2016 peptide between the portion of an immunoglobulin and the biologically active molecule. The linker can for example be synthesized on the N terminus of the biologically active molecule. Linkers can include peptides and/or organic molecules (e.g. polyethylene glycol and/or short amino acid sequences). This combined recombinant and chemical synthesis allows for the rapid screening of biologically active molecules and linkers to optimize desired properties of the chimeric protein of the invention, e.g., viral inhibition, hemostasis, production of red blood cells, biological half-life, stability, binding to serum proteins or some other property of the chimeric protein. The method also allows for the incorporation of non-natural amino acids into the chimeric protein of the invention which may be useful for optimizing a desired property of the chimeric protein of the invention. If desired, the chimeric protein produced by this method can be refolded to a biologically active conformation using conditions known in the art, e.g., reducing conditions and then dialyzed slowly into PBS.

[0178] Alternatively, the N-terminal cysteine can be on the portion of an immunoglobulin constant region, e.g., an Fc fragment. An Fc fragment can be generated with an N- terminal cysteine by taking advantage of the fact that a native Fc has a cysteine at position 226 (see Kabat et al. 1991, Sequences of Proteins of Immunological Interest, U.S. Department of Public Health, Bethesda, MD).

[0179] To expose a terminal cysteine, an Fc fragment can be recombinantly expressed. In one embodiment, the Fc fragment is expressed in a prokaryotic cell, e.g., E.coli. The sequence encoding the Fc portion beginning with Cys 226 (EU numbering) can be placed immediately following a sequence endcoding a signal peptide, e.g., OmpA, PhoA, STII. The prokaryotic cell can be osmotically shocked to

2016244273 13 Oct 2016 release the recombinant Fc fragment. In another embodiment, the Fc fragment is produced in a eukaryotic cel!, e.g., a CHO cell, a BHK cell. The sequence encoding the Fc portion fragment can be placed directly following a sequence encoding a signal peptide, e.g., mouse Igx light chain or MHC class I Kb signal sequence, such that when the recombinant chimeric protein is synthesized by a eukaryotic cell, the signal sequence will be cleaved, leaving an N terminal cysteine which can than be isolated and chemically reacted with a molecule bearing a thioester (e.g. a C terminal thioester if the molecule is comprised of amino acids).

[0180] The N terminal cysteine on an Fc fragment can also be generated using an enzyme that cleaves its substrate at its N terminus, e.g., Factor X^a, enterokinase, and the product isolated and reacted with a molecule with a thioester.

[0181] The recombinantly expressed Fc fragment can be used to make homodimers or monomer-dimer hybrids.

[0182] in a specific embodiment, an Fc fragment is expressed with the human a interferon signal peptide adjacent to the Cys at position 226. When a construct encoding this polypeptide is expressed in CHO cells, the CHO cells cleave the signal peptide at two distinct positions (at Cys 226 and at Val within the signal peptide 2 amino acids upstream in the N terminus direction). This generates a mixture of two species of Fc fragments (one with an N-terminal Val and one with an N-terminal Cys). This in turn results in a mixture of dimeric species (homodimers with terminal Val, homodimers with terminal Cys and heterodimers where one chain has a terminal Cys and the other chain has a terminal Val). The Fc fragments can be reacted with a biologically active molecule having a C terminal thioester and the resulting monomer-dimer hybrid can be isolated from the mixture (e.g. by size

2016244273 13 Oct 2016 exclusion chromatography). It is contemplated that when other signal peptide sequences are used for expression of Fc fragments in CHO cells a mixture of species of Fc fragments with at least two different N termini will be generated.

[0183] In another embodiment, a recombinantly produced Cys-Fc can form a homodimer. The homodimer can be reacted with peptide that has a branched linker on the C terminus, wherein the branched linker has two C terminal thioesters that can be reacted with the Cys-Fc. In another embodiment, the biologically active molecule has a single non-terminal thioester that can be reacted with Cys-Fc. Alternatively, the branched linker can have two C terminal cysteines that can be reacted with an Fc thioester. In another embodiment, the branched linker has two functional groups that can be reacted with the Fc thioester, e.g., 2-mercaptoamine. The biologically active molecule may be comprised of amino acids. The biologically active molecule may include a small organic molecule or a small inorganic molecule.

F. Methods of Using Chimeric Proteins [0184] The chimeric proteins of the invention have many uses as will be recognized by one skilled in the art, including, but not limited to methods of treating a subject with a disease or condition. The disease or condition can include, but is not limited to, a viral infection, a hemostatic disorder, anemia, cancer, leukemia, an inflammatory condition or an autoimmune disease (e.g. arthritis, psoriasis, lupus erythematosus, multiple sclerosis), or a bacterial infection (see, e.g., U.S. Patent Nos. 6,086,875, 6,030,613, 6,485,726; WO 03/077834; US2003-0235536A1).

1. Methods of Treating a Subject with a Red Blood Cell Deficiency [0185] The invention relates to a method of treating a subject having a deficiency of red blood ceils, e.g., anemia, comprising administering a

2016244273 13 Oct 2016 therapeutically effective amount of at least one chimeric protein, wherein the chimeric protein comprises a first and a second polypeptide chain, wherein the first chain comprises at least a portion of an immunoglobulin constant region and at least one agent capable of inducing proliferation of red blood cells, e.g., EPO, and the second polypeptide chain comprises at least a portion of an immunoglobulin without the agent capable of inducing red blood cell proliferation of the first chain.

2. Methods of Trearing a Subject with a Viral Infection [0186] The invention relates to a method of treating a subject having a viral infection or exposed to a virus comprising administering a therapeutically effective amount of at least one chimeric protein, wherein the chimeric protein comprises a first and a second polypeptide chain, wherein the first chain comprises at least a portion of an immunoglobulin constant region and at least one antiviral agent, e.g., a fusion inhibitor or interferon a and the second polypeptide chain comprises at least a portion of an immunoglobulin without the antiviral agent of the first chain. In one embodiment, the subject is infected with a virus which can be treated with IFNa, e.g., hepatitis C virus. In one embodiment, the subject is infected with HIV, such as HIV1 or HIV-2.

[0187] In one embodiment, the chimeric protein of the invention inhibits viral replication. In one embodiment, the chimeric protein of the invention prevents or inhibits viral entry into target cells, thereby stopping, preventing, or limiting the spread of a viral infection in a subject and decreasing the viral burden in an infected subject. By linking a portion of an immunoglobulin to a viral fusion inhibitor the invention provides a chimeric protein with viral fusion inhibitory activity with greater stability and greater bioavailability compared to viral fusion inhibitors alone, e.g.,

Ο Τ20, Τ21, Τ1249. Thus, in one embodiment, the viral fusion inhibitor decreases or

C4 q prevents HIV infection of a target cell, e.g., HIV-1.

o ch a. Conditions That May Be Treated [0188] The chimeric protein of the invention can be used to inhibit or prevent the infection of a target cell by a hepatitis virus, e.g., hepatitis virus C. The chimeric protein may comprise an anti-viral agent which inhibits viral replication.

[0189] in one embodiment, the chimeric protein of the invention comprises a fusion inhibitor. The chimeric protein of the invention can be used to inhibit or prevent the infection of any target cell by any virus (see, e.g., U.S. Patent Nos. 6,086,875, 6,030,613, 6,485,726; WO 03/077834; US2003-0235536A1). In one embodiment, the virus is an enveloped virus such as, but not limited to HIV, SIV, measles, influenza, Epstein-Barr virus, respiratory syncytia virus, or parainfluenza virus. In another embodiment, the virus is a non-enveloped virus such as rhino virus or polio virus [0190] The chimeric protein of the invention can be used to treat a subject already infected with a virus. The subject can be acutely infected with a virus. Alternatively, the subject can be chronically infected with a virus. The chimeric protein of the invention can also be used to prophylactically treat a subject at risk for contracting a viral infection, e.g., a subject known or believed to in close contact with a virus or subject believed to be infected or carrying a virus. The chimeric protein of the invention can be used to treat a subject who may have been exposed to a virus, but who has not yet been positively diagnosed.

[0191] In one embodiment, the invention relates to a method of treating a subject infected with HCV comprising administering to the subject a therapeutically

2016244273 13 Oct 2016 effective amount of a chimeric protein, wherein the chimeric protein comprises an Fc fragment of an IgG and a cytokine, e.g., IFNa.

[0192] In one embodiment, the invention relates to a method of treating a subject infected with HIV comprising administering to the subject a therapeutically effective amount of a chimeric protein wherein the chimeric protein comprises an Fc fragment of an IgG and the viral fusion inhibitor comprises T20.

3, Methods of Treating a Subject Having a Hemostatic Disorder [0193] The invention relates to a method of treating a subject having a hemostatic disorder comprising administering a therapeutically effective amount of at least one chimeric protein, wherein the chimeric protein comprises a first and a second chain, wherein the first chain comprises at least one clotting factor and at least a portion of an immunoglobulin constant region, and the second chain comprises at least a portion of an immunoglobulin constant region.

[0194] The chimeric protein of the invention treats or prevents a hemostatic disorder by promoting the formation of a fibrin clot. The chimeric protein of the invention can activate any member of a coagulation cascade. The clotting factor can be a participant in the extrinsic pathway, the intrinsic pathway or both. In one embodiment, the clotting factor is Factor VII or Factor Vila. Factor Vila can activate Factor X which interacts with Factor Va to cleave prothrombin to thrombin, which in turn cleaves fibrinogen to fibrin. In another embodiment, the clotting factor is Factor IX or Factor IXa. In yet another embodiment, the clotting factor is Factor VIII or Factor Villa, in yet another embodiment, the clotting factor is von Willebrand Factor, Factor XI, Factor XII, Factor V, Factor X or Factor XIII.

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a. Conditions That May Be Treated [0195] The chimeric protein of the invention can be used to treat any hemostatic disorder. The hemostatic disorders that may be treated by administration of the chimeric protein of the invention include, but are not limited to, hemophilia A, hemophilia B, von Willebrand’s disease, Factor XI deficiency (PTA deficiency), Factor XII deficiency, as well as deficiencies or structural abnormalities in fibrinogen, prothrombin, Factor V, Factor Vli, Factor X, or Factor XIII.

[0196] In one embodiment, the hemostatic disorder is an inherited disorder.

In one embodiment, the subject has hemophilia A, and the chimeric protein comprises Factor VIII or Factor Villa. In another embodiment, the subject has hemophilia A and the chimeric protein comprises Factor VII or Factor Vila, in another embodiment, the subject has hemophilia B and the chimeric protein comprises Factor IX or Factor IXa. In another embodiment, the subject has hemophilia B and the chimeric protein comprises Factor VII or Factor Vila. In another embodiment, the subject has inhibitory antibodies to Factor VIII or Factor Villa and the chimeric protein comprises Factor VII or Factor Vila. In yet another embodiment, the subject has inhibitory antibodies against Factor IX or Factor IXa and the chimeric protein comprises Factor VII or Factor Vila.

[0197] The chimeric protein of the invention can be used to prophylactically treat a subject with a hemostatic disorder. The chimeric protein of the invention can be used to treat an acute bleeding episode in a subject with a hemostatic disorder [0198] In one embodiment, the hemostatic disorder is the result of a deficiency in a clotting factor, e.g., Factor IX, Factor VIII. In another embodiment,

2016244273 13 Oct 2016 the hemostatic disorder can be the result of a defective clotting factor, e.g., von Willebrand’s Factor.

[0199] In another embodiment, the hemostatic disorder can be an acquired disorder. The acquired disorder can result from an underlying secondary disease or condition. The unrelated condition can be, as an example, but not as a limitation, cancer, an autoimmune disease, or pregnancy. The acquired disorder can result from old age or from medication to treat an underlying secondary disorder (e.g. cancer chemotherapy).

4. Methods of Treating a Subject In Need of a General Hemostatic Agent [0200] The invention also relates to methods of treating a subject that does not have a hemostatic disorder or a secondary disease or condition resulting in acquisition of a hemostatic disorder. The invention thus relates to a method of treating a subject in need of a general hemostatic agent comprising administering a therapeutically effective amount of at least one chimeric protein, wherein the chimeric protein comprises a first and a second polypeptide chain wherein the first polypeptide chain comprises at least a portion of an immunoglobulin constant region and at least one clotting factor and the second chain comprises at least a portion of an immunoglobulin constant region without the clotting factor of the first polypeptide chain.

a. Conditions That May Be Treated [0201] In one embodiment, the subject in need of a general hemostatic agent is undergoing, or is about to undergo, surgery. The chimeric protein of the invention can be administered prior to or after surgery as a prophylactic. The chimeric protein

2016244273 13 Oct 2016 of the invention can be administered during or after surgery to control an acute bleeding episode. The surgery can include, but is not limited to, liver transplantation, liver resection, or stem cell transplantation.

[0202] The chimeric protein of the invention can be used to treat a subject having an acute bleeding episode who does not have a hemostatic disorder. The acute bleeding episode can result from severe trauma, e.g., surgery, an automobile accident, wound, laceration gun shot, or any other traumatic event resulting in uncontrolled bleeding.

i 5. Treatment Modalities ! [0203] The chimeric protein ofthe invention can be administered ‘ intravenously, subcutaneously, intra-muscuiarly, or via any mucosal surface, e.g., orally, sublingually, buccally, sublingually, nasally, rectaliy, vaginally or via pulmonary route. The chimeric protein can be implanted within or linked to a _; biopoiymer solid support that allows for the slow release of the chimeric protein to j the desired site.

[0204] The dose of the chimeric protein of the invention will vary depending on the subject and upon the particular route of administration used. Dosages can range from 0.1 to 100,000 pg/kg body weight. In one embodiment, the dosing range is 0.1-1,000 pg/kg. The protein can be administered continuously or at specific timed intervals, in vitro assays may be employed to determine optimal dose ranges and/or schedules for administration. Many in vitro assays that measure viral : infectivity are known in the art. For example, a reverse transcriptase assay, or an rt

PCR assay or branched DNA assay can be used to measure HIV concentrations. A

2016244273 13 Oct 2016

StaClot assay can be used to measure clotting activity. Additionally, effective doses may be extrapolated from dose-response curves obtained from animal models.

[0205] The invention also relates to a pharmaceutical composition comprising a viral fusion inhibitor, at least a portion of an immunoglobulin and a pharmaceutically acceptable carrier or excipient Examples of suitable pharmaceutical carriers are described in Remington’s Pharmaceutical Sciences by E.W. Martin. Examples of excipients can include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition can also contain pH buffering reagents, and wetting or emulsifying agents.

[0206] For oral administration, the pharmaceutical composition can take the form of tablets or capsules prepared by conventional means. The composition can also be prepared as a liquid for example a syrup or a suspension. The liquid can include suspending agents (e.g. sorbitol syrup, cellulose derivatives or hydrogenated edible fats), emulsifying agents (lecithin or acacia), non-aqueous vehicles (e.g. almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils), and preservatives (e.g. methyl or propyl -p-hydroxybenzoates or sorbic acid). The preparations can also include flavoring, coloring and sweetening agents. Alternatively, the composition can be presented as a dry product for constitution with water or another suitable vehicle.

[0207] For buccal and sublingual administration the composition may take the form of tablets, lozenges or fast dissolving films according to conventional protocols.

2016244273 13 Oct 2016 [0208] For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray from a pressurized pack or nebulizer (e.g. in PBS), with a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0209] The pharmaceutical composition can be formulated for parenteral administration (i.e. intravenous or intramuscular) by bolus injection. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multidose containers with an added preservative. The compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and contain formuiatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., pyrogen free water.

[0210] The pharmaceutical composition can also be formulated for rectal administration as a suppository or retention enema, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

2016244273 13 Oct 2016

6. Combination Therapy [0211] The chimeric protein of the invention can be used to treat a subject with a disease or condition in combination with at least one other known agent to treat said disease or condition.

[0212] In one embodiment, the invention relates to a method of treating a subject infected with HIV comprising administering a therapeutically effective amount of at least one chimeric protein comprising a first and a second chain, wherein the first chain comprises an HIV fusion inhibitor and at least a portion of an immunoglobulin constant region and the second chain comprises at least a portion of an immunoglobulin without an HIV fusion inhibitor of the first chain, in combination with at least one other anti-HIV agent. Said other anti-HIV agent can be any therapeutic with demonstrated anti-HIV activity. Said other anti-HIV agent can include, as an example, but not as a limitation, a protease inhibitor (e.g.

Amprenavir®, Crixivan®, Ritonivir®), a reverse transcriptase nucleoside analog (e.g. AZT, DDI, D4T, 3TC, Ziagen®), a nonnucleoside analog reverse transcriptase inhibitor (e.g. Sustiva®), another HIV fusion inhibitor, a neutralizing antibody specific to HIV, an antibody specific to CD4, a CD4 mimic, e.g., CD4-lgG2 fusion protein (U.S. Patent Application 09/912,824) or an antibody specific to CCR5, or CXCR4, or a specific binding partner of CCR5, or CXCR4.

[0213] In another embodiment, the invention relates to a method of treating a subject with a hemostatic disorder comprising administering a therapeutically effective amount of at least one chimeric protein comprising a first and a second chain, wherein the first chain comprises at least one clotting factor and at least a portion of an immunoglobulin constant region and the second chain comprises at

2016244273 13 Oct 2016 least a portion of an immunoglobulin constant region without the clotting factor of the first chain, in combination with at least one other clotting factor or agent that promotes hemostasis. Said other clotting factor or agent that promotes hemostasis can be any therapeutic with demonstrated clotting activity. As an example, but not as a limitation, the clotting factor or hemostatic agent can include Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, FactorXII, Factor XIII, prothrombin, or fibrinogen or activated forms of any of the preceding. The clotting factor of hemostatic agent can also include anti-fibrinolytic drugs, e.g., epsilon-amino-caproic acid, tranexamic acid.

7. Methods of Inhibiting Viral Fusion With a Target Cell [0214] The invention also relates to an in vitro method of inhibiting HIV fusion with a mammalian cell comprising combining the mammalian cell with at least one chimeric protein, wherein the chimeric protein comprises a first and a second chain, wherein the first chain comprises at least a portion of an immunoglobulin constant region and an HIV inhibitor and the second chain comprises at least a portion of an immunoglobulin constant region without the HIV inhibitor of the first chain. The mammalian cell can include any cell or cell line susceptible to infection by HIV including but not limited to primary human CD4⁺ T cells or macrophages, MOLT-4 cells, CEM cells, AA5 cells or HeLa cells which express CD4 on the cell surface.

G. Methods of Isolating Chimeric Proteins [0215] Typically, when chimeric proteins of the invention are produced they are contained in a mixture of other molecules such as other proteins or protein fragments. The invention thus provides for methods of isolating any of the chimeric proteins described supra from a mixture containing the chimeric proteins. It has

2016244273 13 Oct 2016 been determined that the chimeric proteins of the invention bind to dye ligands under suitable conditions and that altering those conditions subsequent to binding can disrupt the bond between the dye ligand and the chimeric protein, thereby providing a method of isolating the chimeric protein. In some embodiments the mixture may comprise a monomer-dimer hybrid, a dimer and at least a portion of an immunoglobulin constant region, e.g., an Fc. Thus, in one embodiment, the invention provides a method of isolating a monomer-dimer hybrid. In another embodiment, the invention provides a method of isolating a dimer.

[0216] Accordingly, in one embodiment, the invention provides a method of isolating a monomer-dimer hybrid from a mixture, where the mixture comprises

a) the monomer-dimer hybrid comprising a first and second polypeptide chain, wherein the first chain comprises a biologically active molecule, and at least a portion of an immunoglobulin constant region and wherein the second chain comprises at least a portion of an immunoglobulin constant region without a biologically active molecule or immunoglobulin variable region;

b) a dimer comprising a first and second polypeptide chain, wherein the first and second chains both comprise a biologically active molecule, and at least a portion of an immunoglobulin constant region; and

c) a portion of an immunoglobulin constant region; said method comprising

1) contacting the mixture with a dye ligand linked to a solid support under suitable conditions such that both the monomer-dimer hybrid and the dimer bind to the dye ligand;

2) removing the unbound portion of an immunoglobulin constant region;

2016244273 13 Oct 2016

3) altering the suitable conditions of 1) such that the binding between the monomer-dimer hybrid and the dye ligand linked to the solid support is disrupted;

4) isolating the monomer-dimer hybrid.

In some embodiments, prior to contacting the mixture with a dye ligand, the mixture may be contacted with a chromatographic substance such as protein A sepharose or the like. The mixture is eluted from the chromatographic substance using an appropriate elution buffer (e.g. a low pH buffer) and the eluate containing the mixture is then contacted with the dye ligand.

[0217] Suitable conditions for contacting the mixture with the dye ligand may include a buffer to maintain the mixture at an appropriate pH. An appropriate pH may include a pH of from, 3-10, 4-9, 5-8. In one embodiment, the appropriate pH is 8.0. Any buffering agent known in the art may be used so long as it maintains the pH in the appropriate range, e.g., tris, HEPES, PIPES, MOPS. Suitable conditions may also include a wash buffer to elute unbound species from the dye ligand. The wash buffer may be any buffer which does not disrupt binding of a bound species.

For example, the wash buffer can be the same buffer used in the contacting step.

[0218] Once the chimeric protein is bound to the dye ligand, the chimeric protein is isolated by altering the suitable conditions. Altering the suitable conditions may include the addition of a salt to the buffer. Any salt may be used, e.g., NaCl, KCi. The salt should be added at a concentration that is high enough to disrupt the binding between the dye ligand and the desired species, e.g., a monomer-dimer hybrid.

ο

CM

2016244273 13 Oct [0219] In some embodiments where the mixture is comprised of an Fc, a monomer-dimer hybrid, and a dimer, it has been found that the Fc does not bind to the dye ligand and thus elutes with the flow through. The dimer binds more tightly to the dye ligand than the monomer-dimer hybrid. Thus a higher concentration of salt is required to disrupt the bond (e.g. elute) between the dimer and the dye ligand compared to the salt concentration required to disrupt the bond between the dye ligand and the monomer-dimer hybrid.

[0220] in some embodiments NaCi may be used to isolate the monomerdimer hybrid from the mixture. In some embodiments the appropriate concentration of salt which disrupts the bond between the dye ligand and the monomer-dimer hybrid is from 200-700 mM, 300-600 mM, 4004500 mM. In one embodiment, the concentration of NaCI required to disrupt the binding between the dye ligand the monomer-dimer hybrid is 400 mM.

[0221] NaCi may also be used to isolate the dimer from the mixture. Typically, the monomer-dimer hybrid is isolated from the mixture before the dimer. The dimer is isolated by adding an appropriate concentration of salt to the buffer, thereby disrupting the binding between the dye ligand and the dimer. In some embodiments the appropriate concentration of salt which disrupts the bond between the dye ligand and the dimer is from 800 mM to 2 M, 900 mM to1.5 M, 950 mM to 1.2 M. In one specific embodiment, 1 M NaCi is used to disrupt the binding between the dye ligand and the dimer. , [0222] The dye ligand may be a bio-mimetic. A bio-mimetic is a humanmade substance, device, or system that imitates nature. Thus in some embodiments the dye ligand imitates a molecule’s naturally occurring ligand. The

2016244273 13 Oct 2016 dye ligand may be chosen from Mimetic Red 1™, Mimetic Red 2™, Mimetic Orange 1 ™, Mimetic Orange 2™, Mimetic Orange 3™, Mimetic Yellow 1 ™, Mimetic Yellow 2™, Mimetic Green 1™, Mimetic Blue 1 ™, and Mimetic Blue 2™ (Prometic Biosciences (USA) Inc., Wayne, NJ). In one specific embodiment, the dye ligand is Mimetic Red 2™ (Prometic Biosciences (USA) Inc., Wayne, NJ). In certain embodiments the dye ligand is linked to a solid support, e.g., from Mimetic Red 1A6XL™, Mimetic Red 2 A6XL™, Mimetic Orange 1 A6XL™, Mimetic Orange 2 A6XL™, Mimetic Orange 3 A6XL™, Mimetic Yellow 1 A6XL™, Mimetic Yellow 2 A6XL™, Mimetic Green 1 A6XL™, Mimetic Blue 1 A6XL™, and Mimetic Blue 2 A6XL™ (Prometic Biosciences (USA) Inc., Wayne, NJ).

[0223] The dye ligand may be linked to a solid support. The solid support may be any solid support known in the art (see, e.g., www.seperationsNOW.com). Examples of solid supports may include a bead, a gel, a membrane, a nanoparticle, or a microsphere. The solid support may comprise any material which can be linked to a dye ligand (e.g. agarose, polystyrene, sepharose, sephadex). Solid supports may comprise any synthetic organic polymer such as polyacrylic, vinyl polymers, acrylate, polymethacrylate, and polyacrylamide. Solid supports may also comprise a carbohydrate polymer, e.g., agarose, cellulose, or dextran. Solid supports may comprise inorganic oxides, such as silica, zirconia, titania, ceria, alumina, magnesia (i.e., magnesium oxide), or calcium oxide. Solid supports may also comprise combinations of some of the above-mentioned supports including, but not limited to, dextran-acrylamide.

2016244273 13 Oct 2016

Examples

Example 1: Molecular Weight Affects FcRn Mediated Trancytosis [0224] Chimeric proteins comprised of various proteins of interest and IgG Fc were recombinantly produced (Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed., Cold Spring Harbor Laboratory Press, (1989)) or in the case of contactin-Fc, ΜΑΒ-β-gal, (a complex of a monoclonal antibody bound to β-gal) (Biodesign International, Saco, ME) and MAB-GH (a complex of monoclonal antibody and growth hormone)(Research Diagnostics, Inc. Flanders, NJ) were purchased commercially. Briefly, the genes encoding the protein of interest were . cloned by PCR, and then sub-cloned into an Fc fusion expression plasmid. The plasmids were transfected into DG44 CHO cells and stable transfectants were selected and amplified with methotrexate. The chimeric protein homodimers were purified over a protein A column. The proteins tested included interferon a, growth hormone, erythropoietin, follicle stimulating hormone, Factor IX, beta-galactosidase,

I contactin, and Factor VIII. Linking the proteins to immunoglobulin portions, including the FcRn receptor binding partner, or using commercially available whole antibody (including the FcRn binding region)-antigen complexes permitted the investigation of transcytosis as a function of molecular weight (see U.S. Patent No. 6,030,613). The chimeric proteins were administered to rats orally and serum levels were measured 2-4 hours post administration using an ELISA for recombinantly produced chimeric proteins and both a western blot and ELISA for commercially obtained antibody complexes and chimeric proteins. Additionally, all of the commercially obtained proteins or complexes as well as Factor Vlll-Fc, Factor !X-Fc and Epo-Fc controls were iodinated using IODO beads (Pierce, Pittsburgh, PA). The results indicated

2016244273 13 Oct 2016 serum levels of Fc and monoclonal antibody chimeric proteins orally administered to rats are directly related to the size of the protein. The apparent cutoff point for orally administered Fc chimeric proteins is between 200-285 kD. (Table 2).

TABLE 2

Protein	Size (kD)	Transcytosis
IFNa-Fc	92	++++
GH-Fc	96	+++
Epo-Fc	120	+++
FSH-Fc	170	+++
MAB:GH	172-194	+++
FIX-Fc	200	+
MAB:pGal	285-420	-
Contactin-Fc	300	-
FVII!A-Fc	380	-

Example 2: Cloning of pcDNA 3.1 -Flag-Fc [0225] The sequence for the FLAG peptide (Asp-Tyr-Lys-Asp-Asp-Asp-AspLys), a common affinity tag used to identify or purify proteins, was cloned into the pcDNA 3.1-Fc plasmid, which contains the mouse IgK signal sequence followed by the Fc fragment of human IgG 1 (amino acids 221-447, EU numbering). The construct was created by overlapping PCR using the following primers:

FlagFc-F1: 5’-GCTGGCTAGCCACCATGGA -3’(SEQ ID NO:41)

FlagFc-RT. 5’- CTTGTCATCGTCGTCCTTGTAGTCGTCA

CCAGTGGAACCTGGAAC -3’(SEQ ID NO:42)

FlagFc-F2: 5’- GACTACAAGG ACGACGATGA CAAGGACAAA ACTCACACAT GCCCACCGTG CCCAGCTCCG GAACTCC -3’ (SEQ ID NO:43)

FlagFc-R2: 5’- TAGTGGATCCTCATTTACCCG -3’ (SEQ ID NO:44) [0226] The pcDNA 3.1-Fc template was then added to two separate PCR reactions containing 50 pmol each of the primer pairs FlagFc-F1/R1 or FlagFc-F2/R2 in a 50 μΙ reaction using Pfu Ultra DNA polymerase (Stratagene, CA) according to

2016244273 13 Oct 2016 manufacturer’s standard protocol in a MJ Thermocycler using the following cycles: 95°C 2 minutes; 30 cycles of (95°C 30 seconds, 52°C 30 seconds, 72°C 45 seconds), followed by 72°C for 10 minutes. The products of these two reactions were then mixed in another PCR reaction (2 pi each) with 50 pmol of FlagFc-F1 and FlagFc-R2 primers in a 50 μΙ reaction using Pfu Ultra DNA polymerase (Stratagene, CA) according to manufacturer’s standard protocol in a MJ Thermocycler using the following cycles: 95°C 2 minutes; 30 cycles of (95°C 30 seconds, 52°C 30 seconds, 72°C 45 seconds), followed by 72°C for 10 minutes. The resulting fragment was gel purified, digested and inserted into the pcDNA 3.1-Fc plasmid Nhel-Bam HI. The resulting plasmid contains contains the mouse Igx signal sequence producing the FlagFc protein.

Example 3: Cloning of -Factor Vll-Fc construct [0227] The coding sequence for Factor VII, was obtained by RT-PCR from human fetal liver RNA (Clontech, Palo Alto, CA). The cloned region is comprised of the cDNA sequence from bp 36 to bp 1430 terminating just before the stop codon. A Sbfl site was introduced on the N-terminus. A BspEI site was introduced on the Cterminus. The construct was cloned by PCR using the primers:

Downstream: 5’ GCTACCTGCAGGCCACCATGGTCTCCCAGGCCCTCAGG 3’(SEQ ID NO:45)

Upstream : 5’ CAGTTCCGGAGCTGGGCACGGCGGGCACGTGTGAGTTT TGTCGGGAAAT GG 3’ (SEQ ID NO:46) and the following conditions: 95°C for 5 minutes followed by 30 cycles of 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute and 45 seconds, and a final extension cycle of 72°C for 10 minutes.

2016244273 13 Oct 2016 [0228] The fragment was digested Sbfl - BspE I and inserted into pED.dC-Fc a plasmid encoding for the Fc fragment of an IgG 1.

Example 4: Cloning of Factor IX-Fc construct [0229] The human Factor IX coding sequence, including the prepropeptide sequence, was obtained by RT-PCR amplification from adult human liver RNA using the following primers:

natFIX-F: 5’-TTACTGCAGAAGGTTATGCAGCGCGTGAACATG- 3’(SEQ ID NO:47)

F9-R: 5’-TTTTTCGAATTCAGTGAGCTTTGTTTTTTCCTTAATCC- 3’(SEQ ID NO:48) [0230] 20 ng of adult human liver RNA (Clontech, Palo Alto, CA) and 25 pmol each primer were added to a RT-PCR reaction using the Superscript.™ OneStep RT-PCR with PLATINUM® Taq system (Invitrogen, Carlsbad, CA) according to manufacturers protocol. Reaction was carried out in a MJ Thermocycier using the following cycles: 50°C 30 minutes; 94°C 2 minutes; 35 cycles of (94°C 30 seconds, 58°C 30 seconds, 72°C 1 minute), and a final 72°C 10 minutes. The fragment was gel purified using Qiagen Gel Extraction Kit (Qiagen, Valencia, CA), and digested with Pstl-EcoRI, gel purified, and cloned into the corresponding digest of the pED.dC.XFc plasmid.

Example 5: Cloning of PACE construct [0231] The coding sequence for human PACE (paired basic amino acid cleaving enzyme), an endoprotease, was obtained by RT-PCR. The following primers were used:

PACE-F1: 5’-GGTAAGCTTGCCATGGAGCTGAGGCCCTGGTTGC -3’(SEQ ID NO:49) ο

CM

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PACE-R1: 5’-GTTTTCAATCTCTAGGACCCACTCGCC -3’(SEQ ID NO:50)

PACE-F2: 5’-GCCAGGCCACATGACTACTCCGC -3’(SEQ ID N0:51)

PACE-R2: 5’- GGTGAATTCTCACTCAGGCAGGTGTGAGGGCAGC -3’(SEQ ID NO:52) [0232] The PACE-F1 primer adds a Hind 111 site to the 5’ end of the PACE sequence beginning with 3 nucleotides before the start codon, while the PACE-R2 primer adds a stop codon after amino acid 715, which occurs at the end of the extracellular domain of PACE, as well as adding an EcoRI site to the 3’ end of the stop codon. The PACE-R1 and -F2 primers anneal on the 3’ and 5’ sides of an internal BamHI site, respectively. Two RT-PCR reactions were then set up using 25 pmol each of the primer pairs of PACE-F1/R1 or PACE-F2/R2 with 20 ng of adult human liver RNA (Clontech; Palo Alto, CA) in a 50 pi RT-PCR reaction using the Superscript.™ One-Step RT-PCR with PLATINUM® Taq system (Invitrogen, Carlsbad, CA) according to manufacturers protocol. The reaction was carried out in a MJ Thermocycler using the following cycles: 50°C 30 minutes; 94°C 2 minutes; 30 cycles of (94°C 30 seconds, 58°C 30 seconds, 72°C 2 minutes), followed by 72°C 10 minutes. These fragments were each ligated into the vector pGEM T-Easy (Promega, Madison, Wl) and sequenced fully. The F2-R2 fragment was then subcloned into pcDNA6 V5/His (Invitrogen, Carlsbad, CA) using the BamHI/EcoRI sites, and then the F1-R1 fragment was cloned into this construct using the Hindlll/BamHI sites. The final plasmid, pcDNA6-PACE, produces a soluble form of PACE (amino acids 1-715), as the transmembrane region has been deleted. The sequence of PACE in pcDNA6-PACE is essentially as described in Harrison et al. 1998, Seminars in Hematology 35:4.

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Example 6: Cloning of IFNa-Fc eight amino acid linker construct [0233] The human interferon a 2b (hIFNa) coding sequence, including the signal sequence, was obtained by PCR from human genomic DNA using the following primers:

IFNa-Sig-F: 5’-GCTACTGCAGCCACCATGGCCTTGACCTTTGCTTTAC3’(SEQ ID NO:53)

IFNa-EcoR-R: 5’-CGTTGAATTCTTCCTTACTTCTTAAACTTTCTTGC3’(SEQ ID NO:54) [0234] Genomic DNA was prepared from 373MG human astrocytoma cell line, according to standard methods (Sambrook et al. 1989, Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press). Briefly,

I approximately 2 x 10⁵ ceils were pelleted by centrifugation, resuspended in 100 pi phosphate buffered saline pH 7.4, then mixed with an equal volume of lysis buffer (100 mM Tris pH 8.0/ 200 mM NaCl / 2% SDS / 5 mM EDTA). Proteinase K was added to a final concentration of 100 pg/ml, and the sample was digested at 37°C for 4 hours with occasional gentle mixing. The sample was then extracted twice with phenokchioroform, the DNA precipitated by adding sodium acetate pH 7.0 to 100 mM and an equal volume of isopropanol, and pelleted by centrifugation for 10 min at room temperature. The supernatant was removed and the pellet was washed once with cold 70% ethanol and allowed to air dry before resuspending in TE (10 mM Tris pH 8.0 /1 mM EDTA).

[0235] 100 ng of this genomic DNA was then used in a 25 pi PCR reaction with 25 pmol of each primer using Expand High Fidelity System (Boehringer Mannheim, Indianapolis, IN) according to manufacturer’s standard protocol in a MJ Thermocycler using the following cycles: 94°C 2 minutes; 30 cycles of (94°C 30

2016244273 13 Oct 2016 seconds, 50°C 30 seconds, 72°C 45 seconds), and finally 72°C 10 minutes. The expected sized band (-550 bp) was gel purified with a Gel Extraction kit (Qiagen, Valencia, CA), digested with Psti/EcoRI, gel purified again, and cloned into the Pstl/EcoRI site of pED.dC.XFc, which contains an 8 amino acid linker (EFAGAAAV) followed by the Fc region of human lgG1.

Example 7: Cloning of IFNaFc Alinker construct [0236] 1 pg of purified pED.dC.native human IFNaFc DNA, from Example 6, was then used as a template in a 25 pi PCR reaction with 25 pmol of each primer IFNa-Sig-F and the following primer:

hIFNaNoLinkFc-R: 5’CAGTTCCGGAGCTGGGCACGGCGGG

CACGTGTGAGTTTTGTCTTCCTTACTTCTTAAACTTTTTGCAAGTTTG- 3’(SEQ ID NO:55) [0237] The PCR reaction was carried out using Expand High Fidelity System (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer’s standard protocol in a RapidCycler thermocycler (Idaho Technology, Salt Lake City, UT), denaturing at 94°C for 2 minutes followed by 18 cycles of 95°C for 15 seconds, 55°C for 0 seconds, and 72°C for 1 minute with a slope of 6, followed by 72°C extension for 10 minutes. A PCR product of the correct size (-525 bp) was gel purified using a Gel Extraction kit (Qiagen; Valencia, CA), digested with the Psti and BspEI restriction enzymes, gel purified, and subcloned into the corresponding sites of a modified pED.dC.XFc, where amino acids 231-233 of the Fc region were altered using the degeneracy of the genetic code to incorporate a BspEI site while maintaining the wild type amino acid sequence.

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Example 8: Cloning of IFNaFc GS15 linker construct [0238] A new backbone vector was created using the Fc found in the Alinker construct (containing BspEI and Rsrll sites in the 5’ end using the degeneracy of the genetic code to maintain the amino acid sequence), using this DNA as a template for a PCR reaction with the following primers:

5’ B2xGGGGS: 5’ gtcaggatccggcggtggagggagcgacaaaactcacacgtgccc 3’(SEQ ID NO:56)

3’ GGGGS: 5’ tgacgcggccgctcatttacccggagacaggg 3’(SEQ ID NO:57) [0239] A PCR reaction was carried out with 25 pmol of each primer using Pfu Turbo enzyme (Stratagene, La Jolla, CA) according to manufacturer’s standard protocol in a MJ Thermocycler using the following method: 95°C 2 minutes; 30 cycles of (95°C 30 seconds, 54°C 30 seconds, 72°C 2 minutes), 72°C 10 minutes. The expected sized band (~730 bp) was gel purified with a Gel Extraction kit (Qiagen, Valencia CA), digested BamHI/Notl; gel purified again, and cloned into the BamHI/Notl digested vector of pcDNA6 ID, a version of pcDNA6 with the IRES sequence and dhfr gene inserted into Notl/Xbai site.

[0240] 500 ng of purified pED.dC.native human IFNaFc DNA was then used as a template in a 25 pi PCR reaction with the following primers:

5’ IFNa for GGGGS: 5’ ccgctagcctgcaggccaccatggccttgacc 3’(SEQ ID NO:58)

3’ IFNa for GGGGS: 5’ ccggatccgccgccaccttccttactacgtaaac 3’(SEQ ID NO:59) [0241] A PCR reaction was carried out with 25 pmol of each primer using Expand High Fidelity System (Boehringer Mannheim, Indianapolis, IN) according to manufacturer’s standard protocol in a MJ Thermocycler using the following cycles;

2016244273 13 Oct 2016

95°C 2 minutes; 14 cycles of (94°C 30 seconds, 48°C 30 seconds, 72°C 1 minute),

72°C 10 minutes. The expected sized band (-600 bp) was gel purified with a Gel

Extraction kit (Qiagen, Valencia CA), digested Nhel/BamHI, gel purified again, and cloned into the Nhel/BamHI site of the pcDNA6 ID/Fc vector, above, to create an

IFNa Fc fusion with a 10 amino acid Giy/Ser linker (2xGGGGS), pcDNA6 ID/IFNaGSIO-Fc.

[0242] A PCR reaction was then performed using 500 ng of this pcDNA6 ID/IFNa-GS10-Fc with the following primers

5’ B3XGGGGS:5’(SEQ ID NO:60) gtcaggatccggtggaggcgggtccggcggtggagggagcgacaaaactcacacgtgccc 3’(SEQ ID NO:61) fccIv-R: 5’ atagaagcctttgaccaggc 3’(SEQ ID NO:62) [0243] A PCR reaction was carried out with 25 pmol of each primer using

Expand High Fidelity System (Boehringer Mannheim, Indianapolis, IN) according to manufacturer’s standard protocol in a MJ Thermocycler using the following cycles: 95°C 2 minutes; 14 cycles of (94°C 30 seconds, 48°C 30 seconds, 72°C 1 minute), 72°C 10 minutes. The expected sized band (504 bp) was gel purified with a Gel Extraction kit (Qiagen, Valencia CA), digested BamHI/BspEl, the 68 bp band was gel purified, and cloned into the BamHI/BspEl site of the pcDNA6 ID/IFNa-GS10-Fc vector, above, to create an IFNa Fc fusion with a 15 amino acid Gly/Ser linker (3xGGGGS), pcDNA6 ID/IFNa-GS15-Fc.

Example 9: Cloning of a Basic Peptide Construct [0244] The hinge region of the human IgG 1 Fc fragment from amino acid 221-229 (EU numbering) was replaced with a basic peptide (CCB).

2016244273 13 Oct 2016

Four overlapping oligos were used (IDT, Coralville, IA):

1. CCB-Fc Sense 1:

5’ GCC GGC GAA TTC GGT GGT GAG TAC CAG GCC CTG AAG AAG AAG GTG GCC CAG CTG AAG GCC AAG AAC CAG GCC CTG AAG AAG AAG 3’(SEQ ID NO:63)

2. CCB-Fc Sense 2:

5’ GTG GCC CAG CTG AAG CAC AAG GGC GGC GGC CCC GCC CCA GAG CTC CTG GGC GGA CCG A 3’(SEQ ID NO:64)

3. CCB-Fc Anti-Sense 1:

5’ CGG TCC GCC CAG GAG CTC TGG GGC GGG GCC GCC GCC CTT GTG CTT CAG CTG GGC CAC CTT CTT CTT CAG GGC CTG GTT CTT G 3’(SEQ ID NO:65)

4. CCB-Fc Anti-Sense 2:

5’ GCC TTC AGC TGG GCC ACC TTC TTC TTC AGG GCC TGG TAC TCA CCA CCG AAT TCG CCG GCA 3’(SEQ ID NO:66) [0245] The oligos were reconstituted to a concentration of 50 μΜ with dH₂0.

pi of each oligo were annealed to each other by combining in a thin walled PCR tube with 2.2 pi of restriction buffer #2 (i.e. final concentration of 10 mM Tris HCI pH 7.9, 10 mM MgCI₂, 50 mM Na Cl, 1 mM dithiothreitoi) (New England Biolabs,

Beverly, MA) and heated to 95°C for 30 seconds and then allowed to anneal by cooling slowly for 2 hours to 25°C. 5 pmol of the now annealed oligos were ligated into a pGEM T-Easy vector as directed in the kit manual. (Promega, Madison Wl). The ligation mixture was added to 50 pi of DH5a competent E. co//'cells (invitrogen, Carlsbad, CA) on ice for 2 minutes, incubated at 37°C for 5 minutes, incubated on ice for 2 minutes, and then plated on LB+100 pg/L ampicillin agar plates and placed at 37°C for 14 hours. Individual bacterial colonies were picked and placed in 5 ml of LB+100 pg/L ampicillin and allowed to grow for 14 hours. The tubes were spun

2016244273 13 Oct 2016 down at 2000xg, 4°C for 15 minutes and the vector DNA was isolated using Qiagen miniprep kit (Qiagen, Valencia, CA) as indicated in the kit manual. 2 pg of DNA was digested with NgoM IV-Rsr-il. The fragment was gel purified by the Qiaquick method as instructed in the kit manual (Qiagen, Valencia, CA) and ligated to pED.dcEpoFc with NgoM IV/Rsr il. The ligation was transformed into DH5a competent E. coli cells and the DNA prepared as described for the pGEM T-Easy vector.

Example 10: Cloning of the erythropoietin-acidic peptide Fc construct [0246] The hinge region ofthe human lgG1 Fc fragment in EPO-Fc from amino acid 221-229 (EU numbering) was replaced with an acidic peptide (CCA). Four overlapping oligos were used (IDT, Coralville, IA):

1. Epo-CCA-Fc Sense 1:

5’ CCG GTG ACA GGG AAT TCG GTG GTG AGT ACC AGG CCC TGG AGA AGG AGG TGG CCC AGC TGG AG 3’(SEQ ID NO:67)

2. Epo-CCA-Fc Sense 2:

5’ GCC GAG AAC CAG GCC CTG GAG AAG GAG GTG GCC CAG CTG GAG CAC GAG GGT GGT GGT CCC GCT CCA GAG CTG CTG GGC GGA CA 3’(SEQ IDNO:68)

3. Epo-CCA-Fc Anti-Sense 1:

5’ GTC CGC CCA GCA GCT CTG GAG CGG GAC CAC CAC CCT CGT GCT CCA GCT GGG CCA C 3’(SEQ ID NO:69)

4. Epo-CCA-Fc Anti-Sense 2:

5’ CTC CTT CTC CAG GGC CTG GTT CTC GGC CTC CAG CTG GGC CAC CTC CTT CTC CAG GGC CTG GTA CTC ACC ACC GAA TTC CCT GTC ACC GGA 3’(SEQ ID NO:70)

2016244273 13 Oct 2016 [0247] The oligos were reconstituted to a concentration of 50 μΜ with dHaO. 5 μΙ of each oligo were annealed to each other by combining in a thin walled PCR tube with 2.2 μΙ of restriction buffer No. 2 (New England Biolabs, Beverly, MA) and heated to 95°C for 30 seconds and then allowed to cool slowly for 2 hours to 25°C.

pmol of the now annealed oligos were ligated into a pGEM T-Easy vector as directed in the kit manual. (Promega, Madison, Wl). The ligation mixture was added to 50 μΙ of DH5a competent E. coli cells (Invitrogen, Carlsbad, CA) on ice for 2 minutes, incubated at 37°C 5 minutes, incubated on ice for 2 minutes, and then plated on LB+100 pg/L ampicillin agar plates and placed at 37°C for 14 hours. Individual bacterial colonies were picked and placed in 5 ml of LB+100 pg/L ampicillin and allowed to grow for 14 hours. The tubes were spun down at 2000xg, 4°C for 15 minutes and the vector DNA was prepared using Qiagen miniprep kit (Qiagen, Valencia, CA) as indicated in the kit manual. 2 pg of DNA was digested with Age l-Rsr-il. The fragment was gel purified by the Qiaquick method as instructed in the kit manual (Qiagen, Valencia, CA) and ligated into pED.Epo Fc.1 Age i-Rsr II. The ligation was transformed into DH5a competent E. coli cells and DNA prepped as described above.

Example 11: Cloning of Cys-Fc construct [0248] Using PCR and standard molecular biology techniques (Sambrook et al. 1989, Molecular Cloning: A Laboratory Manual, 2^nd ed., Cold Spring Harbor Laboratory Press), a mammalian expression construct was generated such that the coding sequence for the human IFNa signal peptide was directly abutted against the coding sequence of Fc beginning at the first cysteine residue (Cys 226, EU Numbering). Upon signal peptidase cleavage and secretion from mammalian cells,

2016244273 13 Oct 2016 an Fc protein with an N-terminal cysteine residue was thus generated. Briefly, the primers

IFNa-Sig-F (IFNa-Sig-F: 5’-GCTACTGCAGCCACCATGGCCTTGACCTT TGCTTTAC-3’)(SEQ ID NO:71) and Cys-Fc-R (5’-CAGTTCCGGAGCTGGGCACGGCGGA

GAGCCCACAGAGCAGCTTG-3’) (SEQ ID NO:72) were used in a PCR reaction to create a fragment linking the IFNa signal sequence with the N terminus of Fc, beginning with Cys 226. 500 ng of pED.dC.native hIFNa Alinker was added to 25 pmol of each primer in a PCR reaction with Expand High Fidelity System (Boehringer Mannheim, Indianapolis, IN) according to manufacturer’s standard protocol. The reaction was carried out in a MJ Thermocycler using the following cycles: 94°C 2 minutes; 30 cycles of (94°C 30 seconds, 50°C 30 seconds, 72°C 45 seconds), and finally 72°C 10 minutes. The expected sized band (—112 bp) was gel purified with a Gel Extraction kit (Qiagen, Valencia CA), digested with the Pstl and BspEI restriction enzymes, gel purified, and subcloned into the corresponding sites pED.dC.native hIFNa Alinker to generate pED.dC.Cys-Fc (Figure 5).

Example 12: Protein Expression and Preparation of Fc-MESNA [0249] The coding sequence for Fc (the constant region of human lgG1) was obtained by PCR amplification from an Fc-containing plasmid using standard conditions and reagents, following the manufacturer’s recommended procedure to subclone the Fc coding sequence Nde\/Sap\. Briefly, the primers 5’- GTGGTCATA TGGGCATTGAAGGCAGAGGCGCCGCTGCGGTCG - 3’(SEQ ID NO:73) and 5’ GGTGGTTGC TCTTCCGCAAAAACCCGGAGACAGGGAGAGACTCTTCTGCG - 3’

2016244273 13 Oct 2016 (SEQ ID NO:74)were used to amplify the Fc sequence from 500 ng of the plasmid pED.dC.Epo-Fc using Expand High Fidelity System (Boehringer Mannheim, Basel Switzerland) in a RapidCylcler thermocycler (Idaho Technology Salt Lake City,

Utah), denaturing at 95°C for 2 minutes followed by 18 cycles of 95°C for 0 sec,

55°C for 0 sec, and 72°C for 1 minute with a slope of 4, followed by 72°C extension for 10 minutes. The PCR product was subcloned into an intermediate cloning vector and sequenced fully, and then subcloned using the Ndel and Sapl sites in the pTWINI vector following standard procedures. Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989, Molecular Cloning: A Laboratory Manual, 2^nd ed.; Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. This plasmid was then transformed into BL21(DE3) pLysS cells using standard methods. Id. A 1 liter culture of cells was grown to an absorbance reading of 0.8 AU at 37°C, induced with 1 mM isopropyl beta-D-1-thiogalactopyranoside, and grown overnight at 25°C. Cells were pelleted by centrifugation, lysed in 20 mM Tris 8.8/1% NP40/0.1 mM phenylmethanesulfonyl fluoride/1 pg/ml Benzonase (Novagen Madison, Wl), and bound to chitin beads (New England Biolabs; Beverly, MA) overnight at 4°C. Beads were then washed with several column volumes of 20 mM Tris 8.5/ 500 mM NaCi/1 mM EDTA, and then stored at -80°C. Purified Fc-MESNA was generated by eluting the protein from the beads in 20 mM Tris 8.5/ 500 mM NaCi /1 mM EDTA / 500 mM 2-mercapto ethane sulfonic acid (MESNA), and the eluate was used directly in the coupling reaction, below.

Example 13: Factor Vll-Fc monomer-dimer hybrid expression and purification [0250] CHO DG-44 cells expressing Factor Vli-Fc were established. CHO DG-44 cells were grown at 37°C, 5% CO₂, in MEM Alpha plus nucleoside and

2016244273 13 Oct 2016 ribonucleosides and supplemented with 5% heat-inactivated fetal bovine serum until transfection.

[0251] DG44 cells were plated in 100 mm tissue culture petri dishes and grown to a confluency of 50%- 60%. A total of 10 pg of DNA was used to transfect one 100 mm dish: 7.5 pg of pED.dC.FVIl-Fc + 1.5 pg pcDNA3/Flag-Fc + 1 pg of pcDNA6-PACE. The cells were transfected as described in the Superfect transfection reagent manual (Qiagen, Valencia, CA). The media was removed from transfection after 48 hours and replaced with MEM Alpha without nucleosides plus 5% dialyzed fetal bovine serum and 10 pg/ml of Blasticidin (Invitrogen, Carlsbad,

CA) and 0.2 mg/ml geneticin (Invitrogen, Carlsbad, CA). After 10 days, the cells were released from the plate with 0.25% trypsin and transferred into T25 tissue culture flasks, and the selection was continued for 10-14 days until the cells began to grow well as stable cell lines were established. Protein expression was subsequently amplified by the addition 25 nM methotrexate.

[0252] Approximately 2 x 10⁷ cells were used to inoculate 300 ml of growth medium in a 1700 cm² roller bottle (Corning, Corning, NY) supplemented with 5 pg/ml of vitamin K₃ (menadione sodium bisulfite) (Sigma, St Louis, MO). The roller bottles were incubated in a 5% CO₂ at 37°C for 72 hours. Then the growth medium was exchanged with 300 ml serum-free production medium (DMEM/F12 with 5 pg/ml bovine insulin and 10 pg/ml Gentamicin) supplemented with 5 pg/L of vitamin K₃.

The production medium (conditioned medium) was collected every day for 10 days and stored at 4°C. Fresh production medium was added to the roller bottles after each collection and the bottles were returned to the incubator. Pooled media was first clarified using a Sartoclean glass fiber filter (3.0 pm + 0.2 pm) (Sartorious Corp.

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Gottingen, Germany) followed by an Acropack 500 filter (0.8 pm + 0.2 pm) (Pall Corp., East Hills, NY). The clarified media was then concentrated approximately 20fold using Peiiicon Biomax tangential flow filtration cassettes (10 kDa MWCO) (Millipore Corp., Billerica, MA).

[0253] Fc chimeras were then captured from the concentrated media by passage over a Protein A Sepharose 4 Fast Flow Column (AP Biotech, Piscataway, NJ). A 5 x 5 cm (100 ml) column was loaded with <5 mg Fc protein per mi column volume at a linear flow rate of 100 cm/hour to achieve a residence time of > 3 minutes. The column was then washed with >5 column volumes of 1X DPBS to remove non-specificaliy bound proteins. The bound proteins were eluted with 100 mM Glycine pH 3.0. Elution fractions containing the protein peak were then neutralized by adding 1 part 1 M Tris-HCL, pH 8 to 10 parts elute fraction.

[0254] To remove FLAG-Fc homodimers (that is, chimeric Fc dimers with FLAG peptide expressed as fusions with both Fc molecules) from the preparation, the Protein A Sepharose 4 Fast Flow pool was passed over a Unosphere S cationexchange column (BioRad Corp., Richmond, CA). Under the operating conditions for the column, the FLAG-Fc monomer-dimer hybrid is uncharged (FLAG-Fc theoretical pl=6.19) and flows through the column while the hFVIi-Fc constructs are positively charged, and thus bind to the column and elute at higher ionic strength. The Protein A Sepharose 4 Fast Flow pool was first dialyzed into 20 mM MES, mM NaCI, pH 6.1. The dialyzed material was then loaded onto a 1.1 x 11 cm (9.9 ml) column at 150 cm/hour. During the wash and elution, the flow rate was increased to 500 cm/hour. The column was washed sequentially with 8 column volumes of 20 mM MES, 20 mM NaCI, pH 6.1 and 8 column volumes of 20 mM ο

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MES, 40 mM NaCI, pH 6.1. The bound protein was eluted with 20 mM MES,

750 mM NaCI, pH 6.1. Elution fractions containing the protein peak were pooled and sterile filtered through a 0.2 pm filter disc prior to storage at -80°C.

[0255] An anti-FLAG MAB affinity column was used to separate chimeric Fc dimers with hFVII fused to both Fc molecules from those with one FLAG peptide and one hFVII fusion. The Unosphere S Eluate pool was diluted 1:1 with 20 mM Tris, mM NaCI, 5 mM CaCI₂, pH 8 and loaded onto a 1.6 x 5 cm M2 anti-FLAG sepharose column (Sigma Corp., St. Louis, MO) at a linear flow rate of 60 cm/hour. Loading was targeted to < 2.5 mg monomer-dimer hybrid /ml column volume. After loading the column was washed with 5 column volumes 20 mM Tris, 50 mM NaCI, mM CaCE, pH 8.0, monomer-dimer hybrids were then eluted with 100 mM Glycine, pH 3.0. Elution fractions containing the protein peak were then neutralized by adding 1 part 1 M Tris-HCI, pH 8 to 10 parts eluate fraction. Pools were stored at

-80°C.

Example 14: Factor IX-Fc homodimer and monomer-dimer hybrid expression and purification [0256] CHO DG-44 cells expressing Factor IX-Fc were established. DG44 cells were plated in 100 mm tissue culture petri dishes and grown to a confluency of 50%- 60%. A total of 10 pg of DNA was used to transfect one 100 mm dish: for the homodimer transfection, 8 pg of pED.dC.Factor IX-Fc + 2 pg of pcDNA6-PACE was used; for the monomer-dimer hybrid transfection, 8 pg of pED.dC.Factor IX-Fc + 1 pg of pcDNA3-FlagFc +1 pg pcDNA6-PACE was used. Thecells were transfected as described in the Superfect transfection reagent manual (Qiagen, Valencia, CA). The media was removed from transfection after 48 hours and replaced with MEM

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Alpha without nucleosides plus 5% dialyzed fetal bovine serum and 10 pg/ml of Biasticidin (Invitrogen, Carlsbad, CA) for both transfections, while the monomerdimer hybrid transfection was also supplemented with 0.2 mg/ml geneticin (Invitrogen, Carlsbad, CA). After 3 days, the cells were released from the plate with 0.25% trypsin and transferred into T25 tissue culture flasks, and the selection was continued for 10-14 days until the cells began to grow well as stable ceil lines were established. Protein expression was subsequently amplified by the addition 10 nM or 100 nM methotrexate for the homodimer or monomer-dimer hybrid, respectively.

[0257] For both cell lines, approximately 2 x 10⁷ cells were used to inoculate 300 ml of growth medium in a 1700 cm² roller bottle (Corning, Corning, NY), supplemented with 5 pg/L of vitamin K3 (menadione sodium bisulfite) (Sigma, St Louis, MO). The roller bottles were incubated in a 5% CO2 at 37°C for approximately 72 hours. The growth medium was exchanged with 300 ml serumfree production medium (DMEM/F12 with 5 μg/ml bovine insulin and 10 pg/ml Gentamicin), supplemented with 5 pg/L of vitamin K3. The production medium (conditioned medium) was collected everyday for 10 days and stored at 4°C. Fresh production medium was added to the roller bottles after each collection and the bottles were returned to the incubator. Prior to chromatography, the medium was clarified using a SuporCap-100 (0.8/0.2 pm) filter (Pall Gelman Sciences, Ann Arbor, Ml). Ail of the following steps were performed at 4°C. The clarified medium was applied to Protein A Sepharose, washed with 5 column volumes of 1X PBS (10 mM phosphate, pH 7.4, 2.7 mM KCI, and 137 mM NaCI), eluted with 0.1 M glycine, pH 2.7 , and then neutralized with 1/10 volume of 1 M Tris-HCI, pH 9.0. The protein was then dialyzed into PBS.

2016244273 13 Oct 2016 [0258] The monomer-dimer hybrid transfection protein sample was subject to further purification, as it contained a mixture of FIX-Fc:FIX-Fc homodimer, FIXFc:Flag-Fc monomer-dimer hybrid, and Flag-Fc:Flag-Fc homodimer. Material was concentrated and applied to a 2.6 cm x 60 cm (318 ml) Superdex 200 Prep Grade column at a flow rate of 4 ml/minute (36 cm/hour) and then eluted with 3 column volumes of 1X PBS. Fractions corresponding to two peaks on the UV detector were collected and analyzed by SDS-PAGE. Fractions from the first peak contained either FIX-Fc:FIX-Fc homodimer or FlX-Fc:FlagFc monomer-dimer hybrid, while the second peak contained FlagFc:FlagFc homodimer. All fractions containing the monomer-dimer hybrid but no FlagFc homodimer were pooled and applied directly to a 1.6 x 5 cm M2 anti-FLAG sepharose column (Sigma Corp., St. Louis, MO) at a linear flow rate of 60 cm/hour. After loading, the column was washed with 5 column volumes PBS. Monomer-dimer hybrids were then eluted with 100 mM Glycine, pH 3.0. Elution fractions containing the protein peak were then neutralized by adding 1/10 volume of 1 M Tris-HCI, and analyzed by reducing and nonreducing SDS-PAGE. Fractions were dialyzed into PBS, concentrated to 1-5 mg/ml, and stored at -80°C.

Example 15: IFNa homodimer and monomer-dimer hybrid expression and purification [0259] CHO DG-44 cells expressing hlFNct were established. DG44 cells were plated in 100 mm tissue culture petri dishes and grown to a confluency of 50%60%. A total of 10 pg of DNA was used to transfect one 100 mm dish: for the homodimer transfection, 10 pg of the hIFNaFc constructs; for the monomer-dimer hybrid transfection, 8 pg of the hIFNaFc constructs + 2 pg of pcDNA3-FlagFc. The

2016244273 13 Oct 2016 cells were transfected as described in the Superfect transfection reagent manual (Qiagen, Valencia, CA). The media was removed from transfection after 48 hours and replaced with MEM Alpha without nucleosides plus 5% dialyzed fetal bovine serum, while the monomer-dimer hybrid transfection was also supplemented with 0.2 mg/ml geneticin (Invitrogen, Carlsbad, CA). After 3 days, the cells were released from the plate with 0.25% trypsin and transferred into T25 tissue culture flasks, and the selection was continued for 10-14 days until the cells began to grow well and stable cell lines were established. Protein expression was subsequently amplified by the addition methotrexate: ranging from 10 to 50 nM.

[0260] For all cell lines, approximately 2 x 10⁷ ceils were used to inoculate 300 ml of growth medium in a 1700 cm² roller bottle (Corning, Corning, NY). The roller bottles were incubated in a 5% CO₂ at 37°C for approximately 72 hours. Then the growth medium was exchanged with 300 ml serum-free production medium (DMEM/F12 with 5 pg/ml bovine insulin and 10 pg/ml Gentamicin). The production medium (conditioned medium) was collected every day for 10 days and stored at 4°C. Fresh production medium was added to the roller bottles after each collection and the bottles were returned to the incubator. Prior to chromatography, the medium was clarified using a SuporCap-100 (0.8/0.2 pm) filter from Pall Gelman Sciences (Ann Arbor, Ml). All of the following steps were performed at 4°C. The clarified medium was applied to Protein A Sepharose, washed with 5 column volumes of 1X PBS (10 mM phosphate, pH 7.4, 2.7 mM KCI, and 137 mM NaCI), eluted with 0.1 M glycine, pH 2.7, and then neutralized with 1/10 volume of 1 M TrisHCI, pH 9.0. The protein was then dialyzed into PBS.

2016244273 13 Oct 2016 [0261] The monomer-dimer hybrid transfection protein samples were then subject to further purification, as it contained a mixture of IFNaFc:IFNaFc homodimer, IFNaFc:FIagFc monomer-dimer hybrid, and FlagFc:FlagFc homodimer (or Alinker or GS15 linker). Material was concentrated and applied to a 2.6 cm x 60 cm (318 ml) Superdex 200 Prep Grade column at a flow rate of 4 ml/min (36 cm/hr) and then eluted with 3 column volumes of 1X PBS. Fractions corresponding to two peaks on the UV detector were collected and analyzed by SDS-PAGE. Fractions from the first peak contained either IFNaFc:IFNaFc homodimer or IFNaFc:FlagFc monomer-dimer hybrid, while the second peak contained FlagFc:FlagFc homodimer. All fractions containing the monomer-dimer hybrid, but no FlagFc homodimer, were pooled and applied directly to a 1.6 x 5 cm M2 anti-FLAG sepharose column (Sigma Corp., St. Louis, MO) at a linear flow rate of 60 cm/hour. After loading the column was washed with 5 column volumes PBS monomer-dimer hybrids were then eluted with 100 mM Glycine, pH 3.0. Elution fractions containing the protein peak were then neutralized by adding 1/10 volume of 1 M Tris-HCI, and analyzed by reducing and nonreducing SDS-PAGE. Fractions were dialyzed into PBS, concentrated to 15 mg/ml, and stored at -80°C.

Example 16: Coiled coil protein expression and purification [0262] The plasmids, pED.dC Epo-CCA-Fc and pED.dC CCB-Fc will be transfected either alone or together at a 1:1 ratio into CHO DG44 cells. The cells will be transfected as described in the Superfect transfection reagent manual (Qiagen, Valencia, CA). The media will be removed after 48 hours and replaced with MEM Alpha w/o nucleosides plus 5% dialyzed fetal bovine serum. Purification will be done by affinity chromatography over a protein A column according to methods

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2016244273 13 Oct 2016 known in the art. Alternatively, purification can be achieved using size exclusion chromatography.

Example 17: Cys-Fc expression and purification [0263] CHO DG-44 cells expressing Cys-Fc were established. The pED.dC.Cys-Fc expression plasmid, which contains the mouse dihydrofolate reductase (dhfr) gene, was transfected into CHO DG44 (dhfr deficient) cells using Superfect reagent (Qiagen; Valencia, CA) according to manufacturer’s protocol, followed by selection for stable transfectants in aMEM (without nucleosides) tissue culture media supplemented with 5% dialyzed FBS and penicillin/stroptomycin antibiotics (Invitrogen; Carlsbad, CA) for 10 days. The resulting pool of stably transfected cells were then amplified with 50 nM methotrexate to increase expression. Approximately 2 x 10⁷ cells were used to inoculate 300 mi of growth medium in a 1700 cm² roller bottle (Corning, Corning, NY). The roller bottles were incubated in a 5% CO₂ at 37°C for approximately 72 hours. The growth medium was exchanged with 300 ml serum-free production medium (DMEM/F12 with 5 pg/ml bovine insulin and 10 pg/ml Gentamicin). The production medium (conditioned medium) was collected every day for 10 days and stored at 4°C. Fresh production medium was added to the roller bottles after each collection and the bottles were returned to the incubator. Prior to chromatography, the medium was clarified using a SuporCap-100 (0.8/0.2 pm) filter from Pall Gelman Sciences (Ann Arbor, Ml). All of the following steps were performed at 4°C. The clarified medium was applied to Protein A Sepharose, washed with 5 column volumes of 1X PBS (10 mM phosphate, pH 7.4, 2.7 mM KCi, and 137 mM NaCl), eluted with 0.1 M glycine, pH 2.7, and then

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2016244273 13 Oct 2016 neutralized with 1/10 volume of 1 M Tris-HCI, pH 9.0. Protein was dialyzed into PBS and used directly in conjugation reactions.

Example 18: Coupling of T20-thioesters to Cys-Fc [0264] Cys-Fc (4 mg, 3.2 mg/ml final concentration) and either T20-thioester or T20-PEG-thioester (2 mg, approximately 5 molar equivalents) were incubated for 16 hours at room temperature in 0.1 M Tris 8/10 mM MESNA. Analysis by SDSPAGE (Tris-Gly gel) using reducing sample buffer indicated the presence of a new band approximately 5 kDa larger than the Fc control (>40-50% conversion to the conjugate). Previous N-terminal sequencing of Cys-Fc and unreacted Cys-Fc indicated that the signal peptide is incorrectly processed in a fraction of the molecules, leaving a mixture of (Cys)-Fc, which will react through native ligation with peptide-thioesters, and (Val)-(Gly)-(Cys)-Fc, which will not. As the reaction conditions are insufficient to disrupt the dimerization of the Cys-Fc molecules, this reaction generated a mixture of T20-Cys-Fc:T20-Cys-Fc homodimers, T20-Cys-Fc: Fc monomer-dimer hybrids, and Cys-Fc:Cys-Fc Fc-dimers. This protein was purified using size exclusion chromatography as indicated above to separate the three species. The result was confirmed by SDS-PAGE analysis under nonreducing conditions.

Example 19: Antiviral assay for IFNa activity [0265] Antiviral activity (lU/ml) of IFNa fusion proteins was determined using a CPE (cytopathic effect) assay. A549 cells were plated in a 96 well tissue culture plate in growth media (RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine) for 2 hours at 37°C, 5% CO₂. IFNa standards and IFNa fusion proteins were diluted in growth media and added to cells in triplicate for 20

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2016244273 13 Oct 2016 hours at 37°C, 5% CO₂. Following incubation, all media was removed from wells, encephalomyocarditis virus (EMC) virus was diluted in growth media and added (3000 pfu/well) to each well with the exception of control wells. Plates were incubated at 37°C, 5% CO₂for 28 hours. Living cells were fixed with 10% cold trichloroacetic acid (TCA) and then stained with Sulforhodamine B (SRB) according to published protocols (Rubinstein et al. 1990, J. Natl. Cancer Inst. 82, 1113). The SRB dye was solubilized with 10 mM Tris pH 10.5 and read on a spectrophotometer at 490 nm. Samples were analyzed by comparing activities to a known standard curve World Health Organization IFNcc 2b International Standard ranging from 5 to 0.011 lU/ml. The results are presented below in Table 3 and Figure 6 and demonstrate increased antiviral activity of monomer-dimer hybrids.

TABLE 3: INTERFERON ANTIVIRAL ASSAY

HOMODIMER V. MONOMER-DIMER HYBRID

Protein	Antiviral Activity (lU/nmol)	Std dev
IFNaFc 8aa linker homodimer	0.45 x10^s	0.29 x10^s
IFNaFc 8aa linker:FlagFc monomer-dimer hybrid	4.5 x 10^s	1.2x10^
IFNaFc Δ linker homodimer	0.22 x10^s	0.07 x 10^s
IFNaFc Δ delta linker: FlagFc monomer-dimer hybrid	2.4x10^b	0.0005x10^&
IFNaFc GS15 linker homodimer	2.3x10^b	1.0x10^b
IFNaFc GS15 linker monomer-dimer hybrid	5.3x10^b	0.15x10^b

Example 20: FVIIa Clotting Activity Analysis [0266] The StaClot FVIIa-rTF assay kit was purchased from Diagnostica Stago (Parsippany, NJ) and modified as described in Johannessen et al. 2000,

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Blood Coagulation and Fibrinolysis 11 :S159. A standard curve was preformed with the FViia World Health Organization standard 89/688. The assay was used to compare clotting activity of monomer-dimer hybrids compared to homodimers. The results showed the monomer-dimer hybrid had four times the clotting activity compared to the homodimer (Figure 7).

Example 21: FVHa-Fc Oral dosing in day 10 rats [0267] 25 gram day 9 newborn Sprague Dawley rats were purchased from Charles River (Wilmington, MA) and allowed to acclimate for 24 hours. The rats were dosed orally with FVIIaFc homodimer, monomer-dimer hybrid or a 50:50 mix of the two. A volume of 200 pi of a FVIIaFc solution for a dose of 1 mg/kg was administered. The solution was composed of a Tris-HCI buffer pH 7.4 with 5 mg/ml soybean trypsin inhibitor. The rats were euthanized with CO₂ at several time points, and 200 pi of blood was drawn by cardiac puncture. Plasma was obtained by the addition of a 3.8% sodium citrate solution and centrifugation at room temperature at a speed of 1268xg. The plasma samples were either assayed fresh or frozen at 20°C. Orally dosed monomer-dimer hybrid resulted in significantly higher maximum (C_max) serum concentrations compared to homodimeric Factor VII (Figure 8).

Example 22: Factor IX-Fc Oral dosing of neonatal rats [0268] Ten-day old neonatal Sprague-Dawley rats were dosed p.o. with 200 pi of FIX-Fc homodimer or FiX-Fc: FlagFc monomer-dimer hybrid at approximately equimolar doses of 10 nmol/kg in 0.1 M sodium phosphate buffer, pH 6.5 containing 5 mg/ml soybean trypsin inhibitor and 0.9% NaCI. At 1,2, 4, 8, 24, 48, and 72 hours post injection, animals were euthanized with CO₂, blood was drawn via cardiac puncture and plasma was obtained by the addition of a 3.8%

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2016244273 13 Oct 2016 sodium citrate solution and centrifugation at room temperature at a speed of 1268xg. Samples were then sedimented by centrifugation, serum collected and frozen at -20°C until analysis of the fusion proteins by ELISA.

Example 23: Factor IX-Fc ELISA [0269] A 96-well Immulon 4HBX ELISA plate (Thermo LabSystems, Vantaa, Finland) was coated with 100 μΙ/well of goat anti-Factor IX IgG (Affinity Biologicals, Ancaster, Canada) diluted 1:100 in 50 mM carbonate buffer, pH 9.6. The plates were incubated at ambient temperature for 2 hours or overnight at 4°C sealed with plastic film. The wells were washed 4 times with PBST, 300 μΙ/well using the TECAN plate washer. The wells were blocked with PBST + 6% BSA, 200 μΙ/well, and incubated 90 minutes at ambient temperature. The wells were washed 4 times with PBST, 300 μΙ/well using the TECAN plate washer. Standards and blood samples from rats described in Example 18 were added to the wells, (100 μΙ/well), and incubated 90 minutes at ambient temperature. Samples and standards were diluted in HBET buffer (HBET: 5.95 g HEPES, 1.46 g NaCl, 0.93 g Na₂EDTA, 2.5 g Bovine Serum Albumin, 0.25 ml Tween-20, bring up to 250 ml with dH₂0, adjust pH to 7.2). Standard curve range was from 200 ng/m! to 0.78 ng/ml with 2 fold dilutions in between. Wells were washed 4 times with PBST, 300 μΐ/well using the TECAN plate washer. 100 μΙ/well of conjugated goat anti-human IgG-Fc-HARP antibody (Pierce, Rockford, IL) diluted in HBET 1:25,000 was added to each well. The plates were incubated 90 minutes at ambient temperature. The wells were washed 4 times with PBST, 300 μί/well using the TECAN plate washer. The plates were developed with 100 μΙ/well of tetramethylbenzidine peroxidase substrate (TMB) (Pierce, Rockford, IL) was added according to the manufacturer’s instructions. The plates

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2016244273 13 Oct were incubated 5 minutes at ambient temperature in the dark or until color developed. The reaction was stopped with 100 pl/well of 2 M sulfuric acid. Absorbance was read at 450 nm on SpectraMax plusplate reader (Molecular Devices, Sunnyvale, CA). Analysis of blood drawn at 4 hours indicated more than a fold difference in serum concentration between Factor IX-Fc monomer-dimer hybrids compared to Factor IX Fc homodimers (Figure 9). The results indicated Factor IX-Fc monomer-dimer hybrid levels were consistently higher than Factor IXFc homodimers (Figure 10).

Example 24: Cloning of Epo-Fc [0270] The mature Epo coding region was obtained by PCR amplification from a plasmid encoding the mature erythropoietin coding sequence, originally obtained by RT-PCR from Hep G2 mRNA, and primers hepoxba-F and hepoeco-R, indicated below. Primer hepoxba-F contains an Xbal site, while primer hepoeco-R contains an EcoRI site. PCR was carried out in the Idaho Technology RapidCycler using Vent polymerase, denaturing at 95°C for 15 seconds, followed by 28 cycles with a slope of 6.0 of 95°C for 0 seconds, 55°C for 0 seconds, and 72°C for 1 minute 20 seconds, followed by 3 minute extension at 72°C. An approximately 514 bp product was gel purified, digested with Xbal and EcoRI, gel purified again and directionally subcioned into an Xbal/EcoRI-digested, gel purified pED.dC.XFc vector, mentioned above. This construct was named pED.dC.EpoFc.

[0271] The Epo sequence, containing both the endogenous signal peptide and the mature sequence, was obtained by PCR amplification using an adult kidney QUICK-clone cDNA preparation as the template and primers Epo+Pep-Sbf-F and Epo+Pep-Sbf-R, described below. The primer Epo+Pep-Sbf-F contains an Sbfl site

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2016244273 13 Oct 2016 upstream of the start codon, while the primer Epo+Pep-Sbf-R anneals downstream of the endogenous Sbfl site in the Epo sequence. The PCR reaction was carried out in the PTC-200 MJ Thermocycler using Expand polymerase, denaturing at 94°C for 2 minutes, followed by 32 cycles of 94°C for 30 seconds, 57°C for 30 seconds, and 72°C for 45 seconds, followed by a 10 minute extension at 72°C. An approximately 603 bp product was gel isolated and subcloned into the pGEM-T Easy vector. The correct coding sequence was excised by Sbf\ digestion, gel purified, and cloned into the Psfl-digested, shrimp alkaline phosphatase (SAP)-treated, gel purified pED.dC.EpoFc plasmid. The plasmid with the insert in the correct orientation was initially determined by Kpn] digestion. A Xmn\ and PvuW digestion of this construct was compared with pED.dC.EpoFc and confirmed to be in the correct orientation. The sequence was determined and the construct was named pED.dC.natEpoFc.

PCR Primers:

hepoxba-F (EPO-F): 5’-AATCTAGAGCCCCACCACGCCTCATCTGTGAC~3’(SEQ ID NO:75) hepoeco-R (EPO-R) 5’-TTGAATTCTCTGTCCCCTGTCCTGCAGGCC-3’(SEQ ID NO:76)

Epo+Pep-Sbf-F: 5’-GTACCTGCAGGCGGAGATGGGGGTGCA-3’(SEQ ID

NO:77)

Epo+Pep-Sbf-R: 5’-CCTGGTCATCTGTCCCCTGTCC-3’(SEQ ID NO:78)

Example 25: Cloning of Epo-Fc [0272] An alternative method of cloning EPO-Fc is described herein.

Primers were first designed to amplify the full length Epo coding sequence, including the native signal sequence, as follows:

Epo-F: 5’-GTCCAACCTG CAGGAAGCTTG CCGCCACCAT GGGAGTGCAC GAATGTCCTG CCTGG- 3’(SEQ ID NO:79)

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Epo-R: 5’-GCCGAATTCA GTTTTGTCGA CCGCAGCGG CGCCGGCGAA CTCTCTGTCC CCTGTTCTGC AGGCCTCC- 3’(SEQ ID NO:80) [0273] The forward primer incorporates an Sbfl and HindiII site upstream of a Kozak sequence, while the reverse primer removes the internal Sbfl site, and adds an 8 amino acid linker to the 3’ end of the coding sequence (EFAGAAAV) (SEQ ID NO:81) as well as Sail and EcoRI restriction sites. The Epo coding sequence was then amplified from a kidney cDNA library (BD Biosciences Clontech, Palo Alto, CA) using 25 pmol of these primers in a 25 pi PCR reaction using Expand High Fidelity System (Boehringer Mannheim, Indianapolis, IN) according to manufacturer’s standard protocol in a MJ Thermocycler using the following cycles: 94°C 2 minutes; 30 cycles of (94°C 30 seconds, 58°C 30 seconds, 72°C 45 seconds), followed by 72°C for 10 minutes. The expected sized band (641 bp) was gel purified with a Gel Extraction kit (Qiagen, Valencia, CA) and ligated into the intermediate cloning vector pGEM T-Easy (Promega, Madison, Wl). DNA was transformed into DH5a cells (Invitrogen, Carlsbad, CA) and miniprep cultures grown and purified with a Plasmid Miniprep Kit (Qiagen, Valencia, CA) both according to manufacturer’s standard protocols. Once the sequence was confirmed, this insert was digested out with Sbfl/EcoRI restriction enzymes, gel purified, and cloned into the Pstl/EcoRI sites of the mammalian expression vector pED.dC in a similar manner.

[0274] Primers were designed to amplify the coding sequence for the constant region of human IgG1 (the Fc region, EU numbering 221-447) as follows:

Fc-F: 5’-GCTGCGGTCG ACAAAAC.TCA CACATGCCCA CCGTGCCCAG CTCCGGAACT CCTGGGCGGA CCGTCAGTC- 3’(SEQ ID NO:82)

Fc-R 5’-ATTGGAATTC TCATTTACCC GGAGACAGGG AGAGGC- 3’(SEQ ID NO:83)

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The forward primer incorporates a Sail site at the linker-Fc junction, as well as introducing BspEI and Rsrll sites into the Fc region without affecting the coding sequence, while the reverse primer adds an EcoRI site after the stop codon. The Fc coding sequence was then amplified from a leukocyte cDNA library (BD Biosciences Clontech, Palo Alto, CA) using 25 pmol of these primers in a 25 pi PCR reaction using Expand High Fidelity System (Boehringer Mannheim, Indianapolis, IN) according to manufacturer’s standard protocol in a MJ Thermocycier using the following cycles: 94°C 2 minutes; 30 cycles of (94°C 30 seconds, 58°C 30 seconds, 72°C 45 seconds), followed by 72°C for 10 minutes. The expected sized band (696 bp) was gel purified with a Gel Extraction kit (Qiagen, Valencia, CA) and ligated into the intermediate cloning vector pGEM T-Easy (Promega, Madison, Wl). DNA was transformed into DH5a cells (Invitrogen, Carlsbad, CA) and miniprep cultures grown and purified with a Plasmid Miniprep Kit (Qiagen, Valencia, CA), both according to manufacturer’s standard protocols. Once the sequence was confirmed, this insert was digested out with Sal/EcoRI restriction enzymes, gel purified, and cloned into the Sall/EcoRI sites of the plasmid pED.dC.Epo (above) in a similar manner, to generate the mammalian expression plasmid pED.dC.EpoFc. In another experiment this plasmid was also digested with Rsrll/Xmal, and the corresponding fragment from pSYN-Fc-002, which contains the Asn 297 Ala mutation (EU numbering) was cloned in to create pED.dC.EPO-Fc N297A (pSYN-EPO-004). Expression in mammalian cells was as described in Example 26. The amino acid sequence of EpoFc with an eight amino acid linker is provided in figure 2j. During the process of this alternative cloning method, although the exact EpoFc amino acid sequence was preserved (figure 2J), a number of non-coding changes were made at the nucleotide

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2016244273 13 Oct 2016 level (figure 3J). These are G6A (G at nucleotide 6 changed to A) (eliminate possible secondary structure in primer), G567A (removes endogenous Sbfl site from Epo), A582G (removes EcoRI site from linker), A636T and T639G (adds unique BspEI site to Fc), and G651C (adds unique Rsrli site to Fc). The nucleotide sequence in figure 3J is from the construct made in Example 25, which incorporates these differences from the sequence of the construct from Example 24.

Example 26: EPO-Fc Homodimer And Monomer-dimer Hybrid Expression And

Purification [0275] DG44 cells were plated in 100 mm tissue culture petri dishes and grown to a confluency of 50%-60%. A total of 10 pg of DNA was used to transfect one 100 mm dish: for the homodimer transfection,10 pg of pED.dC.EPO-Fc; for the monomer-dimer hybrid transfection, 8 pg of pED.dC.EPO-Fc + 2 pg of pcDNA3FlagFc. The constructs used were cloned as described in Example 24. The cloning method described in Example 25 could also be used to obtain constructs for use in this example. The cells were transfected as described in the Superfect transfection reagent manual (Qiagen, Valencia, CA). Alternatively, pED.dC.EPO-Fc was cotransfected with pSYN-Fc-016 to make an untagged monomer. The media was removed from transfection after 48 hours and replaced with MEM Alpha without nucleosides plus 5% dialyzed fetal bovine serum for both transfections, while the monomer-dimer hybrid transfection was also supplemented with 0.2 mg/ml geneticin (Invitrogen, Carlsbad, CA). After 3 days, the cells were released from the plate with 0.25% trypsin and transferred into T25 tissue culture flasks, and the selection was continued for 10-14 days until the cells began to grow well as stable cell lines were no

2016244273 13 Oct 2016 established. Protein expression was subsequently amplified by the addition methotrexate.

[0276] For both cell lines, approximately 2 x 10⁷ cells were used to inoculate 300 ml of growth medium in a 1700 cm² roller bottle (Corning, Corning, NY). The roller bottles were incubated in a 5% CO₂ at 37°C for approximately 72 hours. The growth medium was exchanged with 300 ml serum-free production medium (DMEM/F12 with 5 pg/ml bovine insulin and 10 pg/ml Gentamicin). The production medium (conditioned medium) was collected every day for 10 days and stored at 4°C. Fresh production medium was added to the roller bottles after each collection and the bottles were returned to the incubator. Prior to chromatography, the medium was clarified using a SuporCap-100 (0.8/0.2 pm) filter from Pall Gelman Sciences (Ann Arbor, Ml). All ofthe following steps were performed at4°C. The clarified medium was applied to Protein A Sepharose, washed with 5 column volumes of 1X PBS (10 mM phosphate, pH 7.4, 2.7 mM KCI, and 137 mM NaCI), eluted with 0.1 M glycine, pH 2.7, and then neutralized with 1/10 volume of 1 M TrisHCI, pH 9.0. Protein was then dialyzed into PBS.

[0277] The monomer-dimer hybrid transfection protein sample was subject to further purification, as it contained a mixture of EPO-Fc:EPO-Fc homodimer, EPO-Fc:Flag-Fc monomer-dimer hybrid, and Flag-Fc:Flag-Fc homodimer. Material was concentrated and applied to a 2.6 cm x 60 cm (318 mi) Superdex 200 Prep Grade column at a flow rate of 4 ml/min (36 cm/hour) and then eluted with 3 column volumes of 1X PBS. Fractions corresponding to two peaks on the UV detector were collected and analyzed by SDS-PAGE. Fractions from the first peak contained either EPO-Fc:EPO-Fc homodimer or EPO-Fc:FlagFc monomer-dimer hybrid, while

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2016244273 13 Oct the second peak contained FiagFc:FlagFc homodimer. Ail fractions containing the monomer-dimer hybrid but no FlagFc homodimer were pooled and applied directly to a 1.6 x 5 cm M2 anti-FLAG sepharose column (Sigma Corp.) at a linear flow rate of 60 cm/hour. After loading the column was washed with 5 column volumes PBS. Monomer-dimer hybrids were then eluted with 100 mM Glycine, pH 3.0. Elution fractions containing the protein peak were then neutralized by adding 1/10 volume of 1 M Tris-HCI, and analyzed by reducing and nonreducing SDS-PAGE. Fractions were dialyzed into PBS, concentrated to 1-5 mg/ml, and stored at -80°C.

[0278] Alternatively, fractions from first peak of the Superdex 200 were analyzed by SDS-PAGE, and only fractions containing a majority of EpoFc monomer-dimer hybrid, with a minority of EpoFc homodimer, were pooled. This pool, enriched for the monomer-dimer hybrid, was then reapplied to a Superdex 200 column, and fractions containing only EpoFc monomer-dimer hybrid were then pooled, dialyzed and stored as purified protein. Note that this alternate purification method could be used to purify non-tagged monomer-dimer hybrids as well.

Example 27: Administration of EpoFc Dimer and Monomer-Dimer Hybrid With an Eight Amino Acid Linker to Cynomolgus Monkeys [0279] For pulmonary administration, aerosols of either EpoFc dimer or EpoFc monomer-dimer hybrid proteins (both with the 8 amino acid linker) in PBS, pH 7.4 were created with the Aeroneb Pro™ (AeroGen, Mountain View, CA) nebulizer, in-line with a Bird Mark 7A respirator, and administered to anesthetized naive cynomolgus monkeys through endotracheal tubes (approximating normal tidal breathing). Both proteins were also administered to naive cynomolgus monkeys by intravenous injection. Samples were taken at various time points, and the amount of

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Epo-containing protein in the resulting plasma was quantitated using the Quantikine IVD Human Epo Immunoassay (R&D Systems, Minneapolis, MN). Pharmacokinetic parameters were calculated using the software WinNonLin. Table 4 presents the bioavailability results of cynomolgus monkeys treated with EpoFc monomer-dimer hybrid or EpoFc dimer.

TABLE 4: ADMINISTRATION OF EPOFC MONOMER-DIMER

HYBRID AND EPOFC DIMER TO MONKEYS

Protein	Monkey #	Route	Approx. Deposited Dose¹ (pg/kg)	Cmax (ng/ml)	Cmax (fmol/ml)	t-l/2 (hr)	t-i/2 avg (hr)
EpoFc monomer- dimer hybrid	CO6181	pulm	20	72.3	1014	23.6	25.2
CO6214	pulm	20	50.1	703	23.5
C07300	pulm	20	120	1684	36.2
CO7332	pulm	20	100	1403	17.5
CO7285	IV	25	749	10508	21.3	22.6
CO7288	IV	25	566	7941	23
CO7343	IV	25	551	1014	23.5
EpoFc dimer	DD026	pulm	15	10.7	120	11.5	22.1
DD062	pulm	15	21.8	244	27.3
DD046	pulm	15	6.4	72	21.8
DD015	pulm	15	12.8	143	20.9
DD038	pulm	35	27	302	29
F4921	IV	150	3701	41454	15.1	14.6
96Z002	IV	150	3680	41219	15.3
1261CQ	IV	150	2726	30533	23.6
127-107	IV	150	4230	47379	15.0
118-22	IV	150	4500	50403	8.7
126-60	IV	150	3531	39550	9.8

- - ---- Based on 15% deposition fraction of nebulized dose as determined by gamma scintigraphy [0280] The percent bioavailability (F) was calculated for the pulmonary doses using the following equation:

F= (AUC pulmonary / Dose pulmonary) / (AUC IV / Dose IV) * 100

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TABLE 5: CALCULATION OF PERCENT BIOAVAILABiLITY FOR EPOFC

MONOMER-DIMER HYBRID V. DIMER AFTER PULMONARY ADMINISTRATION

TO NAIVE CYNOMOLGUS MONKEYS

Protein	Monkey #	Approx. Dose¹ (deposited)	AUC ng*hr/mL	Bioavailability² (F)	Average Bioavailabiity
EpoFc monomer- dimer hybrid	CO6181	20 pg/kg	3810	25.2%	34.9%
CO6214	20 pg/kg	3072	20.3%
C07300	20 pg/kg	9525	63.0%
CO7332	20 pg/kg	4708	31.1%
EpoFc dimer	DD026	15 pg/kg	361	5.1%	10.0%
DD062	15 pg/kg	1392	19.6%
DD046	15 pg/kg	267	3.8%
DD015	15 pg/kg	647	9.1%
DD038	35 pg/kg	2062	12.4%

¹ Based on 15% deposition fraction of nebulized dose as determined by gamma scintigraphy

Mean AUC for IV EpoFc monomer-dimer hybrid = 18,913 ng-hr/mL (n=3 monkeys), dosed at 25 pg/kg. Mean AUC for IV EpoFc dimer = 70, 967 ng-hr/mL (n=6 monkeys), dosed at 150 pg/kg [0281] The pharmacokinetics of EpoFc with an 8 amino acid linker administered to cynomolgus monkeys is presented in figure 11. The figure compares the EpoFc dimer with the EpoFc monomer-dimer hybrid in monkeys after administration of a single pulmonary dose. Based on a molar comparison significantly higher serum levels were obtained in monkeys treated with the monomer-dimer hybrid compared to the dimer.

Example 28: Subcutaneous Administration of EPOFc Monomer-dimer Hybrid [0282] To compare serum concentrations of known erythropoietin agents with EPOFc monomer-dimer hybrids, both EPOFc monomer-dimer hybrid and Aranesp® (darbepoetin alfa), which is not a chimeric fusion protein, were administered subcutaneously to different monkeys and the serum concentration of both was measured overtime.

[0283] Cynomolgus monkeys (n = 3 per group) were injected subcutaneously with 0.025 mg/kg EpoFc monomer-dimer hybrid. Blood samples

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2016244273 13 Oct 2016 were collected predose and at times up to 144 hours post dose. Serum was prepared from the blood and stored frozen until analysis by ELISA (Human Epo Quantikine Immunoassay) (R & D Systems, Minneapolis, MN). Pharmacokinetic parameters were determined using WinNonLina® software (Pharsight, Mountainview, CA).

[0284] The results indicated the serum concentrations of both EPOFc monomer-dimer hybrid and Aranesp® (darbepoetin alfa) were equivalent overtime, even though the administered molar dose of Aranesp® (darbepoetin alfa) was slightly larger (Table 6) (figure 12).

TABLE 6

	Route	Dose (pg/kg)	Dose (nmol/kg)	Cmax (ng/mL)	AUC (ng*hr«mL'¹)	Tl/2 (hr)	% Bioavaildbility (F)
EpoFc Monomer- dimer hybrid	Subcutaneous	25	0.3	133 ± 34	10,745 + 3,144	26 + 5	57 ±17
Aranesp®	Subcutaneous	20	0.54	83+11	5390 + 747	22 + 2	53 ±8

Example 29: Intravenous Administration of EPOFc Monomer-dimer Hybrid [0285] To compare serum concentrations of known erythropoietin agents with EPOFc monomer-dimer hybrids, EPOFc monomer-dimer hybrid, Aranesp® (darbepoetin alfa), and Epogen® (epoetin alfa), neither of which is a chimeric fusion protein, were administered intravenously to different monkeys and the serum concentration of both was measured overtime.

[0286] Cynomolgus monkeys (n = 3 per group) were injected intravenously with 0.025 mg/kg EpoFc monomer-dimer hybrid. Blood samples were collected predose and at times up to 144 hours post dose. Serum was prepared from the

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2016244273 13 Oct 2016 blood and stored frozen until analysis by ELISA (Human Epo Quantikine Immunoassay) (R & D Systems, Minneapolis, MN). Pharmacokinetic parameters were determined using WinNonLina software (Pharsight, Mountainview, CA).

[0287] The results indicated the serum concentration versus time (AUC) of EPOFc monomer-dimer hybrid was greater than the concentrations of either

Epogen® (epoetin alfa) or Aranesp® (darbepoetin alfa), even though the monkeys received larger molar doses of both Epogen® (epoetin alfa) and Aranesp® (darbepoetin alfa) (Table 7) (Figure 13).

TABLE 7

	Route	Dose (pg/kg)	Dose (nmol/kg)	Cmax (ng/mL)	AUC (ng»hr*mL'¹)	T-l/2 (hr)
EpoFc Monomer- dimer hybrid	Intravenous	25	0.3	622 + 110	18,913 + 3,022	23 ± 1
Aranesp®	Intravenous	20	0.54	521+8	10,219 ± 298	20 ± 1
Epogen	Intravenous	20	0.66	514 + 172	3936 + 636	6.3 + 0.6

Example 30: Alternative Purification of EpoFc Monomer-dimer Hybrid [0288] Yet another alternative for purifying EPO-Fc is described herein. A mixture containing Fc, EpoFc monomer-dimer hybrid, and EpoFc dimer was applied to a Protein A Sepharose column (Amersham, Uppsala, Sweden). The mixture was eluted according to the manufacturer’s instructions. The Protein A Sepharose eluate, containing the mixture was buffer exchanged into 50 mM Tris-CI (pH 8.0). The protein mixture was loaded onto an 8 mL Mimetic Red 2 XL column (ProMetic Life Sciences, Inc., Wayne, NJ) that had been equilibrated in 50 mM Tris-CI (pH 8.0). The column was then washed with 50 mM Tris-CI (pH 8.0); 50 mM NaCl. This

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2016244273 13 Oct 2016 step removed the majority of the Fc. EpoFc monomer-dimer hybrid was specifically eluted from the column with 50 mM Tris-CI (pH 8.0); 400 mM NaCI. EpoFc dimer can be eluted and the column regenerated with 5 column volumes of 1 M NaOH. Eluted fractions from the column were analyzed by SDS-PAGE (Figure 14).

Example 31: Cloning of Igx signal sequence - Fc construct for making untagged Fc alone.

[0289] The coding sequence for the constant region of lgG1 (EU # 221-447; the Fc region) was obtained by PCR amplification from a leukocyte cDNA library (Clontech, CA) using the following primers:

rcFc-F 5’- GCTGCGGTCGACAAAACTCACACATGCCCACCGTGCCCAGCTCC

GGAACTCCTGGGCGGACCGTCAGTC -3’ (SEQ ID NO: 84) rcFc-R 5’-ATTGGAATTCTCATTTACCCGGAGACAGGGAGAGGC -3’ (SEQ ID NO: 85) [0290] The forward primer adds three amino acids (AAV) and a Sail cloning site before the beginning of the Fc region, and also incorporates a BspEI restriction site at amino acids 231-233 and an Rsril restriction site at amino acids 236-238 using the degeneracy of the genetic code to preserve the correct amino acid sequence (EU numbering). The reverse primer adds an EcoRI cloning site after the stop codon of the Fc. A 25 pi PCR reaction was carried out with 25 pmol of each primer using Expand High Fidelity System (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer’s standard protocol in a MJ Thermocycler using the following cycles: 94°C 2 minutes; 30 cycles of (94°C 30 seconds, 58°C 30 seconds, 72°C 45 seconds), 72°C 10 minutes. The expected sized band (-696 bp) was gel

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2016244273 13 Oct 2016 purified with a Gel Extraction kit (Qiagen, Valencia CA), and cloned into pGEM TEasy (Promega, Madison, Wl) to produce an intermediate plasmid pSYN-Fc-001 (pGEM T-Easy/Fc).

[0291] The mouse Igx signal sequence was added to the Fc CDS using the following primers:

rc-lgx sig seq-F: 5’-TTTAAGCTTGCCGCCACCATGGAGACAGACACACTCC

TGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACAAAACT

CACACATGCCCACCG -3’ (SEQ ID NO: 86)

Fc-noXma-GS-R: 5’- GGTCAGCTCATCGCGGGATGGG -3’ (SEQ ID NO: 87)

Fc-noXma-GS-F: 5’- CCCATCCCGCGATGAGCTGACC -3’ (SEQ ID NO: 88) [0292] The rc-lgx signal sequence-F primer adds a Hindi 11 restriction site to the 5’end of the molecule, followed by a Kozak sequence (GCCGCCACC) (SEQ ID NO: 89) followed by the signal sequence from the mouse Igx light chain, directly abutted to the beginning of the Fc sequence (EU# 221). The Fc-noXma-GS-F and R primers remove the internal Xmal site from the Fc coding sequence, using the degeneracy of the genetic code to preserve the correct amino acid sequence. Two 25 pi PCR reactions were carried out with 25 pmol of either rc-lgx signal sequence-F and Fc-noXma-GS-R or Fc-noXma-GS-F and rcFc-R using Expand High Fidelity System (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer’s standard protocol in a MJ Thermocycler. The first reaction was carried out with 500 ng of leukocyte cDNA library (BD Biosciences Clontech, Palo Alto, CA) as a template using the following cycles: 94°C 2 minutes; 30 cycles of (94°C 30 seconds, 55°C 30 seconds, 72°C 45 seconds), 72°C 10 minutes. The second reaction was

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2016244273 13 Oct 2016 carried out with 500 ng of pSYN-Fc-001 as a template (above) using the following cycles: 94°C 2 minutes; 16 cycles of (94°C 30 seconds, 58°C 30 seconds, 72°C 45 seconds), 72°C 10 minutes. The expected sized bands (~495 and 299 bp, respectively) were gel purified with a Gel Extraction kit (Qiagen, Valencia CA), then combined in a PCR reaction with 25 pmol of rc-lgx signal sequence-F and rcFc-R primers and run as before, annealing at 58°C and continuing for 16 cycles. The expected sized band (-772 bp) was gel purified with a Gel Extraction kit (Qiagen, Valencia CA) and cloned into pGEM T-Easy (Promega, Madison, Wl) to produce an intermediate plasmid pSYN-Fc-007 (pGEM T-Easy/lgK sig seq-Fc). The entire igK signal sequence-Fc cassette was then subcloned using the Hindill and EcoRI sites into either the pEE6.4 (Lonza, Slough, UK) or pcDNA3.1 (Invitrogen, Carlsbad, CA) mammalian expression vector, depending on the system to be used, to generate pSYN-Fc-009 (pEE6.4/lgK sig seq-Fc) and pSYN-Fc-015 (pcDNA3/lgK sig seq-Fc).

Example 32: Cloning of Igx signal sequence - Fc N297A construct for making untagged Fc N297A alone.

[0293] In order to mutate Asn 297 (EU numbering) of the Fc to an Ala residue, the following primers were used:

N297A-F 5’- GAGCAGTACGCTAGCACGTACCG -3’ (SEQ ID NO: 90)

N297A-R 5’- GGTACGTGCTAGCGTACTGCTCC -3’ (SEQ ID NO: 91) [0294] Two PCR reactions were carried out with 25 pmol of either rc-lgK signal sequence-F and N297A-R or N297A-F and rcFc-R using Expand High Fidelity System (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer’s standard protocol in a MJ Thermocycler. Both reactions were carried out using 500

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2016244273 13 Oct 2016 ng of pSYN-Fc-007 as a template using the following cycles: 94°C 2 minutes; 16 cycles of (94°C 30 seconds, 48°C 30 seconds, 72°C 45 seconds), 72°C 10 minutes. The expected sized bands (-319 and 475 bp, respectively) were gel purified with a Gel Extraction kit (Qiagen, Valencia CA), then combined in a PCR reaction with 25 pmol of rc-lgx signal sequence-F and rcFc-R primers and run as before, annealing at 58°C and continuing for 16 cycles. The expected sized band (-772 bp) was gel purified with a Ge! Extraction kit (Qiagen, Valencia CA) and cloned into pGEM TEasy (Promega, Madison, Wl) to produce an intermediate plasmid pSYN-Fc-008 (pGEM T-Easy/lgr sig seq-Fc N297A). The entire Igx signal sequence-Fc alone cassette was then subcloned using the Hindlll and EcoRI sites into either the pEE6.4 (Lonza, Slough, UK) or pcDNA3.1 (Invitrogen, Carlsbad, CA) mammalian expression vector, depending on the system to be used, to generate pSYN-Fc-010 (pEE6.4/lgK sig seq-Fc N297A) and pSYN-Fc-016 (pcDNA3/lgK sig seq-Fc N297A).

[0295] These same N297A primers were also used with rcFc-F and rcFc-R primers and pSYN-Fc-001 as a template in a PCR reaction followed by subcloning as indicated above to generate pSYN-Fc-002 (pGEM T Easy/Fc N297A).

Example 33:Cloning of EpoFc and Fc into single plasmid for double gene vectors for making EpoFc wildtype or N297A monomer-dimer hybrids, and expression.

[0296] An alternative to transfecting the EpoFc and Fc constructs on separate plasmids is to clone them into a single plasmid, also called a double gene vector, such as used in the Lonza Biologies (Slough, UK) system. The Rsril/EcoRI fragment from pSYN-Fc-002 was subcloned into the corresponding sites in pEE12.4 (Lonza Biologies, Slough, UK) according to standard procedures to generate pSYNFc-006 (pEE12.4/Fc N297A fragment). The pSYN-EPO-004 plasmid was used as a no

2016244273 13 Oct 2016 template for a PCR reaction using Epo-F primer from Example 25 and the following primer:

EpoRsr-R: 5’- CTGACGGTCCGCCCAGGAGTTCCG

GAGCTGGGCACGGTGGGCATG TGTGAGTTTTGTCGACCGCAGCGG -3’ (SEQ ID NO: 91) [0297] A PCR reaction was carried out using Expand High Fidelity System (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer’s standard protocol in a MJ Thermocycler as indicated above, for 16 cycles with 55°C annealing temperature. The expected sized band (-689 bp) was gel purified with a Gel Extraction kit (Qiagen, Valencia CA) and cloned into pSYN-Fc-006 using the Hindlll/Rsrll restriction sites, to generate pSYN-EPO-005 (pEE12.4/EpoFc N297A). The double gene vector for the EpoFc N297A monomer-dimer hybrid was then constructed by cloning the Notl/BamHI fragment from pSYN-Fc-010 into the corresponding sites in pSYN-EPO-005 to generate pSYN-EPO-008 (pEE12.46.4/EpoFc N297A/FC N297A).

[0298] The wild type construct was also made by subcloning the wild type Fc sequence from pSYN-Fc-001 into pSYN-EPO-005 using the Rsril and EcoRI sites, to generate pSYN-EPO-006 (pEE12.4/EpoFc). The double gene vector for the EpoFc monomer-dimer hybrid was then constructed by cloning the Notl/BamHI fragment from pSYN-Fc-009 into the corresponding sites in pSYN-EPO-006 to generate pSYN-EPO-007 (pEE1,2-4-6.4/EpoFc /Fc).

[0299] Each plasmid was transfected into CHOK1SV cells and positive clones identified and adpated to serum-free suspension, as indicated in the Lonza

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Biologies Manual for Standard Operating procedures (Lonza Biologies, Slough, UK), and purified as indicated for the other monomer-dimer constructs.

Example 34: Cloning of human IFNgFc, IFNP-Fc N297A with eight amino acid linkers and lgK-Fc-6His constructs [0300] 10 ng of a human genomic DNA library from Clontech (BD Biosciences Clontech, Palo Alto, CA) was used as a template to isolate human IFNp with its native signal sequence using the following primers:

IFNP-F H3/Sbfl:

5’- CTAGCCTGCAGGAAGCTTGCCGCCACCATGACCA

ACAAGTGTCTCCTC -3’ (SEQ ID NO: 92)

IFNp-R (EFAG) Sal:

5’TTTGTCGACCGCAGCGGCGCCGGCGAACTCGTTTCGG

AGGTAACCTGTAAG -3’ (SEQ ID NO: 93) [0301] The reverse primer was also used to create an eight amino acid linker sequence (EFAGAAAV) (SEQ ID NO: 94) on the 3’ end of the human IFNp sequence. The PCR reaction was carried out using the Expand High Fidelity System (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer’s standard protocol in a Rapid Cycler thermocycler (Idaho Technology, Salt Lake City, UT). A PCR product of the correct size (-607 bp) was gel purified using a Gel Extraction kit (Qiagen; Valencia, CA), cloned into TA cloning vector (Promega, Madison, Wl) and sequenced. This construct was named pSYN-IFNp-002. pSYNIFNp-002 was digested with Sbfl and Sail and cloned into pSP72 (Promega) at Pstl and Sail sites to give pSYN-IFNp-005.

[0302] Purified pSYN-Fc-001 (0.6 pg) was digested with Sail and EcoRI and cloned into the corresponding sites of pSYN-IFNp-005 to create the plasmid pSYN-IFNp-006 which contains human IFNp linked to human Fc through an eight

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2016244273 13 Oct 2016 amino acid linker sequence. pSYN-IFNp-006 was then digested with Sbfl and EcoRI and the full-length IFNp-Fc sequence cloned into the Pstl and EcoRI sites of pEDdC.sig to create plasmid pSYN-IFNp-008.

[0303] pSYN-Fc-002 containing the human Fc DNA with a single amino acid change from asparagine to alanine at position 297 (N297A; EU numbering) was digested with BspEI and Xmal to isolate a DNA fragment of -365 bp containing the N297A mutation. This DNA fragment was cloned into the corresponding sites in pSYN-IFNp-008 to create plasmid pSYN-IFNp-009 that contains the IFNp-Fc sequence with an eight amino acid linker and an N297A mutation in Fc in the expression vector, pED.dC.

[0304] Cloning of Igx signal sequence-Fc N297A - 6His. The following primers were used to add a 6xHis tag to the C terminus ofthe Fc N297A coding sequence:

Fc GS-F: 5’- GGCAAGCTTGCCGCCACCATGGAGACAGACACACTCC -3' (SEQ ID NO: 95)

Fc.6His-R: 5’- TCAGTGGTGATGGTGATGATGTTTACCCGGAGACAGGGAG -3’ (SEQ ID NO: 96)

Fc.6His-F: 5’- GGTAAACATCATCACCATCACCACTGAGAATTCC

AATATCACTAGTGAATTCG -3’ (SEQ ID NO: 97) .

Sp6+T-R: 5’- GCTATTTAGGTGACACTATAGAATACTCAAGC -3’ (SEQ ID NO: 98) [0305] Two PCR reactions were carried out with 50 pmol of either Fc GS-F and Fc.6His-R or Fc.6His-F and Sp6+T-R using the Expand High Fidelity System (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer’s standard protocol in a MJ Thermocycler. Both reactions were carried out using 500 ng of

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2016244273 13 Oct pSYN-Fc-008 as a template in a 50 μΙ reaction, using standard cycling conditions. The expected sized bands (-780 and 138 bp, respectively) were gel purified with a Gel Extraction kit (Qiagen, Valencia CA), then combined in a 50 μΐ PCR reaction with 50 pmol of Fc GS-F and Sp6+T-R primers and run as before, using standard cycling conditions. The expected sized band (-891 bp) was gel purified with a Gel Extraction kit (Qiagen, Valencia CA) and cloned into pcDNA6 V5-His B using the HindllI and EcoRI sites to generate pSYN-Fc-014 (pcDNA6/lgK sig seq-Fc N297A-6 His).

Example 35: Expression and purification of IFN8Fc, IFN8-Fc N297A homodimer and 1FN8-Fc N297A monomer-dimer hybrid [0306] CHO DG44 cells were plated in 100 mm tissue culture dishes and grown to a confiuency of 50%-60%. A total of 10 pg of DNA was used to transfect a single 100 mm dish. For the homodimer transfection, 10 pg of the ρδΥΝ-ΡΝβ-ΟΟδ or pSYN-IFNp-009 construct was used; for the monomer-dimer hybrid transfection, 8 pg of the pSYN-lFNp-009 + 2 pg of pSYN-Fc-014 construct was used. The cells were transfected using Superfect transfection reagents (Qiagen, Valencia, CA) according to the manufacturer’s instructions. 48 to 72 hours post-transfection, growth medium was removed and cells were released from the plates with 0.25% trypsin and transferred to T75 tissue culture flasks in selection medium (MEM Alpha without nucleosides plus 5% dialyzed fetal bovine serum). The selection medium for the monomer-dimer hybrid transfection was supplemented with 5 pg/ml Blasticidin (Invitrogen, Carlsbad, CA). Selection was continued for 10-14 days until the cells began to grow well and stable cell lines were established. Protein expression was subsequently amplified by the addition methotrexate: ranging from 10 to 50 nM.

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2016244273 13 Oct 2016 [0307] For all cell lines, approximately 2 x 10⁷ cells were used to inoculate 300 ml of growth medium in a 1700 cm² roller bottle (Corning, Corning, NY). The roller bottles were incubated in a 5% CO₂ incubator at 37°C for approximately 72 hours. The growth medium was then exchanged with 300 ml serum-free production medium (DMEM/F12 with 5 pg/ml human insulin). The production medium (conditioned medium) was collected every day for 10 days and stored at 4°C. Fresh production medium was added to the roller bottles after each collection and the bottles were returned to the incubator. Prior to chromatography, the medium was clarified using a SuporCap-100 (0.8/0.2 pm) filter from Pall Gelman Sciences (Ann Arbor, Ml). All of the following steps were performed at 4°C. The clarified medium was applied to Protein A Sepharose, washed with 5 column volumes of 1X PBS (10 mM phosphate, pH 7.4, 2.7 mM KCI, and 137 mM NaCI), eluted with 0.1 M glycine, pH 2.7, and then neutralized with 1/10 volume of 1 M Tris-HCI pH 8.0, 5 M NaCI. The homodimer proteins were further purified over a Superdex 200 Prep Grade sizing column run and eluted in 50 mM sodium phosphate pH 7.5, 500 mM NaCI, 10% glycerol.

[0308] The monomer-dimer hybrid protein was subject to further purification since it contained a mixture of IFNpFc. N297A:IFNpFc N297A homodimer, IFNpFc N297A: Fc N297A His monomer-dimer hybrid, and Fc N297A His: Fc N297A His homodimer. Material was applied to a Nickel chelating column in 50 mM sodium phosphate pH 7.5, 500 mM NaCI. After loading, the column was washed with 50 mM imidazole in 50 mM sodium phosphate pH 7.5, 500 mM NaCI and protein was eluted with a gradient of 50 - 500 mM imidazole in 50 mM sodium phosphate pH 7.5, 500 mM NaCI. Fractions corresponding to elution peaks on a UV detector were

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2016244273 13 Oct 2016 collected and analyzed by SDS-PAGE. Fractions from the first peak contained IFNpFc N297A; Fc N297A His monomer-dimer hybrid, while the second peak contained Fc N297A His: Fc N297A His homodimer. All fractions containing the monomer-dimer hybrid, but no Fc homodimer, were pooled and applied directly to a Superdex 200 Prep Grade sizing column, run and eluted in 50 mM sodium phosphate pH 7.5, 500 mM NaCI, 10% glycerol. Fractions containing IFNp-Fc N297A:Fc N297A His monomer-dimer hybrids were pooled and stored at -80°C.

Example 36: Antiviral assay for IFN8 activity [0309] Antiviral activity (lU/ml) of IFNp fusion proteins was determined using a CPE (cytopathic effect) assay. A549 cells were plated in a 96 well tissue culture plate in growth media (RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine) for 2 hours at 37°C, 5% CO₂. IFNp standards and IFNp fusion proteins were diluted in growth media and added to cells in triplicate for 20 hours at 37°C, 5% CO₂. Following incubation, al! media was removed from wells, encephaiomyocarditis virus (EMCV) was diluted in growth media and added (3000 pfu/well) to each well with the exception of control wells. Plates were incubated at 37°C, 5% CO₂for 28 hours. Living cells were fixed with 10% cold trichloroacetic acid (TCA) and then stained with Sulforhodaimine B (SRB) according to published protocols (Rubinstein etal. 1990, J. Natl. Cancer Inst. 82,1113). The SRB dye was solubilized with 10 mM Tris pH 10.5 and read on a spectrophotometer at 490 nm. Samples were analyzed by comparing activities to a known standard curve ranging from 10 to 0.199 lU/ml. The results are presented below in Table 8 and demonstrate increased antiviral activity of monomer-dimer hybrids.

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TABLE 8: INTERFERON BETA ANTIVIRAL ASSAY

HOMODIMER V. MONOMER-DIMER HYBRID

Protein	Antiviral Activity (Ili/nmoi)	Std dev
IFN3-Fc 8aa linker homodimer	4.5x10^b	0.72 x10^s
IFNpFc N297A 8aa linker homodimer	3.21 x 10^s	0.48 x 10*
IFNpFc N297A 8aa linker: Fc His monomer-dimer hybrid	12.2 x 10*	2 x10^s

Example 37: Administration of IFNBFc Homodimer and Monomer-Dimer

Hybrid With an Eight Amino Acid Linker to Cynomolgus Monkeys [0310] For pulmonary administration, aerosols of either IFNpFc homodimer or IFNpFc N297A monomer-dimer hybrid proteins (both with the 8 amino acid linker) in PBS, pH 7.4, 0.25% HSA were created with the Aeroneb Pro™ (AeroGen, Mountain View, CA) nebulizer, in-line with a Bird Mark 7A respirator, and administered to anesthetized na'ive cynomolgus monkeys through endotracheal tubes (approximating norma! tidal breathing). Blood samples were taken at various time points, and the amount of IFNp-containing protein in the resulting serum was quantitated using a human IFNp Immunoassay (Biosource International, Camarillo, CA). Pharmacokinetic parameters were calculated using the software WinNonLin. Table 9 presents the results of cynomolgus monkeys treated with IFNpFc N297A monomer-dimer hybrid or IFNpFc homodimer.

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TABLE 9: ADMINISTRATION OF IFNBFC N297A MONOMER-DIMER

HYBRID AND IFNBFC HOMODIMER TO MONKEYS

Protein	Monkey #	Route	Approx. Deposited Dose¹ (pg/kg)	Cmax (ng/ml)	AUC (hr*ng/ml)	t-1/2 (hr)	ti/2 avg (hr)
IFNpFc N297A monomerdimer hybrid	CO7308	pulm	20	23.3	987.9	27.6	27.1
CO7336	pulm	20	22.4	970.6	25.6
CO7312	pulm	20	21.2	1002.7	28.0
IFNpFc homodimer	CO7326	pulm	20	2.6	94.6	11.1	11.4
CO7338	pulm	20	5.0	150.6	11.7

Based on 15% deposition fraction of nebulized dose as determined by gamma scintigraphy [0311] The pharmacokinetics of IFNpFc with an 8 amino acid linker administered to cynomolgus monkeys is presented in figure 15. The figure compares the IFNpFc homodimer with the IFNpFc N297A monomer-dimer hybrid in monkeys after administration of a single pulmonary dose. Significantly higher serum levels were obtained in monkeys treated with the monomer-dimer hybrid compared to the homodimer.

[0312] Serum samples were also analyzed for neopterin levels (a biomarker of IFNp activity) using a neopterin immunoassay (MP Biomedicals, Orangeburg,

NY). The results for this analysis are shown in figure 16. The figure compares neopterin stimulation in response to the IFNp-Fc homodimer and the IFNp-Fc N297A monomer-dimer hybrid. It can be seen that significantly higher neopterin levels were detected in monkeys treated with IFNp-Fc N297A monomer-dimer hybrid as compared to the IFNp-Fc homodimer.

[0313] All numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being

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2016244273 13 Oct 2016 modified in all instances by the term about. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

[0314] All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supercede and/or take precedence over any such contradictory material.

[0315] Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only and are not meant to be limiting in any way. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

[0316] The term ‘comprise’ and variants of the term such as ‘comprises’ or ‘comprising’ are used herein to denote the inclusion of a stated integer or stated integers but not to exclude any other integer or any other integers, unless in the context or usage an exclusive interpretation of the term is required.

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2016244273 13 Oct 2016 [0317] Any reference to publications cited in this specification is not an admission that the disclosures constitute common general knowledge in Australia.

[Text continues on page 130.]

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2016244273 29 Sep 2017

Claims

CLAIMS:

1. A chimeric protein comprising one biologically active molecule and two immunoglobulin constant regions or portions thereof, wherein the chimeric protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the biologically active molecule and an immunoglobulin constant region, or a portion thereof, that is an Fc neonatal receptor (FcRn) binding partner and wherein the biologically active molecule is a protein selected from the group consisting of a cytokine and a hormone; and wherein the second polypeptide chain comprises an immunoglobulin constant region, or a portion thereof, that is an FcRn binding partner, but does not comprise a biologically active molecule.
2. The chimeric protein according to claim 1, wherein the first polypeptide chain further comprises a linker between the protein and the immunoglobulin constant region or portion thereof.
3. The chimeric protein according to claim 1 or claim 2, wherein the portion of the immunoglobulin constant region of the first polypeptide chain is an Fc fragment and the portion of the immunoglobulin constant region of the second polypeptide chain is an Fc fragment.
4. The chimeric protein according to any one of claims 1 to 3, wherein the immunoglobulin constant region is an lgG1 or lgG2 constant region.
5. The chimeric protein according to any one of claims 1 to 4, wherein the protein in the first polypeptide chain is a hormone.
6. The chimeric protein according to claim 5, wherein the hormone is erythropoietin (EPO).
7. The chimeric protein according to claim 5, wherein the hormone is growth hormone.
8. The chimeric protein according to claim 5, wherein the hormone is gonadotropin releasing hormone (GnRH), leuprolide, follicle stimulating hormone, progesterone, estrogen, or testosterone.

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2016244273 29 Sep 2017
9. The chimeric protein according to any one of claims 1 to 4, wherein the protein in the first polypeptide chain is a cytokine.
10. The chimeric protein according to claim 9, wherein the cytokine is interferon a or interferon β.
11. The chimeric protein according to claim 9, wherein the cytokine is RANTES, ΜΙΡ1α, MIP1 β, IL-2, IL-3, or GM-CSF.
12. A pharmaceutical composition comprising the chimeric protein according to any one of claims 1 to 11 and a pharmaceutically acceptable excipient.
13. A first polynucleotide encoding the first polypeptide chain of the chimeric protein according to any one of claims 1 to 11, and a second polynucleotide encoding the second polypeptide chain of said chimeric protein.
14. A method of treating a disease or condition in a subject in need thereof comprising administering the chimeric protein according to any one of claims 1 to 11 or the pharmaceutical composition of claim 12.
15. The method according to claim 14, wherein the chimeric protein is administered intravenously, intramuscularly, subcutaneously, orally, bucally, sublingually, nasally, parenterally, rectally, vaginaliy, via a mucosal surface, via an aerosol, or via a pulmonary route.
16. A method of making the chimeric protein according to any one of claims 1 to 11 wherein the method comprises:

transfecting a host cell with a first polynucleotide encoding the first polypeptide chain and a second polynucleotide encoding the second polypeptide chain;

culturing the cell under conditions such that the first and second polypeptide chains are expressed; and isolating the chimeric protein from said host cell.

Date: 29 September 2017

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Fig. 2A

Factor VII-Fc amino acid sequence {signal peptide underlined, propeptide in bold)

101

151

201

251

301

351

401

451

501

551

601

651

MVSQALRLLC LLLGLQGCLA AVFVTQEEAH GVLHRRRRAN AFLEELRP.GS LERECKEEQC SFEEAREIFK DAERTKLFWI SYSDGDQCAS SPCQNGGSCK dqlqsyicfc LPAFEGRNCE thkddqlicv nenggceqyc sdhtgtkrsc rchegyslla dgvsctptve ypcgkipile krnaskpqgr ivggkvcpkg ECPWQVLLLV NGAQLCGGTL INTIWWSAA HCFDKIKNWR NLIAVLGEHD lsehdgdeqs rrvaqviips tyvpgttnhd iallrlhqpv vltdhwplc lpertfsert lafvrfslvs gwgqlldrga talelmvlnv prlmtqdclq QSRKVGDSPN ITEYMFCAGY SDGSKDSCKG DSGGPHATHY RGTWYLTGIV SWGQGCATVG hfgvytrvsq yiewlqklmr seprpgvllr apfpdkthtc PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYWGVEVHN AKTKPREEQY NSTYRWSVL TVLHQDWLNGKEYKCKVSNK ALPAPIEKTI skakgqprep qvytlppsrd eltknqvslt clvkgfypsd IAVEWESNGQ .PENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K

Fig. 2B

Factor IX -Fc amino acid sequence (signal peptide propeptide in bold) ¹ MQRVNMIMAB SPGLITICLL GYLLSAECTV FLDHENANKI

51 KLEEFVQGNL ERECMEEKCS FEEAREVFEN TERTTEFWKQ

101 PCLNGGSCKD DINSYECWCP FGFEGKNCEL DVTCNIKNGR

151 NKWCSCTEG YRLAENQKSC EPAVPFPCGR VSVSQTSKLT

201 YVNSTEAETI LDNITQSTQS FNDFTRWGG EDAKPGQFPW

251 FCGGSIVNEK WIVTAAHCVE TGVKITWAG EHNIEETEHT

301 PHHNYNAAIN KYNHDIALLE LDEPLVLNSY VTPICIADKE

351 GYVSGWGRVF HKGRSALVLQ YLRVPLVDRA TCLRSTKFT1

401 EGGRDSCQGD SGGPHVTEVE GTSFLTGIIS WGEECAMKGK

451 VNWIKEKTKL TEFAGAAAVD KTHTCPPCPA PELLGGPSVF

501 MISRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKTKP

551 WSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG

601 PPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY

651 GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL underlined,

LNRPKRYNSG

YVDGDQCESN

CEQFCKNSAD

RAETVFPDVD

QWLNGKVDA

EQKRNVIRII

YTNIFLKFGS

YNNMFCAGFH

YGIYTKVSRY

LFPPKPKDTL

REEQYNSTYR

QPREPQVYTL

KTTPPVLDSD

SLSPGK

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Fig. 2C

2016244273 13 Oct 2016

IFNa-Fc amino acid sequence (8 amino acid linker) (signal sequence underlined)

1 maltfallva llvlsckssc svgcdlpqth slgsrrtlml laqmrrislf

51 SCLKDRHDFG FPQEEFGNQF QKAETIPVLH EMIQQIFNLF STKDSSAAWD

101 ETLLDKFYTE LYQQLNDLEA CVIQGVGVTE TPLMKEDSIL AVRKYPQRIT

151 LYLKEKKYSP CAWEWRAEI MRSFSLSTNL QESLRSKEEF AGAAAVDKTH

201 TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVW DVSHEDPEVK

251 FNWYVDGVEV HNAKTKPREE QYNSTYRWS VLTVLHQDWL NGKEYKCKVS

301 NKALPAPIEK TISKAKGQPR EPQVYTLPPS RDELTKNQVS LTCLVKGFYP

351 SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS

401 CSVMHEALHN HYTQKSLSLS PGK

Fig. 2D

IFNa-Fc Δ linker amino acid sequence (signal sequence underlined)

1 MALTFALLVA LLVLSCKSSC SVGCDLPQTH SLGSRRTLML LAQMRRISLF 51 SCLKDRHDFG FPQEEFGNQF QKAETIPVLH EMIQQIFNLF STKDSSAAWD

101 ETLLDKFYTE LYQQLNDLEA CVIQGVGVTE TPLMKEDSIL AVRKYFQRIT 151 LYLKEKKYSP CAWEWRAEI MRSFSLSTNL QESLRSKEDK THTCPPCPAP 201 ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV WDVSHEDPE VKFNWYVDGV 251 EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI 301 EKT1SKAKGQ PREPQVYTLP PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE 351 SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL 401 HNHYTQKSLS LSPGK

Fig. 2E

FlagFc amino acid seguence (signal sequence underlined)

101

151

201

251

METDTLLLWV

LFPPKPKDTL

REEQYNSTYR

QPREPQVYTL

KTTPPVLDSD

SLSPGK

LLLWVPGSTG

MISRTPEVTC

WSVLTVLHQ

PPSRDELTKN

GSFFLYSKLT

DDYKDDDDKD

WVDVSHEDP

DWLNGKEYKC

QVSLTCLVKG

VDKSRWQQGN

KTHTCPPCPA

EVKFNWYVDG

KVSNKALPAP

FYPSDIAVEW

VFSCSVMHEA

PELLGGPSVF

VEVHNAKTKP

IEKTISKAKG

ESNGQPENNY

LHNHYTQKSL

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Fig. 2F

Epo-CCA-Fc amino acid sequence (K^b signal sequence underlined, acidic coiled coil in bold)

1 MVPCTLLLLL AAALAPTQTR AGSRAPPRLI CDSRVLQRYL LEAKEAENIT 51 TGCAEHCSLN ENITVPDTKV NFYAWKRMEV GQQAVEVWQG LALLSEAVLR

101 GQALLVNSSQ PWEPLQLHVD KAVSGLRSLT TLLRALGAQK EAISPPDAAS 151 AAPLRTITAD TFRKLFRVYS NFLRGKLKLY TGEACRTGDR EFGGEYQALE 201 KEVAQLEAEN QALEKEVAQL EHEGGGPAPE LLGGPSVFLF PPKPKDTLMI 251 SRTPEVTCW VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRW 301 SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP 351 SRDELTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS 4 01 FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK

Fig. 2G

CCB-Fc amino acid sequence (K^b signal sequence underlined; basic coiled coil in bold)

1 MVPCTLLLLL AAALAPTQTR AGEFGGEYQA LKKKVAQLKA KNQALKKKVA 51 QLKHKGGGPA PELLGGPSVF LFPPKPKDTL MISRTPEVTC WVDVSHEDP

101 EVKFNWYVDG VEVHNAKTKP REEQYNSTYR WSVLTVLHQ DWLNGKEYKC

151 KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSRDELTKN QVSLTCLVKG

201 FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN

251 VFSCSVMHEA LHNHYTQKSL SLSPGK

Fig. 2H

CysFc amino acid sequence (hIFNa signal sequence underlined)

1 MALTFALLVA LLVLSCKSSC SVGCPPCPAP ELLGGPSVFL FPPKPKDTLM 51 ISRTPEVTCV WDVSHEDPE VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV

101 VSVLTVLHQD WLNGKEYKCK VSNKALPAP2 EKTISKAKGQ PREPQVYTLP 151 PSRDELTKNQ VSLTCLVKGF YPSD2AVEWE SNGQPENNYK TTPPVLDSDG 201 SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGK

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Fig. 21

IFNa GS15 Fc protein sequence (signal sequence underlined):

1 MALTFALLVA LLVLSCKSSC SVGCDLPQTH SLGSRRTLML LAQMRRISLF

51 SCLKDRHDFG FPQEEFGNQF QKAETIPVLH EMIQQIFNLF STKDSSAAWD

101 ETLLDKFYTE LYQQLNDLEA CVIQGVGVTE TPLMKEDSIL AVRKYFQRIT

151 LYLKEKKYSP CAWEWRAEI MRSFSLSTNL QESLRSKEGG GGSGGGGSGG

201 GGSDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCWVDVS

251 HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRWSVLT VLHQDWLNGK

301 EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC

351 LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW

401 QQGNVFSCSV MHEALHNHYT QKSLSLSPGK

Fig. 2J

EpoFc amino acid sequence (signal sequence underlined, linker in bold)

1 MGVHECPAWL WLLLSLLSLP LGLPVLGAPP RLICDSRVLE RYLLEAKEAE 51 NITTGCAEHC SLNENITVPD TKVNFYAWKR MEVGQQAVEV WQGLALLSEA

101 VLRGQALLVN SSQPWEPLQL HVDKAVSGLR SLTTLLRALG AQKEAISPPD 151 AASAAPLRTI TADTFRKLFR VYSNFLRGKL KLYTGEACRT GDREFAGAAA 201 VDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCWVDVSHE 251 DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRWSVLTVL HQDWLNGKEY 301 KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV 351 KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ 401 GNVFSCSVMH EALHNHYTQK SLSLSPGK

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Fig. 3A

Factor VII-Fc nucleotide sequence {signal peptide underlined, propeptide in bold) atggtctcccaggccctcaggctcctctgccttctgcttgggcttcagggctgcctggctgcag tcttcgtaacccaggaggaagcccacggcgtcctgcaecggcgccggcgcgccaacgcgttcct ggaggagctgcggccgggctccctggagagggagtgcaaggaggagcagtgctccttcgaggag gcccgggagatcttcaaggacgcggagaggacgaagctgttctggatttcttacagtgatgggg accagtgtgcctcaagtccatgccagaatgggggctcctgcaaggaccagctccagtcctatat ctgcttctgcctecctgccttcgagggccggaactgtgagacgcacaaggatgaccagctgatc tgtgtgaacgagaacggcggctgtgagcagtactgcagtgaccacacgggcaccaagcgctcct gtcggtgccacgaggggtactctctgctggcagacggggtgtcctgcacacccacagttgaata tccatgtggaaaaatacctattctagaaaaaagaaatgccagcaaaccccaaggccgaattgtg gggggcaaggtgtgccccaaaggggagtgtccatggcaggtcotgttgttggtgaatggagctc agttgtgtggggggaccctgatcaacaccatotgggtggtctccgcggcccactgtttcgacaa aatcaagaactggaggaacctgatcgcggtgctgggcgagcacgaectcagcgagcacgacggg gatgagcagagccggcgggtggcgcaggtcatcatccccagcacgtacgtcccgggcaccacca accacgacatcgcgctgctccgcctgcaccagcccgtggtcctcactgaccatgtggtgcccct ctgcctgcccgaacggacgttctctgagaggacgctggccttcgtgcgcttctcattggtcagc ggctqgggccagctgctggaccgtggcgccacggccctggagctcatggtcctcaacgtgcccc ggctgatgacccaggactgcctgcagcagtcacggaaggtgggagactccccaaatatcacgga gtacatgttctgtgccggctactcggatggcagcaaggactcctgcaagggggacagtggaggc ccacatgccacccactaccggggcacgtggtacctgacgggcatcgtcagctggggccagggct gcgcaaccgtgggccactttggggtgtacaccagggtctcccagtacatcgagtggctgcaaaa getcatgcgctcagagccacgcccaggagtcctcctgcgagccccatttcccgacaaaactcac acgtgcccgccgtgcccagctccggaactgctgggcggaccgtcagtcttcctcttccccccaa aacccaaggacsccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgag ccacgaagaccctgaggtcaagttcaactgatacgtggacggcgtggaggtgoataatgccaag acaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgc accaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccc catcgagaaaaccatctccaaagccaaagggcaaccccgagaaccacaggtgtacaccctgccc ccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctcgtcaaaggcttctatc ccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcc tcccgtgttggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcagg tggcagcaggggaacgtcttctcatgetccgtgatgcatgaggctctgcacaaccactacacgc agaagagcctctccotgtctccgggtaaatga

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Fig. 3B

Factor IX-Fc nucleotide sequence (signal peptide underlined, propeptide in bold) atgcagcgcgtgaacatgatcatggcagaatcaccaggcctcatcaccatctgccttttaggat atctactcagtgctgaatgtacagtttttcttgatcatgaaaacgccaacaaaattctgaatcg gccaaagaggtataattcaggtaaattggaagagtttgttcaagggaaccttgagagagaatgt atggaagaaaagtgtagttttgaagaagcacgagaagtttttgaaaacactgaaagaacaactg aattttggaagcagtatattgatggagatcagtgtgagtccaatccatgtttaaatggcggcfcg ttgcaaggatgacattaattcctatgaatgttggtgtccctttggatttgaaggaaagaactgt gaattagatgtaacatgtaacattaagaatggcagatgcgagcsgttttgtaaaaatagtgctg ataacaaggtggtttgctcctgtactgagggatatcgacttgcagaaaaccagaagtcctgtga accagcagtgccatttccatgtggaagagtttctgtttcacaaacttctaagctcacccgtgct gagactgtttttcctgatgtggactatgtaaattctactgaagctgaaaccattttggataaca tcactcaaagcacccaatcatttaatgacttcactcgggttgttggtggagaagatgccaaacc aggtcaattcccttggcaggttgttttgaatggtaaagttgatgcattctgtggaggctctatc gttaatgaaaaatggattgtaactgctgcccactgtgttgaaactggtgttaaaattacagttg tcgcaggtgaacataatattgaggagacagaacatacagagcaaaagcgaaatgtgattcgaat tattcctcaccacaactacaatgcagctattaataagtacaaccatgacattgcccttctggaa ctggscgaacccttagtgctaaacagctacgttacacctatttqcattgctgacaaggaataca cgaacatcttcctcaaatttggatctggctatgtaagtggctggggaagagtcttccacaaagg gsgatcagctttagttcttcagtaccttagagttccacttgttgaccgagccacatgtcttcga tctacaaagttcaccatctataacaacatgttctgtgctggcttccatgaaggaggtagagatt catgtcaaggagatagtgggggaccccatgttactgaagtggaagggaccagtttcttaactgg aattattagctggggtgaagagtgtgcaatgaaaggcaaatatggaatatataccaaggtatcc cggtatgtcaactggattasggaaaaaacaaagctcactgaattcgccggcgccgctgcggtcg acaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcct cttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtg gtggacgtgagccacqaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgc ataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcct caccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagcc ctcccagcccccatcgagaaaaccatctccaaagccaaagagcagccccgagaaccacaggtgt acaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaa R sggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactac ) aagaccacgcctcccgtgttggactccgacggctccttcttcctctacagcaagctcaccgtgg acaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaa j ccactacacgcagaagagcctctccctgtctccgggtaaatga

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Fig. 3C

IFNa-Fc nucleotide sequence (8 amino acid linker) atggccttgacctttgctttactggtggccctcctggtgctcagctgcaagtcaagctgctctg tggcictgtgatctgcctcaaacccacagcctgggtagcaggaggaccttgatgctcctggcaca gatgaggagaatctctcttttctcctgcttgaaggacagacatgactttggatttccccaggag gagtttggcaaccagttccaaaaggctgaaaccatccctgtcctccatgagatgatccagcaga tcttcaatctcttcagcacaaaggactcatctgctgcttgggatgagaccctcctagacaaatt ctacactgaactctaccagcagctgaatgacctggaagcctgtgtgatacagggggtgggggtg acagagactcccctgatgaaggaggactccattctgqctgtgaggaaatacttccaaagaatca ctctctatctgaaagagaagaaatacagcccttgtgcctgggaggttgtcagagcagaaatcat gagatctttttctttgtcaacaaacttgcaagaaagtttaagaagtaaggaagaattcgccggc gccgctgcggtcgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggac cgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggt cacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggac ggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtg tggtcagcgtcctcaccgtcctgcaccaggactaqctgaatggcaaggagtacaagtgcaaggt ctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccga gaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctga cctgcctggtcaaaggcttctatcccagcgacatcgccgtggsgtgggagagcaatgggcagcc ggagaacaactacaagaccacgcctcccgtgttggactccgacggctccttcttcctctacagc aagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatg agg'ctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga

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2016244273 13 Oct 2016

Fig. 3D

IFNtt-Fc Δ linker nucleotide sequence atggccttgacctttgctttactggtggccctcctggtgctcagctgcaagtcaagctgctctg tcjcjcictgtgatctgcctcaaacccacagcctgggtagcaggaggaccttgatgctcctggcaca gatgaggagaatctctcttttctcctgcttgaaggacagacatgactttggatttccccaggag gagtttggcaaccagttccaaaaggctgaaaccatccctgtcctccatgagatgatccagcaga tcttcaatctcttcagcacaaaggactcatctgctgcttgagatgagaccctcctagacaaatt ctacactgaactctaccagcagctgaatgacctggaagcctgtgtgatacagggggtgggggtg acagagactcccctgatgaaggaggactccattctggctgtgaggaaatacttccaaagaatca ctctctatctgaaagagaagaaatacagcccttgtgcctgggaggttgtcagagcagaaatcat gagatctttttctttgtca-acaaacttgcaagaaagtttaagaagtaaggaagacaaaactcac acgtgcccgccgtgcccagctccggaactgctgggcggaccgtcagtcttcctcttccccccaa aacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgag ccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaag acaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgc accaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccc catcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgccc ccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatc ccagcgacatcgccgtggagtgggagaacaataggcagccggagaacaactacaagaccacgcc tcccgtgttggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcagg tggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgc sgaagagcctctccctgtctccgggtaaatga

Fig. 3E

FlagFc nucleotide sequence atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgacg actacaaggacgacgatgacaaggacaaaactcacacatgcccaccgtgcccagctccggaact cctggggggaccgtcagtcttcctctt ccccccaaaacccaaggacaccctcatgatctcccgg acccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaact ggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacag cacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtac aagtgcaaggtctccaacsaagccctcccagcccccatcgagaaaaccatctccaaagccaaag ggcagccccgagaaccacaggtgtacaccctgcccccatcccgggstgagctgaccaagaacca ggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgagagagc aatgggcagccggagaacaactacaagaccacgcctcccgtgttggactccgacggctccttct tcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctc cgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa tga

9/27

2016244273 13 Oct

O pig. 3F

CM

Epo-CCA-Fc nucleotide sequence (K^b signal sequence'underlined, acidic coiled coil in bold) atggtaccgtgcacgctgctcctgctgttggcggccgccctggctccgactcagacccgcgccg gctctagagccccaccacgcctcatctgtgacagccgagtcctgcagaggtacctcttggaggc caaggaggccgagaatatcacgacgggctgtgctgaacactgcagcttgaatgagaatatcact gtcccagacaccaaagttaatttctatgcctggaagaggatggaggtcgggcagcaggccgtag aagtctggcagggcctggccctgctgtcggaagctgtcctgcggggccaggccctgttggtcaa ctcttcccagccgtgggagcccctgcagctgcatgtggataaagccgtcagtggccttcgcagc ctcaccactctgcttcgggctctgggagcccagaaggaagccatctcccctccagatgcggcct cagctgctccactccgaacaatcactgctgacactttccgcaaactcttccgagtctactccaa tttcctccggggaaagctgaagctgtacacaggggaggcctgcaggaccggtgacagggaattc ggtggtgagtaccaggccctggagaaggaggtggcccagctggaggccgagaaccaggccctgg agaaggaggtggcccagctggagcacgagggtggtggtcccgcacccgagctgctgggcggacc gtcagtcttcctcttccccccaaaacccsaggacaccctcatgatctcccggacccctgaggtc acatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacg gcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgt ggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtc tccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgag aaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgac ctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccg gagaacaactacaagaccacgcctcccgtgttggactccgacggctccttcttcctctacagca agctcaccgtagacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatga ggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga

Fig. 3G

CCB-Fc nucleotide seguence (signal sequence underlined, basic coiled coil in bold) atggtaccgtqcacgctgctcctgctgttggcggccgccctggctccgactcagacccgcgccg gcgaattcggtggtgagtaccaggccctgaagaagaaggtggcccagctgaaggccaagaacca ggccctgaagaagaaggtggcccagctgaagcacaagggcggcggccccgccccagagctcctg ggcggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggaccc ctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggta cgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacg taccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagt gcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggca gccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtc agcctgacctgcctggtcasaggcttctatcccagcgacatcgccgtggagtgggagagcaatg ggcagccggagaacaactacaagaccacgcctcccgtgttggactccgacggctccttcttcct ctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtg atgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga

10/27

2016244273 13 Oct 2016

Fig. 3H

CysF.c nucleotide sequence (hIFNa signal sequence underlined) atggccttgacctttgctttactggtggccctcctggtgctcagctgcaagtcaagctgctctg tgggctgcccgccgtgcccagctccggaactgctgggcggaccgtcagtcttcctcttcccccc aaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtg agccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgcca agacaaagccgcgggeggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcct gcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcc cccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgc cc'ccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtca'aaggcttcta tcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacg cctcccgtgttggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagca ggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacac gcagaagagcctctccctgtctccgggtaaatga

Fig. 31

IFNa GS15 Fc nucleotide sequence (signal sequence underlined):

atggccttgacctttgctttactggtggccctcctggtactcagctgcaagtcaagctgctctg tqqqctgtgatctgcctcaaacccacagcctgggtagcaggaggaccttgatgctcctggcaca gatgaggagaatctctcttttctcctgcttaaaggacagacatgactttggatttccccaggag gagtttggcaaccagttccaaaaggctgaaaccatccctgtcctccatgagatgatccagcaga tcttcaatctcttcagcacaaaggactcatctgctgcttgggatgagaccctcctagacaaatt ctacactgaactctaccagcagctgaatgacctggaggcctgtgtgatacagggggtgggggtg acagagactcccctgatgaaggaggactccattctggctgtgaggaaatacttccaaagaatca ctctctatctgaaagagaaqaaatacagcccttgtgcctgggaggttqtcagagcagaaatcat gagatctttttctttgtcaacaaacttgcaagaaagtttacgtagtaagaaaggtggcggcgga tccggtggaggcgggtccggcggtggagggagcgacaaaactcacacgtgcccgccgtgcccag ctccggaactgctgggcggaccgtcagtettcctcttccccccaaaacccaaggacaccctcat gatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtc aagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagc agtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatgg caaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctcc aaagecaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctga ccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtgga gtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgttggactccgac ggctccttcttcctctacagcaagctcaccgtggacaagagcsggtggcagcaggggaacgtct tctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtc tccgggtaaatga

11/27

2016244273 13 Oct 2016

Fig. 3J

EpoFc nucleotide sequence (signal sequence underlined, linker in bold) atgggagtgcacgaatgtcctgcctggctgtggcttctcctgtccctgctgtcgctccctctgg gcctcccagtcctgggcgccccaccacgcctcatctgtgacagccgagtcctggagaggtacct cttggaggccaaggaggccgagaatatcacgacgggctgtgctgaacactgcagcttgaatgag aatatcactgtcccagacaccaaagttaatttctatgcctggaagaggatggaggtcgggcagc aggccgtagaagtctggcagggcctggccctgctgtcggaagctgtcctgcggggccaggccct gttggtcaactcttcccagccgtgggagcccctgcagctgcatgtggataaagccgtcagtggc cttcgcagcctcaccactctgcttcgggctctgggagcccagaaggaagccatctcccctccag atgcggcctcagctgctccactccgaacaatcactgctgacactttccgcaaactcttccgagt ctactccaatttcctccggggaaagctgaagctgtacacaggggaggcctgcagaacaggggac agagagttcgccggcgccgctgcggtcgacaaaactcacacatgcccaccgtgcccagctccgg aactcctgggcggaccgtcagtcttcctcttceccccaaaacccaaggacaccctcatgatctc ccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttc aactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtaca acagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaagga gtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagcc aaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaaga accaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtggga gagcaatgggcagccggagaacaactacaagaccacgcctcccgtgttggactccgacggctcc ttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcat gctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccggg taaatga

12/27

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Figure 5a

2016244273 13 Oct 2016

Amino acid sequence of Fc-MESNA (produced in pTWINI vector from NEB; when Fc-Intein-CBD is eluted from chitin beads with MESNA, produces the following protein with a C-terminal thioester on the final Phe residue)

1 MG1EGRGAAA VDTSHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV 51 TCVWDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRWSVLTVL

101 HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT 151 KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK 201 LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGF

Figure 5 b

Nucleotide sequence of Fc CDS in pTWINI (the final F residue, ttt, directly abuts the Mxe GyrA intern CDS in pTWINI) atgggcattgaaggcagaggcgccgctgcggtcgatactagtcacacatgcccacc'gtgcccag cacct gaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcat gatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtc aagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagc agt'acaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatgg caaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctcc aaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctga ccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtgga gt gggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgttggactccgac ggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtct tctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagtctctccctgtc tccgggtttt

14/27

2016244273 13 Oct 2016

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18/27

2016244273 13 Oct 2016

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19/27

2016244273 13 Oct 2016

Fig- Π

Pharmacokinetics of EpoFc Dimer v. Monomer-Dimer Hybrid in Cynomolgus Monkeys After a Single Pulmonary Dose

Molar Comparison ••o··

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monkey 1/1: monkey 42: monkey 43: monkey 44: monkey 45: monkey -#6: monkey 47: monkey «8: monkey 49:

monomer-dimer, 281 pmol/kg monomer-dimer, 281 pmol/kg monomer-dimer, 281 pmol/kg monomer-dimer, 281 pmol/kg dimer, 168 pmol/kg dimer, 168 pmol/kg dimer, 392 pmol/kg dimer, 168 pmol/kg dimer, 168 pmol/kg
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2016244273 13 Oct 2016

Fig 17a.

IFNP-Fc nucleotide sequence (signal peptide underlined) atgaccaacaagtgtctcctccaaattgctctcctgttgtgcttctccactacagctctttcca tgagctacaacttgcttggattcctacaaagaagcagcaattttcagtgtcagaagctcctgtg gcaattgaatgggaggcttgaatattgcctcaaggacaggatgaactttgacatccctgaggag attaagcagctgcagcagttccagaaggaggacgccgcattgaccatctatgagatgctccaga acatctttgctattttcagacaagattcatctagcactggctggaatgagactattgttgagaa cctcctggctaatgtctatcatcagataaaccatctgaagacagtcctggaagaaaaactggag aaagaagatttcaccaggggaaaactcatgagcagtctgcacctgaaaagatattatgggagga ttctgcattacctgaaggccaaggagtacagtcactgtgcctggaccatagtcagagtggaaat cctaaggaacttttacttcattaacagacttacaggttacctccgaaacgagttcgccggcgcc gctgcggtcgacaaaactcacacatgcccaccgtgcccagctccggaactcctgggcggaccgt cagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcac atgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggc gtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtgg tcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctc caacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaa ccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacct gcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccgga gaacaactacaagaccacgcctcccgtgttggactccgacggctccttcttcctctacagcaag ctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgagg ctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga

Fig 17b.

IFNp-Fc amino acid sequence (signal sequence underlined, linker sequence in bold, N297 in bold underlined).

1 MTNKCLLQIA LLLCFSTTAL SMSYNLLGFL QRSSNFQCQK LLWQLNGRLE 51 YCLKDRMNFD IPEEIKQLQQ FQKEDAALTI YEMLQNIFAI FRQDSSSTGW 101 NETIVENLLA NVYHQINHLK TVLEEKLEKE DFTRGKLMSS LHLKRYYGRI 151 LHYLKAKEYS HCAWTIVRVE ILRNFYFINR LTGYLRNEFA GAAAVDKTHT

2 01 CPPCPAPELL GGPSVFLPPP KPKDTLMISR TPEVTCVWD VSHEDPEVKF 251 NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKEYKCKVSN 301 KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS 351 DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC 401 SVMHEALHNH YTQKSLSLSP GK
26/27

2016244273 13 Oct 2016

Fig. 18A

YTSLiHSLIEESQNQQEKNEQELLELDKWASLWNWF (SEQ ID NO: 99).

Fig. 18B

NNLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQ (SEQ ID NO: 100)

Fig. 18C

WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF (SEQ ID NO: 101)

27/27

2016244273 13 Oct 201

SEQUENCE LISTING <110> Syntonix Pharmaceuticals, Inc.

PETERS, ROBERT T.

MEZO, ADAM R.

RIVERA, DANIEL S.

BITONTI, ALAN J.

LOW, SUSAN C.

STATTEL, JAMES <120> IMMUNOGLOBULIN CHIMERIC MONOMER-DIMER HYBRIDS <130> 2159.240AU14/EKS/C-K <140> 2012200470 <141> 2004-05-06 <150> 2004252422 <151> 2004-05-06 <150> PCT/US04/14064 <151> 2004-05-06 <150> US 60/539,207 <151> 2004-01-26 <150> US 60/487,964 <151> 2003-07-17 <150> US 60/469,600 <151> 2003-05-06 <160> 107 <170> Patentln version 3.5 <210> 1 <211> 36 <212> PRT <213> Human immunodeficiency virus <400> 1

Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gin Asn Gin Gin 15 10 15

Glu Lys Asn Glu Gin Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu 20 25 30

Trp Asn Trp Phe 35 <210> 2 <211> 37 <212> PRT

2016244273 13 Oct 2016 <213> Human immunodeficiency virus <400> 2

Asn Asn Leu Arg Ala Ile Glu Ala Gin Gin His Leu Leu Gin Leu Thr 15 10 15

Val Trp Gly Ile Lys Gin Leu Gin Ala Arg Ile Leu Ala Val Glu Arg 20 25 30

Tyr Leu Lys Asp Gin 35 <210> 3 <211> 39 <212> PRT <213> Human immunodeficiency virus <400> 3

Trp Gin Glu Trp Glu Gin Lys Ile Thr Ala Leu Leu Glu Gin Ala Gin 15 10 15

Ile Gin Gin Glu Lys Asn Glu Tyr Glu Leu Gin Lys Leu Asp Lys Trp 20 25 30

Ala Ser Leu Trp Glu Trp Phe 35 <210> 4 <211> 238 <212> PRT <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic construct <400> 4

Met Gly Ile Glu Gly Arg Gly Ala Ala Ala Val Asp Thr Ser His Thr 15 10 15

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 20 25 30

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 35 40 45

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

65 70 75 80

2016244273 13 Oct 2016

Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 85 90 95

Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 100 105 110

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 115 120 125

Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro 130 135 140

Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val 145 150 155 160

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 165 170 175

Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 180 185 190

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 195 200 205

Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 210 215 220

Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Phe 225 230 235 <210> 5 <211> 714 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic construct <400> 5 atgggcattg aaggcagagg cgccgctgcg gtcgatacta gtcacacatg cccaccgtgc 60

2016244273 13 Oct 2016 ccagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa acccaaggac

120 accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 180 gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 240 aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg 300 caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca 360 gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac 420 accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc 480 aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac 540 aactacaaga ccacgcctcc cgtgttggac tccgacggct ccttcttcct ctacagcaag 600 ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat 660 gaggctctgc acaaccacta cacgcagaag agtctctccc tgtctccggg tttt 714 <210> 6 <211> 671 <212> PRT <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic construct <400> 6

Met Val Ser Gin Ala Leu Arg Leu Leu Cys Leu Leu Leu Gly Leu Gin 15 10 15

Gly Cys Leu Ala Ala Val Phe Val Thr Gin Glu Glu Ala His Gly Val 20 25 30

Leu His Arg Arg Arg Arg Ala Asn Ala Phe Leu Glu Glu Leu Arg Pro 35 40 45

Gly Ser Leu Glu Arg Glu Cys Lys Glu Glu Gin Cys Ser Phe Glu Glu

2016244273 13 Oct 2016

50 55

Ala Arg Glu Ile Phe Lys Asp Ala 65 70

Ser Tyr Ser Asp Gly Asp Gin Cys 85

Gly Ser Cys Lys Asp Gin Leu Gin 100

Ala Phe Glu Gly Arg Asn Cys Glu 115 120

Glu Arg Thr Lys Leu Phe Trp Ile 75 80

Ala Ser Ser Pro Cys Gin Asn Gly 90 95

Ser Tyr lie Cys Phe Cys Leu Pro 105 110

Thr His Lys Asp Asp Gin Leu Ile 125

Cys Val Asn Glu Asn Gly Gly Cys 130 135

Glu Gin Tyr Cys Ser Asp His Thr 140

Gly Thr Lys Arg Ser Cys Arg Cys 145 150

His Glu Gly Tyr Ser Leu Leu Ala 155 160

Asp Gly Val Ser Cys Thr Pro Thr 165

Val Glu Tyr Pro Cys Gly Lys Ile 170 175

Pro Ile Leu Glu Lys Arg Asn Ala 180

Ser Lys Pro Gin Gly Arg Ile Val 185 190

Gly Gly Lys Val Cys Pro Lys Gly 195 200

Glu Cys Pro Trp Gin Val Leu Leu 205

Leu Val Asn Gly Ala Gin Leu Cys 210 215

Gly Gly Thr Leu Ile Asn Thr Ile 220

Trp Val Val Ser Ala Ala His Cys 225 230

Phe Asp Lys Ile Lys Asn Trp Arg 235 240

Asn Leu Ile Ala Val Leu Gly Glu 245

His Asp Leu Ser Glu His Asp Gly 250 255

Asp Glu Gin Ser Arg Arg Val Ala 260

Gin Val Ile Ile Pro Ser Thr Tyr 265 270

Val Pro Gly Thr Thr Asn His Asp 275 280

Ile Ala Leu Leu Arg Leu His Gin 285

2016244273 13 Oct 2016

Pro Val Val Leu Thr Asp His Val 290 295

Thr Phe Ser Glu Arg Thr Leu Ala 305 310

Gly Trp Gly Gin Leu Leu Asp Arg 325

Val Leu Asn Val Pro Arg Leu Met 340

Arg Lys Val Gly Asp Ser Pro Asn 355 360

Val Pro Leu Cys Leu Pro Glu Arg 300

Phe Val Arg Phe Ser Leu Val Ser 315 320

Gly Ala Thr Ala Leu Glu Leu Met 330 335

Thr Gin Asp Cys Leu Gin Gin Ser 345 350

Ile Thr Glu Tyr Met Phe Cys Ala 365

Gly Tyr Ser Asp Gly Ser Lys Asp 370 375

Ser Cys Lys Gly Asp Ser Gly Gly 380

Pro His Ala Thr His Tyr Arg Gly 385 390

Thr Trp Tyr Leu Thr Gly Ile Val 395 400

Ser Trp Gly Gin Gly Cys Ala Thr 405

Val Gly His Phe Gly Val Tyr Thr 410 415

Arg Val Ser Gin Tyr Ile Glu Trp 420

Leu Gin Lys Leu Met Arg Ser Glu 425 430

Pro Arg Pro Gly Val Leu Leu Arg 435 440

Ala Pro Phe Pro Asp Lys Thr His 445

Thr Cys Pro Pro Cys Pro Ala Pro 450 455

Glu Leu Leu Gly Gly Pro Ser Val 460

Phe Leu Phe Pro Pro Lys Pro Lys 465 470

Asp Thr Leu Met Ile Ser Arg Thr 475 480

Pro Glu Val Thr Cys Val Val Val 485

Asp Val Ser His Glu Asp Pro Glu 490 495

Val Lys Phe Asn Trp Tyr Val Asp 500

Gly Val Glu Val His Asn Ala Lys 505 510

Thr Lys Pro Arg Glu Glu Gin Tyr

Asn Ser Thr Tyr Arg Val Val Ser

2016244273 13 Oct 2016

515 520 525 Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys 530 535 540 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 545 550 555 560 Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro 565 570 575 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys Leu 580 585 590 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 595 600 605 Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 610 615 620 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 625 630 635 640 Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 645 650 655 His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 660 665 670 <210> 7 <211> 2016 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic construct <400> 7 atggtctccc aggccctcag gctcctctgc cttctgcttg ggcttcaggg ctgcctggct 60 gcagtcttcg taacccagga ggaagcccac ggcgtcctgc accggcgccg gcgcgccaac 120 gcgttcctgg aggagctgcg gccgggctcc ctggagaggg agtgcaagga ggagcagtgc

180

2016244273 13 Oct 2016

tccttcgagg 240 aggcccggga gatcttcaag gacgcggaga ggacgaagct gttctggatt tcttacagtg 300 atggggacca gtgtgcctca agtccatgcc agaatggggg ctcctgcaag gaccagctcc 360 agtcctatat ctgcttctgc ctccctgcct tcgagggccg gaactgtgag acgcacaagg 420 atgaccagct gatctgtgtg aacgagaacg gcggctgtga gcagtactgc agtgaccaca 480 cgggcaccaa gcgctcctgt cggtgccacg aggggtactc tctgctggca gacggggtgt 540 cctgcacacc cacagttgaa tatccatgtg gaaaaatacc tattctagaa aaaagaaatg 600 ccagcaaacc ccaaggccga attgtggggg gcaaggtgtg ccccaaaggg gagtgtccat 660 ggcaggtcct gttgttggtg aatggagctc agttgtgtgg ggggaccctg atcaacacca 720 tctgggtggt ctccgcggcc cactgtttcg acaaaatcaa gaactggagg aacctgatcg 780 cggtgctggg cgagcacgac ctcagcgagc acgacgggga tgagcagagc cggcgggtgg 840 cgcaggtcat catccccagc acgtacgtcc cgggcaccac caaccacgac atcgcgctgc 900 tccgcctgca ccagcccgtg gtcctcactg accatgtggt gcccctctgc ctgcccgaac 960 ggacgttctc tgagaggacg ctggccttcg tgcgcttctc attggtcagc ggctggggcc 1020 agctgctgga ccgtggcgcc acggccctgg agctcatggt cctcaacgtg ccccggctga 1080 tgacccagga ctgcctgcag cagtcacgga aggtgggaga ctccccaaat atcacggagt 1140 acatgttctg tgccggctac tcggatggca gcaaggactc ctgcaagggg gacagtggag 1200 gcccacatgc cacccactac cggggcacgt ggtacctgac gggcatcgtc agctggggcc 1260 agggctgcgc aaccgtgggc cactttgggg tgtacaccag ggtctcccag tacatcgagt 1320 ggctgcaaaa gctcatgcgc tcagagccac gcccaggagt cctcctgcga

2016244273 13 Oct 2016 gccccatttc ccgacaaaac tcacacgtgc ccgccgtgcc cagctccgga actgctgggc

1380 ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 1440 cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 1500 tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 1560 aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1620 aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1680 tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1740 gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1800 atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1860 gtgttggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1920 tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1980 acgcagaaga gcctctccct gtctccgggt aaatga

2016 <210> 8 <211> 696 <212> PRT <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic construct <400> 8

Met Gin Arg Val Asn Met Ile Met Ala Glu Ser Pro Gly Leu Ile Thr 15 10 15

Ile Cys Leu Leu Gly Tyr Leu Leu Ser Ala Glu Cys Thr Val Phe Leu 20 25 30

Asp His Glu Asn Ala Asn Lys Ile Leu Asn Arg Pro Lys Arg Tyr Asn 35 40 45

Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu Arg Glu Cys

50 55 60

2016244273 13 Oct 2016

Met Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu Val Phe Glu Asn 65 70 75 80

Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val Asp Gly Asp Gln 85 90 95

Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser Cys Lys Asp Asp Ile 100 105 110

Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly Phe Glu Gly Lys Asn Cys 115 120 125

Glu Leu Asp Val Thr Cys Asn Ile Lys Asn Gly Arg Cys Glu Gln Phe 130 135 140

Cys Lys Asn Ser Ala Asp Asn Lys Val Val Cys Ser Cys Thr Glu Gly 145 150 155 160

Tyr Arg Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val Pro Phe 165 170 175

Pro Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr Arg Ala 180 185 190

Glu Thr Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu Ala Glu 195 200 205

Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser Phe Asn Asp Phe 210 215 220

Thr Arg Val Val Gly Gly Glu Asp Ala Lys Pro Gly Gln Phe Pro Trp 225 230 235 240

Gln Val Val Leu Asn Gly Lys Val Asp Ala Phe Cys Gly Gly Ser Ile 245 250 255

Val Asn Glu Lys Trp Ile Val Thr Ala Ala His Cys Val Glu Thr Gly 260 265 270

Val Lys Ile Thr Val Val Ala Gly Glu His Asn Ile Glu Glu Thr Glu

2016244273 13 Oct 2016

275 280

His Thr Glu Gin Lys Arg Asn Val 290 295

Tyr Asn Ala Ala Ile Asn Lys Tyr 305 310

Leu Asp Glu Pro Leu Val Leu Asn 325

Ala Asp Lys Glu Tyr Thr Asn Ile 340

285

Ile Arg Ile Ile Pro His His Asn 300

Asn His Asp Ile Ala Leu Leu Glu 315 320

Ser Tyr Val Thr Pro Ile Cys Ile 330 335

Phe Leu Lys Phe Gly Ser Gly Tyr 345 350

Val Ser Gly Trp Gly Arg Val Phe 355 360

His Lys Gly Arg Ser Ala Leu Val 365

Leu Gin Tyr Leu Arg Val Pro Leu 370 375

Val Asp Arg Ala Thr Cys Leu Arg 380

Ser Thr Lys Phe Thr Ile Tyr Asn 385 390

Asn Met Phe Cys Ala Gly Phe His 395 400

Glu Gly Gly Arg Asp Ser Cys Gin 405

Gly Asp Ser Gly Gly Pro His Val 410 415

Thr Glu Val Glu Gly Thr Ser Phe 420

Leu Thr Gly Ile Ile Ser Trp Gly 425 430

Glu Glu Cys Ala Met Lys Gly Lys 435 440

Tyr Gly Ile Tyr Thr Lys Val Ser 445

Arg Tyr Val Asn Trp Ile Lys Glu 450 455

Lys Thr Lys Leu Thr Glu Phe Ala 460

Gly Ala Ala Ala Val Asp Lys Thr 465 470

His Thr Cys Pro Pro Cys Pro Ala 475 480

Pro Glu Leu Leu Gly Gly Pro Ser 485

Val Phe Leu Phe Pro Pro Lys Pro 490 495

Lys Asp Thr Leu Met Ile Ser Arg 500

Thr Pro Glu Val Thr Cys Val Val 505 510

2016244273 13 Oct 2016

Val Asp Val Ser His Glu Asp Pro 515 520

Asp Gly Val Glu Val His Asn Ala 530 535

Tyr Asn Ser Thr Tyr Arg Val Val 545 550

Asp Trp Leu Asn Gly Lys Glu Tyr 565

Glu Val Lys Phe Asn Trp Tyr Val 525

Lys Thr Lys Pro Arg Glu Glu Gin 540

Ser Val Leu Thr Val Leu His Gin 555 560

Lys Cys Lys Val Ser Asn Lys Ala 570 575

Leu Pro Ala Pro Ile Glu Lys Thr 580

Ile Ser Lys Ala Lys Gly Gin Pro 585 590

Arg Glu Pro Gin Val Tyr Thr Leu 595 600

Pro Pro Ser Arg Asp Glu Leu Thr 605

Lys Asn Gin Val Ser Leu Thr Cys 610 615

Leu Val Lys Gly Phe Tyr Pro Ser 620

Asp Ile Ala Val Glu Trp Glu Ser 625 630

Asn Gly Gin Pro Glu Asn Asn Tyr 635 640

Lys Thr Thr Pro Pro Val Leu Asp 645

Ser Asp Gly Ser Phe Phe Leu Tyr 650 655

Ser Lys Leu Thr Val Asp Lys Ser 660

Arg Trp Gin Gin Gly Asn Val Phe 665 670

Ser Cys Ser Val Met His Glu Ala 675 680

Leu His Asn His Tyr Thr Gin Lys 685

Ser Leu Ser Leu Ser Pro Gly Lys 690 695 <210> 9 <211> 2091 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic construct <400> 9

2016244273 13 Oct 2016

atgcagcgcg 60 tgaacatgat catggcagaa tcaccaggcc tcatcaccat ctgcctttta ggatatctac 120 tcagtgctga atgtacagtt tttcttgatc atgaaaacgc caacaaaatt ctgaatcggc 180 caaagaggta taattcaggt aaattggaag agtttgttca agggaacctt gagagagaat 240 gtatggaaga aaagtgtagt tttgaagaag cacgagaagt ttttgaaaac actgaaagaa 300 caactgaatt ttggaagcag tatgttgatg gagatcagtg tgagtccaat ccatgtttaa 360 atggcggcag ttgcaaggat gacattaatt cctatgaatg ttggtgtccc tttggatttg 420 aaggaaagaa ctgtgaatta gatgtaacat gtaacattaa gaatggcaga tgcgagcagt 480 tttgtaaaaa tagtgctgat aacaaggtgg tttgctcctg tactgaggga tatcgacttg 540 cagaaaacca -gaagtcctgt gaaccagcag tgccatttcc atgtggaaga gtttctgttt 600 cacaaacttc taagctcacc cgtgctgaga ctgtttttcc tgatgtggac tatgtaaatt 660 ctactgaagc tgaaaccatt ttggataaca tcactcaaag cacccaatca tttaatgact 720 tcactcgggt tgttggtgga gaagatgcca aaccaggtca attcccttgg caggttgttt 780 tgaatggtaa agttgatgca ttctgtggag gctctatcgt taatgaaaaa tggattgtaa 840 ctgctgccca ctgtgttgaa actggtgtta aaattacagt tgtcgcaggt gaacataata 900 ttgaggagac agaacataca gagcaaaagc gaaatgtgat tcgaattatt cctcaccaca 960 actacaatgc agctattaat aagtacaacc atgacattgc ccttctggaa ctggacgaac 1020 ccttagtgct aaacagctac gttacaccta tttgcattgc tgacaaggaa tacacgaaca 1080 tcttcctcaa atttggatct ggctatgtaa gtggctgggg aagagtcttc cacaaaggga 1140 gatcagcttt agttcttcag taccttagag ttccacttgt tgaccgagcc

2016244273 13 Oct 2016

acatgtcttc gatctacaaa gttcaccatc tataacaaca tgttctgtgc tggcttccat 1200 gaaggaggta 1260 gagattcatg tcaaggagat agtgggggac cccatgttac tgaagtggaa gggaccagtt 1320 tcttaactgg aattattagc tggggtgaag agtgtgcaat gaaaggcaaa tatggaatat 1380 ataccaaggt atcccggtat gtcaactgga ttaaggaaaa aacaaagctc actgaattcg 1440 ccggcgccgc tgcggtcgac aaaactcaca catgcccacc gtgcccagca cctgaactcc 1500 tggggggacc gtcagtcttc ctcttccccc caaaacccaa ggacaccctc atgatctccc 1560 ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt 1620 tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg cgggaggagc 1680 agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag gactggctga 1740 atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagccccc atcgagaaaa 1800 ccatctccaa agccaaaggg cagccccgag aaccacaggt gtacaccctg cccccatccc 1860 gggatgagct gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc ttctatccca 1920 gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac aagaccacgc 1980 ctcccgtgtt ggactccgac ggctccttct tcctctacag caagctcacc gtggacaaga 2040 gcaggtggca gcaggggaac gtcttctcat gctccgtgat gcatgaggct ctgcacaacc 2091 actacacgca gaagagcctc tccctgtctc cgggtaaatg a

<210> 10 <211> 423 <212> PRT <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic construct <400> 10

Met Ala Leu Thr Phe Ala Leu Leu Val Ala Leu Leu Val Leu Ser Cys

15 10 15

2016244273 13 Oct 2016

Lys Ser Ser Cys Ser Val Gly Cys Asp Leu Pro Gin Thr His Ser Leu 20 25 30

Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gin Met Arg Arg Ile Ser 35 40 45

Leu Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly Phe Pro Gin Glu 50 55 60

Glu Phe Gly Asn Gin Phe Gin Lys Ala Glu Thr Ile Pro Val Leu His 65 70 75 80

Glu Met Ile Gin Gin Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser 85 90 95

Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr 100 105 110

Gin Gin Leu Asn Asp Leu Glu Ala Cys Val Ile Gin Gly Val Gly Val 115 120 125

Thr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys 130 135 140

Tyr Phe Gin Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro 145 150 155 160

Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu 165 170 175

Ser Thr Asn Leu Gin Glu Ser Leu Arg Ser Lys Glu Glu Phe Ala Gly 180 185 190

Ala Ala Ala Val Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 195 200 205

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 210 215 220

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

225

230

235

240

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

245 250 255

2016244273 13 Oct 2016

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr 260 265 270

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp 275 280 285

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 290 295 300

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gin Pro Arg 305 310 315 320

Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 325 330 335

Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 340 345 350

Ile Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys 355 360 365

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 370 375 380

Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser 385 390 395 400

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser 405 410 415

Leu Ser Leu Ser Pro Gly Lys 420 <210> 11 <211> 1272 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic construct

2016244273 13 Oct 2016

<400> 11 atggccttga 60 cctttgcttt actggtggcc ctcctggtgc tcagctgcaa gtcaagctgc tctgtgggct 120 gtgatctgcc tcaaacccac agcctgggta gcaggaggac cttgatgctc ctggcacaga 180 tgaggagaat ctctcttttc tcctgcttga aggacagaca tgactttgga tttccccagg 240 aggagtttgg caaccagttc caaaaggctg aaaccatccc tgtcctccat gagatgatcc 300 agcagatctt caatctcttc agcacaaagg actcatctgc tgcttgggat gagaccctcc 360 tagacaaatt ctacactgaa ctctaccagc agctgaatga cctggaagcc tgtgtgatac 420 agggggtggg ggtgacagag actcccctga tgaaggagga ctccattctg gctgtgagga 480 aatacttcca aagaatcact ctctatctga aagagaagaa atacagccct tgtgcctggg 540 aggttgtcag agcagaaatc atgagatctt tttctttgtc aacaaacttg caagaaagtt 600 taagaagtaa ggaagaattc gccggcgccg ctgcggtcga caaaactcac acatgcccac 660 cgtgcccagc acctgaactc ctggggggac cgtcagtctt cctcttcccc ccaaaaccca 720 aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg gacgtgagcc 780 acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg cataatgcca 840 agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg 900 tcctgcacca ggactggctg aatggcaagg agtacaagtg caaggtctcc aacaaagccc 960 tcccagcccc catcgagaaa accatctcca aagccaaagg gcagccccga gaaccacagg 1020 tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc 1080 tggtcaaagg cttctatccc agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg cctcccgtgt tggactccga cggctccttc

1140

2016244273 13 Oct 2016 ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca

1200 tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct 1260 ccgggtaaat ga

1272 <210> 12 <211> 415 <212> PRT <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic construct <400> 12

Met 1 Ala Leu Thr Phe 5 Ala Leu Leu Val Ala 10 Leu Leu Val Leu Ser 15 Cys Lys Ser Ser Cys Ser Val Gly Cys Asp Leu Pro Gin Thr His Ser Leu 20 25 30 Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gin Met Arg Arg Ile Ser 35 40 45 Leu Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly Phe Pro Gin Glu 50 55 60 Glu Phe Gly Asn Gin Phe Gin Lys Ala Glu Thr Ile Pro Val Leu His 65 70 75 80 Glu Met Ile Gin Gin Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser 85 90 95 Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr 100 105 110 Gin Gin Leu Asn Asp Leu Glu Ala Cys Val Ile Gin Gly Val Gly Val 115 120 125 Thr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys 130 135 140 Tyr Phe Gin Arg lie Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro

145 150

155 160

2016244273 13 Oct 2016

Cys Ala Trp Glu Val Val Arg Ala 165

Ser Thr Asn Leu Gin Glu Ser Leu 180

Thr Cys Pro Pro Cys Pro Ala Pro 195 200

Phe Leu Phe Pro Pro Lys Pro Lys 210 215

Glu Ile Met Arg Ser Phe Ser Leu 170 175

Arg Ser Lys Glu Asp Lys Thr His 185 190

Glu Leu Leu Gly Gly Pro Ser Val 205

Asp Thr Leu Met Ile Ser Arg Thr 220

Pro Glu Val Thr Cys Val Val Val 225 230

Asp Val Ser His Glu Asp Pro Glu 235 240

Val Lys Phe Asn Trp Tyr Val Asp 245

Gly Val Glu Val His Asn Ala Lys 250 255

Thr Lys Pro Arg Glu Glu Gin Tyr 260

Asn Ser Thr Tyr Arg Val Val Ser 265 270

Val Leu Thr Val Leu His Gin Asp 275 280

Trp Leu Asn Gly Lys Glu Tyr Lys 285

Cys Lys Val Ser Asn Lys Ala Leu 290 295

Pro Ala Pro Ile Glu Lys Thr Ile 300

Ser Lys Ala Lys Gly Gin Pro Arg 305 310

Glu Pro Gin Val Tyr Thr Leu Pro 315 320

Pro Ser Arg Asp Glu Leu Thr Lys 325

Asn Gin Val Ser Leu Thr Cys Leu 330 335

Val Lys Gly Phe Tyr Pro Ser Asp 340

Ile Ala Val Glu Trp Glu Ser Asn 345 350

Gly Gin Pro Glu Asn Asn Tyr Lys 355 360

Thr Thr Pro Pro Val Leu Asp Ser 365

Asp Gly Ser Phe Phe Leu Tyr Ser 370 375

Lys Leu Thr Val Asp Lys Ser Arg 380

Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

385 390 395 400

2016244273 13 Oct 2016

His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 405 410 415 <210> 13 <211> 1248 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic construct <400> 13

atggccttga 60 cctttgcttt actggtggcc ctcctggtgc tcagctgcaa gtcaagctgc tctgtgggct 120 gtgatctgcc tcaaacccac agcctgggta gcaggaggac cttgatgctc ctggcacaga 180 tgaggagaat ctctcttttc tcctgcttga aggacagaca tgactttgga tttccccagg 240 aggagtttgg caaccagttc caaaaggctg aaaccatccc tgtcctccat gagatgatcc 300 agcagatctt caatctcttc agcacaaagg actcatctgc tgcttgggat gagaccctcc 360 tagacaaatt ctacactgaa ctctaccagc agctgaatga cctggaagcc tgtgtgatac 420 agggggtggg ggtgacagag actcccctga tgaaggagga ctccattctg gctgtgagga 480 aatacttcca aagaatcact ctctatctga aagagaagaa atacagccct tgtgcctggg 540 aggttgtcag agcagaaatc atgagatctt tttctttgtc aacaaacttg caagaaagtt 600 taagaagtaa ggaagacaaa actcacacgt gcccgccgtg cccagctccg gaactgctgg 660 gcggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg atctcccgga 720 cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg

780

2016244273 13 Oct 2016 gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac

840 tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 900 gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc 960 ccatcccggg atgagctgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 1020 tatcccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 1080 accacgcctc ccgtgttgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg 1140 gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg 1200 cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtaaatga

1248 <210> 14 <211> 256 <212> PRT <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic construct <400> 14

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 15 10 15

Gly Ser Thr Gly Asp Asp Tyr Lys Asp Asp Asp Asp Lys Asp Lys Thr 20 25 30

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 35 40 45

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg 50 55 60

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 65 70 75 80

Giu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 85 90 95

Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val

100 105 110

2016244273 13 Oct 2016

Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr 115 120 125

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 130 135 140 lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu 145 150 155 160

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys 165 170 175

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 180 185 190

Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 195 200 205

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 210 215 220

Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 225 230 235 240

Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 245 250 255 <210> 15 <211> 771 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic construct <400> 15 atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60 gacgactaca aggacgacga tgacaaggac aaaactcaca catgcccacc gtgcccagct 120 ccggaactcc tggggggacc gtcagtcttc ctcttccccc caaaacccaa ggacaccctc 180

2016244273 13 Oct 2016 atgatctccc ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct

240 gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg 300 cgggaggagc agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag 360 gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagccccc 420 atcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggt gtacaccctg 480 cccccatccc gggatgagct gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc 540 ttctatccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac 600 aagaccacgc ctcccgtgtt ggactccgac ggctccttct tcctctacag caagctcacc 660 gtggacaaga gcaggtggca gcaggggaac gtcttctcat gctccgtgat gcatgaggct 720 ctgcacaacc actacacgca gaagagcctc tccctgtctc cgggtaaatg a

771 <210> 16 <211> 444 <212> PRT <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic construct <400> 16

Met Val Pro Cys Thr Leu Leu Leu Leu Leu Ala Ala Ala Leu Ala Pro 15 10 15

Thr Gin Thr Arg Ala Gly Ser Arg Ala Pro Pro Arg Leu Ile Cys Asp 20 25 30

Ser Arg Val Leu Gin Arg Tyr Leu Leu Glu Ala Lys Glu Ala Glu Asn 35 40 45

Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu Asn Ile Thr 50 55 60

Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg Met Glu Val

65 70 75 80

2016244273 13 Oct 2016

Gly Gin Gin Ala Val Glu Val Trp Gin Gly Leu Ala Leu Leu Ser Glu 85 90 95

Ala Val Leu Arg Gly Gin Ala Leu Leu Val Asn Ser Ser Gin Pro Trp 100 105 110

Glu Pro Leu Gin Leu His Val Asp Lys Ala Val Ser Gly Leu Arg Ser 115 120 125

Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gin Lys Glu Ala Ile Ser 130 135 140

Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile Thr Ala Asp 145 150 155 160

Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu Arg Gly Lys 165 170 175

Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp Arg Glu Phe 180 185 190

Gly Gly Glu Tyr Gin Ala Leu Glu Lys Glu Val Ala Gin Leu Glu Ala 195 200 205

Glu Asn Gin Ala Leu Glu Lys Glu Val Ala Gin Leu Glu His Glu Gly 210 215 220

Gly Gly Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285

Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300

Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

2016244273 13 Oct 2016

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335

Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg 340 345 350

Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gin Pro 370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin 405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430

Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 <210> 17 <211> 1335 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic construct <400> 17 atggtaccgt gcacgctgct cctgctgttg gcggccgccc tggctccgac tcagacccgc 60 gccggctcta gagccccacc acgcctcatc tgtgacagcc gagtcctgca gaggtacctc 120 ttggaggcca aggaggccga gaatatcacg acgggctgtg ctgaacactg cagcttgaat 180 gagaatatca ctgtcccaga caccaaagtt aatttctatg cctggaagag gatggaggtc 240

2016244273 13 Oct 2016

gggcagcagg 300 ccgtagaagt ctggcagggc ctggccctgc tgtcggaagc tgtcctgcgg ggccaggccc 360 tgttggtcaa ctcttcccag ccgtgggagc ccctgcagct gcatgtggat aaagccgtca 420 gtggccttcg cagcctcacc actctgcttc gggctctggg agcccagaag gaagccatct 480 cccctccaga tgcggcctca gctgctccac tccgaacaat cactgctgac actttccgca 540 aactcttccg agtctactcc aatttcctcc ggggaaagct gaagctgtac acaggggagg 600 cctgcaggac cggtgacagg gaattcggtg gtgagtacca ggccctggag aaggaggtgg 660 cccagctgga CJCJCGCJ3Cf3clC caggccctgg agaaggaggt ggcccagctg gagcacgagg 720 gtggtggtcc cgcacccgag ctgctgggcg gaccgtcagt cttcctcttc cccccaaaac 780 ccaaggacac cctcatgatc tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga 840 gccacgaaga ccctgaggtc aagttcaact ggtacgtgga cggcgtggag gtgcataatg 900 ccaagacaaa gccgcgggag gagcagtaca acagcacgta ccgtgtggtc agcgtcctca 960 ccgtcctgca ccaggactgg ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag 1020 ccctcccagc ccccatcgag aaaaccatct ccaaagccaa agggcagccc cgagaaccac 1080 aggtgtacac cctgccccca tcccgggatg agctgaccaa gaaccaggtc agcctgacct 1140 gcctggtcaa aggcttctat cccagcgaca tcgccgtgga gtgggagagc aatgggcagc 1200 cggagaacaa ctacaagacc acgcctcccg tgttggactc cgacggctcc ttcttcctct 1260 acagcaagct caccgtggac aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg 1320 tgatgcatga ggctctgcac aaccactaca cgcagaagag cctctccctg tctccgggta 1335 aatga

13 Oct 2016 <N <N o

<N <210> 18 <211> 276 <212> PRT <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic construct <400> 18

Met Val Pro Cys Thr Leu Leu Leu Leu Leu Ala Ala Ala Leu Ala Pro 15 10 15

Thr Gin Thr Arg Ala Gly Glu Phe Gly Gly Glu Tyr Gin Ala Leu Lys 20 25 30

Lys Lys Val Ala Gin Leu Lys Ala Lys Asn Gin Ala Leu Lys Lys Lys 35 40 45

Val Ala Gin Leu Lys His Lys Gly Gly Gly Pro Ala Pro Glu Leu Leu 50 55 60

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 65 70 75 80

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 85 90 95

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 100 105 110

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr 115 120 125

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn 130 135 140

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 145 150 155 160

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin 165 170 175

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val 180 185 190

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

195 200 205

2016244273 13 Oct 2016

Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro 210 215 220

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 225 230 235 240

Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val 245 250 255

Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu 260 265 270

Ser Pro Gly Lys 275 <210> 19 <211> 831 <212> DNA <213> Artificial· Sequence <220>

<223> Description of Artificial Sequence: Synthetic construct <400> 19 atggtaccgt gcacgctgct cctgctgttg gcggccgccc tggctccgac tcagacccgc 60 gccggcgaat tcggtggtga gtaccaggcc ctgaagaaga aggtggccca gctgaaggcc 120 aagaaccagg ccctgaagaa gaaggtggcc cagctgaagc acaagggcgg cggccccgcc 180 ccagagctcc tgggcggacc gtcagtcttc ctcttccccc caaaacccaa ggacaccctc 240 atgatctccc ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct 300 gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg 360 cgggaggagc agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag 420 gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagccccc 480

2016244273 13 Oct 2016 atcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggt gtacaccctg

540 cccccatccc gggatgagct gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc 600 ttctatccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac 660 aagaccacgc ctcccgtgtt ggactccgac ggctccttct tcctctacag caagctcacc 720 gtggacaaga gcaggtggca gcaggggaac gtcttctcat gctccgtgat gcatgaggct 780 ctgcacaacc actacacgca gaagagcctc tccctgtctc cgggtaaatg a

831

<210> 20 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic construct <400> 20 Met Ala Leu Thr Phe Ala Leu Leu Val Ala Leu Leu Val Leu Ser Cys 1 5 10 15

Lys Ser Ser Cys Ser Val Gly Cys Pro Pro Cys Pro Ala Pro Glu Leu 20 25 30

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 35 40 45

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 50 55 60

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 65 70 75 80

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 85 90 95

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 100 105 110

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

2016244273 13 Oct 2016

115 120

125

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 130 135

Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 145 150 155

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 165 170

Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn 180 185

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 195 200

Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn 210 215

Val Met His Glu Ala Leu His Asn His Tyr Thr 225 230 235

Leu Ser Pro Gly Lys 245 <210> 21 <211> 738 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence:

<400> 21 atggccttga cctttgcttt actggtggcc ctcctggtgc 60 tctgtgggct gcccgccgtg cccagctccg gaactgctgg 120 ttccccccaa aacccaagga caccctcatg atctcccgga 180 gtggtggacg tgagccacga agaccctgag gtcaagttca 240 gaggtgcata atgccaagac aaagccgcgg gaggagcagt 300

Gin Pro Arg Glu Pro 140

Leu Thr Lys Asn Gin 160

Pro Ser Asp Ile Ala 175

Asn Tyr Lys Thr Thr 190

Leu Tyr Ser Lys Leu 205

Val Phe Ser Cys Ser 220

Gin Lys Ser Leu Ser 240

Synthetic construct tcagctgcaa gcggaccgtc cccctgaggt actggtacgt acaacagcac gtcaagctgc agtcttcctc cacatgcgtg ggacggcgtg gtaccgtgtg

2016244273 13 Oct 2016 gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag

360 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 420 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 480 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 540 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgttgga ctccgacggc 600 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 660 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 720 ctgtctccgg gtaaatga

738 <210> 22 <211> 430 <212> PRT <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic construct <400> 22

Met Ala Leu Thr Phe Ala Leu Leu Val Ala Leu Leu Val Leu Ser Cys 15 10 15

Lys Ser Ser Cys Ser Val Gly Cys Asp Leu Pro Gin Thr His Ser Leu 20 25 30

Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gin Met Arg Arg Ile Ser 35 40 45

Leu Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly Phe Pro Gin Glu 50 55 60

Glu Phe Gly Asn Gin Phe Gin Lys Ala Glu Thr Ile Pro Val Leu His 65 70 75 80

Glu Met Ile Gin Gin Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser 85 90 95

2016244273 13 Oct 2016

Ala Ala Trp Asp Glu Thr Leu Leu 100

Gin Gin Leu Asn Asp Leu Glu Ala 115 120

Thr Glu Thr Pro Leu Met Lys Glu 130 135

Tyr Phe Gin Arg Ile Thr Leu Tyr 145 150

Cys Ala Trp Glu Val Val Arg Ala 165

Ser Thr Asn Leu Gin Glu Ser Leu 180

Ser Gly Gly Gly Gly Ser Gly Gly 195 200

Cys Pro Pro Cys Pro Ala Pro Glu 210 215

Leu Phe Pro Pro Lys Pro Lys Asp 225 230

Glu Val Thr Cys Val Val Val Asp 245

Asp Lys Phe Tyr Thr Glu Leu Tyr 105 110

Cys Val Ile Gin Gly Val Gly Val 125

Asp Ser Ile Leu Ala Val Arg Lys 140

Leu Lys Glu Lys Lys Tyr Ser Pro 155 160

Glu Ile Met Arg Ser Phe Ser Leu 170 175

Arg Ser Lys Glu Gly Gly Gly Gly 185 190

Gly Gly Ser Asp Lys Thr His Thr 205

Leu Leu Gly Gly Pro Ser Val Phe 220

Thr Leu Met Ile Ser Arg Thr Pro 235 240

Val Ser His Glu Asp Pro Glu Val 250 255

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 260 265 270

Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 275 280 285

Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 290 295 300

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 305 310 315 320

Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro

325 330 335

2016244273 13 Oct 2016

Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val 340 345 350

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 355 360 365

Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 370 375 380

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 385 390 395 400

Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 405 410 415

Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 420 425 430 <210> 23 <211> 1291 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic construct <400> 23 atggccttga cctttgcttt actggtggcc ctcctggtgc tcagctgcaa gtcaagctgc 60 tctgtgggct gtgatctgcc tcaaacccac agcctgggta gcaggaggac cttgatgctc 120 ctggcacaga tgaggagaat ctctcttttc tcctgcttga aggacagaca tgactttgga 180 tttccccagg aggagtttgg caaccagttc caaaaggctg aaaccatccc tgtcctccat 240 gagatgatcc agcagatctt caatctcttc agcacaaagg actcatctgc tgcttgggat 300 gagaccctcc tagacaaatt ctacactgaa ctctaccagc agctgaatga cctggaggcc 360 tgtgtgatac agggggtggg ggtgacagag actcccctga tgaaggagga ctccattctg 420

2016244273 13 Oct 2016

gctgtgagga 480 aatacttcca aagaatcact ctctatctga aagagaagaa atacagccct tgtgcctggg 540 aggttgtcag agcagaaatc atgagatctt tttctttgtc aacaaacttg caagaaagtt 600 tacgtagtaa ggaaggtggc ggcggatccg gtggaggcgg gtccggcggt ggagggagcg 660 acaaaactca cacgtgcccg ccgtgcccag ctccggaact gctgggcgga ccgtcagttt 720 cctcttcccc ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg 780 cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg 840 cgtggaggtg cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg 900 tgtggtcagc gtcctcaccg tcctgcacca ggactggctg aatggcaagg agtacaagtg 960 caaggtctcc aacaaagccc tcccagcccc catcgagaaa accatctcca aagcaaaggg 1020 cagccccgag aaccacaggt gtacaccctg cccccatccc gggatgagct gaccaagaac 1080 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 1140 gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgtt ggactccgac 1200 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac 1260 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 1291 tccctgtctc cgggtaaatg a

<210> 24 <211> 428 <212> PRT <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic construct <400> 24

Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu 15 10 15

2016244273 13 Oct 2016

Leu Ser Leu Pro Leu Gly Leu Pro 20

Ile Cys Asp Ser Arg Val Leu Glu 35 40

Ala Glu Asn Ile Thr Thr Gly Cys 50 55

Asn Ile Thr Val Pro Asp Thr Lys 65 70

Val Leu Gly Ala Pro Pro Arg Leu 25 30

Arg Tyr Leu Leu Glu Ala Lys Glu 45

Ala Glu His Cys Ser Leu Asn Glu 60

Val Asn Phe Tyr Ala Trp Lys Arg 75 80

Met Glu Val Gly Gln Gln Ala Val 85

Glu Val Trp Gln Gly Leu Ala Leu 90 95

Leu Ser Glu Ala Val Leu Arg Gly 100

Gln Ala Leu Leu Val Asn Ser Ser 105 110

Gln Pro Trp Glu Pro Leu Gln Leu 115 120

His Val Asp Lys Ala Val Ser Gly 125

Leu Arg Ser Leu Thr Thr Leu Leu 130 135

Arg Ala Leu Gly Ala Gln Lys Glu 140

Ala Ile Ser Pro Pro Asp Ala Ala 145 150

Ser Ala Ala Pro Leu Arg Thr Ile 155 160

Thr Ala Asp Thr Phe Arg Lys Leu 165

Phe Arg Val Tyr Ser Asn Phe Leu 170 175

Arg Gly Lys Leu Lys Leu Tyr Thr 180

Gly Glu Ala Cys Arg Thr Gly Asp 185 190

Arg Glu Phe Ala Gly Ala Ala Ala 195 200

Val Asp Lys Thr His Thr Cys Pro 205

Pro Cys Pro Ala Pro Glu Leu Leu 210 215

Gly Gly Pro Ser Val Phe Leu Phe 220

Pro Pro Lys Pro Lys Asp Thr Leu 225 230

Met Ile Ser Arg Thr Pro Glu Val 235 240

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

245 250 255

2016244273 13 Oct 2016

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 260 265 270

Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 275 280 285

Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 290 295 300

Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Ly3 Thr lie Scr Lys Ala 305 310 315 320

Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg 325 330 335

Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly 340 345 350

Phe Tyr Pro Ser Asp Xie Ala Val Glu Trp Glu Ser Asn Gly Gin Pro 355 360 365

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 370 375 380

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin 385 390 395 400

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 405 410 415

Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 420 425 <210> 25 <211> 1287 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic construct <400> 25

2016244273 13 Oct 2016

atgggagtgc 60 acgaatgtcc tgcctggctg tggcttctcc tgtccctgct gtcgctccct ctgggcctcc 120 cagtcctggg cgccccacca cgcctcatct gtgacagccg agtcctggag aggtacctct 180 tggaggccaa ggaggccgag aatatcacga cgggctgtgc tgaacactgc agcttgaatg 240 agaatatcac tgtcccagac accaaagtta atttctatgc ctggaagagg atggaggtcg 300 ggcagcaggc cgtagaagtc tggcagggcc tggccctgct gtcggaagct gtcctgcggg 360 gccaggccct gttggtcaac tcttcccagc cgtgggagcc cctgcagctg catgtggata 420 aagccgtcag tggccttcgc agcctcacca ctctgcttcg ggctctggga gcccagaagg 480 aagccatctc ccctccagat gcggcctcag ctgctccact ccgaacaatc actgctgaca 540 ctttccgcaa actcttccga gtctactcca atttcctccg gggaaagctg aagctgtaca 600 caggggaggc ctgcagaaca ggggacagag agttcgccgg cgccgctgcg gtcgacaaaa 660 ctcacacatg cccaccgtgc ccagctccgg aactcctggg cggaccgtca gtcttcctct 720 tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc acatgcgtgg 780 tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg 840 aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg taccgtgtgg 900 tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac aagtgcaagg 960 tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc aaagggcagc 1020 cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc aagaaccagg 1080 tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg gagtgggaga 1140 gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgttggac

2016244273 13 Oct 2016 tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag

1200 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1260 agcctctccc tgtctccggg taaatga

1287 <210> 26 <211> 11 <212> PRT <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic peptide <400> 26

Pro Lys Asn Ser Ser Met Ile Ser Asn Thr Pro

15 10 <210> 27 <211> 7 <212> PRT <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic peptide <400> 27

His Gln Ser Leu Gly Thr Gln

1 5 <210> 28 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic peptide <400> 28

His Gln Asn Leu Ser Asp Gly Lys

1 5 <210> 29 <211> 8 <212> PRT <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic peptide <400> 29

His Gin Asn Ile Ser Asp Gly Lys

2016244273 13 Oct 2016

1 5 <210> <211> <212> <213> 30 8 PRT Artificial Sequence

<220> <223> Description of Artificial Sequence: Synthetic peptide <400> 30

Val Ile Ser Ser His Leu Gly Gin

1 5 <210> <211> <212> <213> 31 8 PRT Artificial Sequence

<220> <223> Description of Artificial Sequence: Synthetic linker peptide <400> 31

Glu Phe Ala Gly Ala Ala Ala Val

1 5 <210> <211> <212> <213> 32 20 PRT Artificial Sequence

<220> <223> Description of Artificial Sequence: Synthetic linker peptide

<220> <223> This sequence may encompass 1-10 Gly-Ala repeats <400> 32

Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala 15 10 15

Gly Ala Gly Ala 20

2016244273 13 Oct 2016 <210> 33 <211> 30 <212> PRT <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic linker peptide <220>

<223> This sequence may encompass 1-10 Gly-Gly-Ser repeats <400> 33

Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly 15 10 15

Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser 20 25 30 <210> 34 <211> 80 <212> PRT <213> Artificial Sequence <220>

<223> Description of Artificial <220>

Sequence: Synthetic linker peptide

<221> misc feature <222> (1) · · ’ (30) <223> This region may encompass <220> <221> misc feature <222> (31)7 ’· (80) <223> This region may encompass <400> 34

1-10 Gly-Gly-Ser repeats

1-10 Gly-Gly-Gly-Gly-Ser repeats

I

-i

Gly Gly Ser Gly Gly Ser Gly Gly 1 5

Gly Ser Gly Gly Ser Gly Gly Ser 20

Gly Gly Ser Gly Gly Gly Gly Ser 35 40

Ser Gly Gly Ser Gly Gly Ser Gly 10 15 Gly Gly Ser Gly Gly Ser Gly Gly 25 30 Gly Gly Gly Gly Ser Gly Gly Gly

Gly Ser Gly Gly Gly Gly 50

Ser Gly Gly Gly Gly 55

Ser Gly Gly Gly Gly 60 ^;i

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

65 70 75 80

2016244273 13 Oct 2016

<210> <211> <212> <213> 35 3 PRT Artificial Sequence <220> <223> Description of Artificial <400> 35 Gly Gly Gly

Sequence: Synthetic linker peptide

<210> <211> <212> <213> 36 7 PRT Artificial Sequence <220> <223> Description of Artificial <400> 36

Sequence: Synthetic linker peptide

Ser Gly Gly Ser Gly Gly Ser 1 5 <210> 37 <211> 15 <212> PRT <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic linker peptide <400> 37

Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gly 15 10 15 <210> 38 <211> 16 <212> PRT <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic linker peptide <400> 38

Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 '5 10 15

2016244273 13 Oct 2016 <210> 39 <211> 18 <212> PRT <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic linker peptide <400> 39

Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly 15 10 15

Gly Ser <210> 40 <211> 50 <212> PRT <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic linker peptide <220>

<223> This sequence may encompass 1-10 Gly-Gly-Gly-Gly-Ser repeats <400> 40

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25 30 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

35 40 45

Gly Ser 50 <210> 41 <211> 19 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic primer

2016244273 13 Oct 2016 <400> 41 gctggctagc caccatgga 19

<210> <211> <212> <213> 42 45 DNA Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer

<400> 42 cttgtcatcg tcgtccttgt agtcgtcacc agtggaacct ggaac 45

<210> <211> <212> <213> 43 67 DNA Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer

<400> 43 gactacaagg acgacgatga caaggacaaa actcacacat gcccaccgtg cccagctccg 60 gaactcc

<210> <211> <212> <213> 44 21 DNA Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer

<400> 44 tagtggatcc tcatttaccc g 21

<210> <211> <212> <213> 45 38 DNA Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 45

2016244273 13 Oct 2016 gctacctgca ggccaccatg gtctcccagg ccctcagg 38

<210> <211> <212> <213> 46 51 DNA Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer

<400> 46 cagttccgga gctgggcacg gcgggcacgt gtgagttttg tcgggaaatg g 51

<210> <211> <212> <213> 47 33 DNA Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer

<400> 47 ttactgcaga aggttatgca gcgcgtgaac atg 33

<210> <211> <212> <213> 48 38 DNA Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer

<400> 48 tttttcgaat tcagtgagct ttgttttttc cttaatcc 38

<210> <211> <212> <213> 49 34 DNA Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer

<400> 49 ggtaagcttg ccatggagct gaggccctgg ttgc 34 <210> 50 <211> 27

2016244273 13 Oct 2016

<212> <213> DNA Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer

<400> 50 gttttcaatc tctaggaccc actcgcc 27

<210> <211> <212> <213> 51 23 DNA Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 51

gccaggccac atgactactc cgc 23

<210> <211> <212> <213> 52 34 DNA Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer

<400> 52 ggtgaattct cactcaggca ggtgtgaggg cage 34

<210> <211> <212> <213> 53 37 DNA Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer

<400> 53 gctactgcag ccaccatggc cttgaccttt gctttac 37

<210> <211> <212> <213> 54 35 DNA Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer

2016244273 13 Oct 2016

<400> cgttga 35 54 attc ttccttactt cttaaacttt cttgc <210> 55 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer

<400> 55 cagttccgga gctgggcacg gcgggcacgt gtgagttttg tcttccttac ttcttaaact 60 ttttgcaagt ttg 73 <210> 56 <211> 45 <212> DNA <213> Artificial Sequence

<220> <223> Description of Artificial Sequence: Synthetic primer <400> 56 gtcaggatcc ggcggtggag ggagcgacaa aactcacacg tgccc 45

<210> 57 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 57 tgacgcggcc gctcatttac ccggagacag gg 32

<210> 58 <211> 32 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic primer <400> 58 ccgctagcct gcaggccacc atggccttga cc

2016244273 13 Oct 2016

<210> <211> <212> <213> 59 34 DNA Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer

<400> 59 ccggatccgc cgccaccttc cttactacgt aaac 34

<210> <211> <212> <213> 60 15 PRT Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic linker peptide <400> 60

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 15 10 15

<210> <211> <212> <213> 61 60 DNA Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer

<400> 61 gtcaggatcc ggtggaggcg ggtccggcgg tggagggagc gacaaaactc acacgtgccc 60

<210> <211> <212> <213> 62 20 DNA Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer

<400> 62 atagaagcct ttgaccaggc 20 <210> 63 <211> 84 <212> DNA

2016244273 13 Oct 2016

<213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide

<400> 63 gccggcgaat tcggtggtga gtaccaggcc ctgaagaaga aggtggccca gctgaaggcc 60 aagaaccagg ccctgaagaa gaag

<210> <211> <212> <213> 64 58 DNA Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide

<400> 64 gtggcccagc tgaagcacaa gggcggcggc cccgccccag agctcctggg cggaccga 58

<210> <211> <212> <213> 65 82 DNA Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide

<400> 65 cggtccgccc aggagctctg gggcggggcc gccgcccttg tgcttcagct gggccacctt 60 cttcttcagg gcctggttct tg

<210> <211> <212> <213> 66 60 DNA Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide

<400> 66 gccttcagct gggccacctt cttcttcagg gcctggtact caccaccgaa ttcgccggca 60 <210> 67 <211> 62 <212> DNA

2016244273 13 Oct 2016

<400> 67 ccggtgacag ggaattcggt ggtgagtacc aggccctgga gaaggaggtg gcccagctgg 60 ag

<210> <211> <212> <213> 68 83 DNA Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 68

gccgagaacc aggccctgga gaaggaggtg gcccagctgg agcacgaggg tggtggtccc 60 gctccagagc tgctgggcgg aca

<210> <211> <212> <213> 69 55 DNA Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide

<400> 69 gtccgcccag cagctctgga gcgggaccac caccctcgtg ctccagctgg gccac 55

<210> <211> <212> <213> 70 90 DNA Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide

<400> 70 ctccttctcc agggcctggt tctcggcctc cagctgggcc acctccttct ccagggcctg 60 gtactcacca ccgaattccc tgtcaccgga

2016244273 13 Oct 2016 <210> 71 <211> 37 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic primer <400> 71 gctactgcag ccaccatggc cttgaccttt gctttac <210> 72 <211> 44 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic primer <400> 72 cagttccgga gctgggcacg gcggagagcc cacagagcag cttg <210> 73 <211> 42 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic primer <400> 73 gtggtcatat gggcattgaa ggcagaggcg ccgctgcggt eg <210> 74 <211> 50 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic primer <400> 74 ggtggttgct cttccgcaaa aacccggaga cagggagaga ctcttctgcg

50 <210> 75 <211> 32 <212> DNA <213> Artificial Sequence <220>

o <223> Description of Artificial Sequence: Synthetic primer <400> 75 aatctagagc cccaccacgc ctcatctgtg ac 32

2016244273 13 Oct 2 <210> 76 <211> 30 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic primer <400> 76 ttgaattctc tgtcccctgt cctgcaggcc <210> 77 <211> 27 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic primer <400> 77 gtacctgcag gcggagatgg gggtgca <210> 78 <211> 22 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic primer <400> 78 cctggtcatc tgtcccctgt cc <210> 79 <211> 56 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic primer <400> 79 gtccaacctg caggaagctt gccgccacca tgggagtgca cgaatgtcct gcctgg 56

2016244273 13 Oct 2016 <210> 80 <211> 67 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic primer <400> 80 gccgaattca gttttgtcga ccgcagcggc gccggcgaac tctctgtccc ctgttctgca 60 ggcctcc

<210> <211> <212> <213> 81 5 PRT Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic linker peptide <400> 81

Gly Gly Gly Gly Ser 1 5

<210> <211> <212> <213> 82 69 DNA Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer

<400> 82 gctgcggtcg acaaaactca cacatgccca ccgtgcccag ctccggaact cctgggcgga 60 ccgtcagtc

<210> <211> <212> <213> 83 36 DNA Artificial Sequence

<220> <223> Description of Artificial Sequence: Synthetic primer

<400> 83 attggaattc tcatttaccc ggagacaggg agaggc 36

2016244273 13 Oct 2016

<210> <211> <212> <213> 84 4 PRT Homo sapiens <400> 84

Glu Leu Leu Gly

<210> <211> <212> <213> 85 10 PRT Artificial Sequence

<220> <223> Description of Artificial Sequence: Synthetic linker peptide

<220> <223> This sequence may encompass 1-10 Gly repeats <400> 85

Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 15 10

<210> <211> <212> <213> 86 10 PRT Artificial Sequence

<220> <223> Description of Artificial Sequence: Synthetic linker peptide <400> 86

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 15 10

<210> <211> <212> <213> 87 7 PRT Artificial Sequence

<220> <223> Description of Artificial Sequence: Synthetic peptide fragment <400> 87

Ser Val Gly Cys Pro Pro Cys 1 5

2016244273 13 Oct 2016

<210> <211> <212> <213> 88 6 PRT Artificial Sequence

<220> <223> Description of Artificial Sequence: Synthetic peptide fragment <400> 88

Val Gly Cys Pro Pro Cys 1 5

<210> <211> <212> <213> 89 7 PRT Artificial Sequence

<220> <223> Description of Artificial Sequence: Synthetic peptide fragment <400> 89

Ser Thr Gly Cys Pro Pro Cys

1 5 <210> <211> <212> <213> 90 4 PRT Artificial Sequence

<220> <223> Description of Artificial Sequence: Synthetic peptide fragment <400> 90

Cys Pro Pro Cys

1 <210> <211> <212> <213> 91 8 PRT Artificial Sequence

<220> <223> Description of Artificial Sequence: Synthetic FLAG peptide <400> 91

Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 <210> 92

2016244273 13 Oct 2016

<211> <212> <213> 48 DNA Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer

<400> 92 ctagcctgca ggaagcttgc cgccaccatg accaacaagt gtctcctc 48

<210> <211> <212> <213> 93 51 DNA Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer

<400> 93 tttgtcgacc gcagcggcgc cggcgaactc gtttcggagg taacctgtaa g 51

<210> <211> <212> <213> 94 37 DNA Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer

<400> 94 ggcaagcttg ccgccaccat ggagacagac acactcc 37

<210> <211> <212> <213> 95 40 DNA Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer

<400> 95 tcagtggtga tggtgatgat gtttacccgg agacagggag 40

<210> <211> <212> <213> 96 53 DNA Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer

<400> 96 ggtaaacatc atcaccatca ccactgagaa ttccaatatc actagtgaat teg

2016244273 13 Oct 2016 <210> 97 <211> 32 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic primer <400> 97 getatttagg tgacactata gaatactcaa gc <210> 98 <211> 1269 <212> DNA <213> Homo sapiens <400> 98

atgaccaaca 60 agtgtctcct ccaaattgct ctcctgttgt gcttctccac tacagctctt tccatgagct 120 acaacttgct tggattccta caaagaagca gcaattttca gtgtcagaag ctcctgtggc 180 aattgaatgg gaggettgaa tattgcctca aggacaggat gaactttgac atccctgagg 240 agattaagca gctgcagcag ttccagaagg aggacgccgc attgaccatc tatgagatgc 300 tccagaacat etttgetatt ttcagacaag attcatctag cactggctgg aatgagacta 360 ttgttgagaa cctcctggct aatgtctatc atcagataaa ccatctgaag acagtcctgg 420 aagaaaaact ggagaaagaa gatttcacca ggggaaaact catgagcagt ctgcacctga 480 aaagatatta tgggaggatt ctgcattacc tgaaggccaa ggagtacagt cactgtgcct 540 ggaccatagt cagagtggaa atcctaagga acttttactt cattaacaga cttacaggtt 600 acctccgaaa cgagttcgcc ggcgccgctg cggtcgacaa aactcacaca tgcccaccgt 660 gcccagctcc ggaactcctg ggcggaccgt cagtcttcct cttcccccca

2016244273 13 Oct 2016 aaacccaagg acaccctcat gatctcccgg acccctgagg tcacatgcgt ggtggtggac

720 gtgagccacg aagaccctga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat 780 aatgccaaga caaagccgcg ggaggagcag tacaacagca cgtaccgtgt ggtcagcgtc 840 ctcaccgtcc tgcaccagga ctggctgaat ggcaaggagt acaagtgcaa ggtctccaac 900 aaagccctcc cagcccccat cgagaaaacc atctccaaag ccaaagggca gccccgagaa 960 ccacaggtgt acaccctgcc cccatcccgg gatgagctga ccaagaacca ggtcagcctg 1020 acctgcctgg tcaaaggctt ctatcccagc gacatcgccg tggagtggga gagcaatggg 1080 cagccggaga acaactacaa gaccacgcct cccgtgttgg actccgacgg ctccttcttc 1140 ctctacagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt cttctcatgc 1200 tccgtgatgc atgaggctct gcacaaccac tacacgcaga agagcctctc cctgtctccg 1260 ggtaaatga

1269 <210> 99 <211> 422 <212> PRT <213> Homo sapiens <400> 99

Met Thr Asn Lys Cys Leu Leu Gin lie Ala Leu Leu Leu Cys Phe Ser 15 10 15

Thr Thr Ala Leu Ser Met Ser Tyr Asn Leu Leu Gly Phe Leu Gin Arg 20 25 30

Ser Ser Asn Phe Gin Cys Gin Lys Leu Leu Trp Gin Leu Asn Gly Arg 35 40 45

Leu Glu Tyr Cys Leu Lys Asp Arg Met Asn Phe Asp He Pro Glu Glu 50 55 60 lie Lys Gin Leu Gin Gin Phe Gin Lys Glu Asp Ala Ala Leu Thr He

65 70 75 80

2016244273 13 Oct 2016

Tyr Glu Met Leu Gin Asn Ile Phe Ala Ile Phe Arg Gin Asp Ser Ser 85 90 95

Ser Thr Gly Trp Asn Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val 100 105 110

Tyr His Gin Ile Asn His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu 115 120 125

Lys Glu Asp Phe Thr Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys 130 135 140

Arg Tyr Tyr Gly Arg lie Leu His Tyr Leu Lys Ala Ly3 Glu Tyr Ser 145 150 155 160

His Cys Ala Trp Thr Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr 165 170 175

Phe Ile Asn Arg Leu Thr Gly Tyr Leu Arg Asn Glu Phe Ala Gly Ala 180 185 190

Ala Ala Val Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 195 200 205

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 210 215 220

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 225 230 235 240

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 245 250 255

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn 260 265 270

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp 275 280 285

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 290 295 300

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gin Pro Arg Glu

305 310 315 320

2016244273 13 Oct 2016

Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 325 330 335

Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 340 345 350

Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr 355 360 365

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 370 375 380

Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys 385 390 395 400

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu 405 410 415

Ser Leu Ser Pro Gly Lys 420 <210> 100 <211> 102 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic primer <400> 100 tttaagcttg ccgccaccat ggagacagac acactcctgc tatgggtact gctgctctgg 60 gttccaggtt ccactggtga caaaactcac acatgcccac eg

102 <210> 101 <211> 22 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic primer <400> 101

2016244273 13 Oct 2016 ggtcagctca tcgcgggatg gg 22

<210> <211> <212> <213> 102 22 DNA Artificial Sequence <220> <223> Description of Artificial <400> 102 cccatcccgc gatgagctga cc 22

Sequence: Synthetic primer

<210> <211> <212> <213> 103 9 DNA Artificial Sequence <220> <223> Description of Artificial <400> 103 gccgccacc 9

Sequence: Synthetic Kozak sequence

<210> <211> <212> <213> 104 23 DNA Artificial Sequence <220> <223> Description of Artificial <400> 104 gagcagtacg ctagcacgta ccg 23

Sequence: Synthetic primer

<210> <211> <212> <213> 105 23 DNA Artificial Sequence <220> <223> Description of Artificial <400> 105 ggtacgtgct agcgtactgc tcc 23

Sequence: Synthetic primer

<210> 106 <211> 69

2016244273 13 Oct 2016 <212> DNA <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic primer <400> 106 ctgacggtcc gcccaggagt tccggagctg ggcacggtgg gcatgtgtga gttttgtcga 60 ccgcagcgg <210> 107 <211> 6 <212> PRT <213> Artificial Sequence <220>

<223> Description of Artificial Sequence: Synthetic 6xHis tag <400> 107

His His His His His His

1 5