NZ618897B2 - Tnf-alpha antigen-binding proteins with increased fcrn binding - Google Patents

Abstract

Disclosed is an antigen binding protein which specifically binds to TNF-alpha comprising: (i) CDRH1 of DYAMH, CDRH2 of AITWNSGHIDYADSVEG, CDRH3 of VSYLSTASSLDY, CDRL1 of RASQGIRNYLA, CDRL2 of AASTLQS, and CDRL3 of QRYNRAPYT; and (ii) a neonatal Fc receptor (FcRn) binding portion of a human IgG1 constant domain comprising amino acid substitutions relative to the human IgG1 constant domain wherein the amino acid substitutions are at amino acid residues 252, 254 and 256 numbered according to EU index of Kabat and the substitution at residue 252 is a substitution of met with tyr; residue 254 is a substitution of ser with thr and residue 256 is a substitution of thr with glu: and wherein the antigen binding protein has an increased FcRn binding affinity at pH 6 and/or increased half-life as compared to an IgG comprising the light chain sequence of SEQ ID No. 2 and the heavy chain sequence of SEQ ID No. 12. Also disclosed it the use of such an antigen binding protein in the manufacture of a medicament for the treatment of rheumatoid arthritis, polyarticular juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease or Psoriasis. nstant domain comprising amino acid substitutions relative to the human IgG1 constant domain wherein the amino acid substitutions are at amino acid residues 252, 254 and 256 numbered according to EU index of Kabat and the substitution at residue 252 is a substitution of met with tyr; residue 254 is a substitution of ser with thr and residue 256 is a substitution of thr with glu: and wherein the antigen binding protein has an increased FcRn binding affinity at pH 6 and/or increased half-life as compared to an IgG comprising the light chain sequence of SEQ ID No. 2 and the heavy chain sequence of SEQ ID No. 12. Also disclosed it the use of such an antigen binding protein in the manufacture of a medicament for the treatment of rheumatoid arthritis, polyarticular juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease or Psoriasis.

Description

TNF-Alpha n-binding ns with increased FcRn binding Field The invention s to novel variants of anti-TNF antibodies.

Background Antibodies are multimeric glycoproteins comprising at least two heavy and two light chains.

Aside from IgM, intact antibodies are usually heterotetrameric glycoproteins of approximately 150Kda, composed of two identical light (L) chains and two cal heavy (H) chains. Each heavy chain has at one end a variable domain (VH) ed by a number of constant regions. Each light chain has a le domain (VL) and a constant region at its other end; the constant region of the light chain is aligned with the first constant region of the heavy chain and the light chain variable domain is aligned with the variable domain of the heavy chain. Depending on the amino acid sequence of the constant region of their heavy , human antibodies can be assigned to five different classes, IgA, IgD, IgE, IgG and IgM. IgG and IgA can be further subdivided into subclasses, IgG1, IgG2, IgG3 and IgG4; and IgA1 and IgA2. The variable domain of the antibody confers binding specificity upon the antibody with certain regions displaying particular variability called complementarity ining regions (CDRs). The more conserved portions of the variable region are called Framework regions (FR). The variable domains of intact heavy and light chains each se four FR connected by three CDRs. The constant regions are not directly involved in the binding of the antibody to the antigen but exhibit various effector functions such as participation in antibody dependent cellmediated cytotoxicity (ADCC), phagocytosis via binding to Fc receptor, half-life/clearance rate via neonatal Fc receptor (FcRn) and ment dependent cytotoxicity via the C1q component of the complement cascade. The nature of the structure of an IgG antibody is such that there are two antigen-binding sites, both of which are specific for the same epitope. They are therefore, monospecific.

In adult mammals, FcRn, also known as the neonatal Fc receptor, plays a key role in maintaining serum antibody levels by acting as a tive receptor that binds and es antibodies of the IgG isotype from degradation. IgG molecules are endocytosed by endothelial cells, and if they bind to FcRn, are recycled out into circulation. In contrast, IgG molecules that do not bind to FcRn enter the cells and are targeted to the lysosomal y where they are degraded.

The neonatal FcRn receptor is believed to be involved in both antibody clearance and the transcytosis across tissues (see Junghans R.P (1997) Immunol.Res 16. 29-57 and Ghetie et al (2000) Annu.Rev.Immunol. 18, 739-766).

WO 9734631 discloses a composition comprising a mutant IgG molecule having sed serum half-life and at least one amino acid substitution in the Fc-hinge region. Amino acid substitution at one or more of the amino acids selected from number 252, 254, 256, 309, 311 or 315 in the CH2 domain or 433 or 434 in the CH3 domain is disclosed.

WO 00/42072 discloses a polypeptide comprising a variant Fc region with altered FcRn binding ty, which polypeptide comprises an amino acid modification at any one or more of amino acid ons 238, 252, 253, 254, 255, 256, 265, 272, 286, 288, 303, 305, 307, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 386,388, 400, 413, 415, 424,433, 434,435, 436, 439, and 447 of the Fc region.

WO 02/060919 discloses a modified lgG comprising an lgG constant domain comprising amino acid modifications at one or more of positions 251, 253, 255, 285-290, 4, 385-389, and 428-435.

WO 2004035752 discloses a modified antibody of class lgG wherein at least one amino acid residue from the heavy chain constant region selected from the group consisting of amino acid residues 250, 314, and 428 is different from that present in an unmodified class lgG antibody.

Shields et al. (2001, J Biol Chem ; 276:6591-604) used alanine scanning mutagenesis to alter residues in the Fc region of a human lgG1 antibody and then assessed the binding to human FcRn.

Positions that effectively abrogated binding to FcRn when changed to e include I253, S254, H435, and Y436. Other positions showed a less pronounced reduction in binding as follows: E233- G236, R255, K288, L309, S415, and H433. Several amino acid positions exhibited an improvement in FcRn binding when changed to alanine. cqua et al. (2002, J lmmunol.;169:5171-80) described random mutagenesis and screening of human lgG1 hinge-Fc fragment phage display libraries against mouse FcRn. They disclosed random mutagenesis of positions 251, 252, 254-256, 308, 309, 311, 312, 314, 7, 389, 428, 433, 434, and 436.

WO2006130834 discloses ed lgG comprising an lgG comprising an lgG nt domain sing amino acid modifications at one or more positions of 252, 254, 256, 433, 434 and 436.

Therefore, modification of Fc domains of lgG dies has been discussed as a means of increasing the serum half- life of therapeutic antibodies. However, numerous such modifications have been suggested with varying and sometimes contradictory results in different antibodies.

The administration of n binding proteins as therapeutics requires injections with a prescribed ncy relating to the clearance and ife characteristics of the protein.

Adalimumab is a monoclonal antibody against TNF-alpha which is used for treatment of rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and Crohn's e. It is produced by recombinant DNA technology using a mammalian cell expression system. It consists of 330 amino acids and has a molecular weight of approximately 148 kilodaltons. See United States Patent 6090382. At doses of 0.5 mg/kg (~40 mg), clearance for adalimumab is said to range from 11 to 15 ml/hour, the distribution volume (Vss) ranges from 5 to 6 litres and the mean terminal phase half-life was approximately two weeks ry of Product teristics available from www.medicines.org.uk). These half life and clearance properties mean that currently adalimumab needs to be administered once every two weeks. In some patients depending on disease it may be necessary to administer a loading dose such as for e in psoriasis patients. This dosage may differ from the maintenance dose.

Summary of invention In one aspect, the invention relates to an antigen binding n which specifically binds to TNF-alpha comprising CDRH1 of SEQ ID NO: 27, CDRH2 of SEQ ID NO: 28, CDRH3 of SEQ ID No: 29, CDRL1 of SEQ ID NO: 30, CDRL2 of SEQ ID NO: 31, and CDRL3 of SEQ ID NO: 32; and a neonatal Fc receptor (FcRn) binding portion of a human IgG1 constant domain comprising amino acid substitutions ve to the human IgG1 constant domain, wherein the amino acid substitutions are at amino acid residues 252, 254 and 256 numbered according to EU index of Kabat and the substitution at residue 252 is a substitution of met with tyr; residue 254 is a substitution of ser with thr and residue 256 is a substitution of thr with glu; and wherein the antigen binding protein has an increased FcRn binding affinity at pH 6 and/ or increased half-life as ed to an IgG comprising the light chain sequence of SEQ ID No. 2 and the heavy chain sequence of SEQ ID No.12.

Throughout the specification the term “human IgG1 constant domain” encompasses all pes and variants thereof known to a person skilled in the art.

In one aspect, the invention relates to an n binding protein which specifically binds to TNF-alpha comprising CDRH1 (SEQ ID NO: 27), CDRH2 (SEQ ID NO: 28), CDRH3 (SEQ ID No: 29), CDRL1 (SEQ ID NO: 30), CDRL2 (SEQ ID NO: 31), and CDRL3 (SEQ ID NO: 32); or variants thereof wherein said variants may contain 1, 2, 3 or 4 amino acid substitutions, insertions or ons as compared to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3; and a neonatal Fc receptor (FcRn) binding portion of a human IgG1 constant domain comprising one of more amino acid substitutions relative to the human IgG1 constant domain, wherein the antigen binding protein has an increased half life as ed to an IgG sing the light chain sequence of SEQ ID No. 2 and heavy chain sequence of SEQ ID No.12 and the antigen binding protein can be administered no more than once every four weeks to achieve comparable mean steady-state trough concentration as that ed by the same dose of IgG comprising the light chain ce of SEQ ID No. 2 and the heavy chain sequence of SEQ ID No.12 administered once every two weeks.

In one aspect, the invention relates to an antigen binding protein which specifically binds to TNF-alpha comprising CDRH1 (SEQ ID NO: 27), CDRH2 (SEQ ID NO: 28), CDRH3 (SEQ ID No: 29), CDRL1 (SEQ ID NO: 30), CDRL2 (SEQ ID NO: 31), and CDRL3 (SEQ ID NO: 32) or variants thereof n said ts may contain 1, 2, 3 or 4 amino acid substitutions, insertions or deletions as compared to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3; and an FcRn binding portion of a human IgG1 constant domain comprising one of more amino acid substitutions relative to the human IgG1 constant domain, wherein the antigen binding n has an affinity for FcRn of 2 fold, or 3 fold, or 4 fold or 5 fold, or 6 fold or 8 fold or greater than an anti-TNF antigen binding protein with the same CDR’s without such modifications at pH 6 as assessed by ProteOn XPR36 protein interaction array system at 25°C, the array system having antigen binding proteins immobilised on the chip.

In one aspect, the invention relates to an antigen binding protein which is a variant of an lgG comprising the light chain sequence of SEQ ID No. 2 and the heavy chain sequence of SEQ ID No.12, wherein the antigen binding protein variant ses one or more substitutions in the neonatal Fc receptor (FcRn) binding portion of the IgG constant domain to increase the half-life of the n binding protein variant ed with the IgG t such substitutions wherein when the variant is stered to patients at a single dose of 40 mg at a four to eight weekly interval, the mean steady-state trough concentration in the patient population does not fall below 4pg/ml or does not fall below 5 pg /m| between dosing intervals. Preferably, the mean serum trough antibody tration in the patient population does not fall below 6 pg /m| between dosing als.

Preferably, the mean serum trough antibody concentration in the patient population does not fall below 5 pg /m| between dosing intervals when the variant is administered to patients at a single dose of 40 mg at an eight weekly interval. .Preferably, the mean serum trough antibody concentration in the patient population does not fall below 4 pg /m| between dosing intervals whilst still ing the optimal cy when the variant is administered to patients at a single dose of 40 mg at an eight weekly interval. Preferably, the mean serum trough antibody concentration in the t population does not fall below 3 pg /m| n dosing intervals whilst still providing the optimal efficacy when the variant is administered to patients at a single dose of 40 mg at an eight weekly interval.

In one aspect, the invention relates to an antigen binding protein as disclosed herein for treatment of a e wherein the antigen binding protein can be administered to patients no more than once every four weeks to e comparable mean steady-state trough tration as that achieved by the same dose of an lgG comprising light chain sequence of SEQ ID No.2 and heavy chain sequence of SEQ ID No.12 administered once every two weeks.

In one aspect, the invention relates to a method of treating a patient with a disease, the method comprising administering an antigen binding protein according to the invention.

In one aspect, the invention relates to a nucleic acid sequence encoding the antigen binding protein according to the ion, or a part thereof such as a heavy or light chain. In one aspect, the invention relates to an expression vector encoding the antigen binding protein according to the invention, or a part thereof such as a heavy or light chain.

In one aspect, the invention relates to a host cell comprising the nucleic acid sequence ng the antigen binding protein ing to the invention. In one aspect, the ion relates to an antigen binding protein according to the invention for use in the treatment of Psoriasis or rheumatoid arthritis.

In one aspect, the invention relates to a kit comprising the antigen binding protein according to the invention, and optionally sing methotrexate for concomitant delivery of antigen binding protein according to the invention and methotrexate.

In one aspect, the invention relates to an n binding protein as sed herein for treatment of Rheumatoid arthritis in an individual who is already being treated with methotrexate, and to an antigen binding protein in combination with methotrexate for treatment of Rheumatoid arthritis, wherein the combination is delivered simultaneously, ntially simultaneously, or tially.

In one aspect, the invention relates to an antigen binding protein as disclosed herein for treatment of Psoriasis in an individual who is already being d with methotrexate, and to an antigen binding n in combination with methotrexate for treatment of Psoriasis, wherein the combination is delivered simultaneously, substantially aneously, or sequentially.

Brief Description of Figures Figure 1 - Binding of anti-TNFq antibodies to human TNFq Figure 2 — Analysis of binding activity of anti-TNFor antibodies to human TNFq following an accelerated stressor study Figure 3 — g of anti-TNFor dies to human TNFq following incubation in 25% human serum for 2 weeks Figure 4 — Binding of anti-TNFor antibodies to human TNFq following freeze-thaw Figure 5 — Analysis of NFor antibodies to Ia receptors (a) Binding to human chRllla (valine 158 variant) (b) Binding to human FCyRIIIA (phenylalanine 158 variant) Figure 6 - Average dose normalised plasma concentrations of BPC2604 in female cynomolgus monkeys and pascolizumab in male cynomolgus monkeys following a single intravenous (1 hr infusion) Detailed Description of Invention The invention relates to novel n binding proteins binding specifically to TNF-alpha. In particular, the invention relates to novel variants of anti-TNF antibodies such as adalimumab which show increased binding to the FcRn receptor and/ or increased half life as compared to adalimumab.

Adalimumab is an lgG monoclonal antibody comprising the light chain sequence of SEQ ID No. 2 and heavy chain sequence of SEQ ID No.12.

The inventors have found that specific modifications to adalimumab as described herein show particular improvements in FcRn binding as shown in the examples below. Affinity matured variants of adalimumab also show improvement in anti-TNF-alpha binding and/or neutralisation activity.

The novel antigen binding proteins of the invention have an increased binding to the FcRn receptor and/ or increased half life and/ or increased Mean Residence Time and/ or decreased Clearance. It is considered that binding to FcRn results in longer serum retention in vivo. In order to increase the retention of the Fc proteins in vivo, the increase in binding affinity is ed around pH 6. In one aspect, the present invention therefore provides an n binding protein with optimised binding to FcRn.

In one embodiment, the ife of the antigen binding protein of the t ion is increased 2 to 6 fold, such as 2 fold, 3 fold, 4 fold, 5 fold or 6 fold as compared to an lgG comprising the light chain sequence of SEQ ID No. 2 and heavy chain sequence of SEQ ID No.12. Preferably, the half- Iife of the antigen binding protein of the invention is increased 3 fold, 4 fold, or more compared to an lgG sing the light chain sequence of SEQ ID No. 2 and heavy chain sequence of SEQ ID No.12. For example, if the IgG is umab having a half life of 10 days or in the range of 10 to 20 days then in one embodiment an antigen binding protein of the t invention shows a half life of about 40 to 80 days. For example an antigen binding protein comprising a heavy chain ce selected from SEQ ID NO:5 or SEQ ID NO:9 or SEQ ID NO:15 or SEQ ID NO:18. or SEQ ID NO:21. or SEQ ID NO:24 or SEQ ID NO:163, or SEQ ID NO:165, or SEQ ID NO:167, or SEQ ID NO:169.

In one embodiment, the antigen binding protein of the invention administered no more than once every four weeks in patients, achieves mean steady-state trough concentrations between about 2 ug/ml to about 7 ug/ml. Preferably, the mean steady-state trough concentrations are between about 4 ug/ml to about 7 ug/ml and more preferably between about 5 ug/ml to about 6 ug/ml.

In one embodiment, the antigen binding protein of the invention administered no more than once every 28 days in patients, achieves mean steady-state trough concentrations n about 2 ug/ml to about 7 ug/ml. Preferably, the mean steady-state trough concentrations are between about 4 ug/ml to about 7 ug/ml and more preferably between about 5 ug/ml to about 6 ug/ml.

In one embodiment of the invention, the antigen binding protein of the invention can be administered once every 4, 5, 6, 7 or 8 weeks to achieve comparable mean steady-state trough concentrations as those achieved by umab, when administered once every two weeks at the same dose.

In a preferred embodiment of all aspects of the invention, the antigen binding protein of the invention can be administered once every 7 or 8 weeks.

In one ment of the invention, the antigen binding protein of the invention can be administered once every 25-80 days for e once every 40-60 days, or for example once every 28, 35, 42, 49 or 56 days to achieve comparable mean steady-state trough concentrations as those achieved by adalimumab, when administered once every 14 days at the same dose.

In one embodiment of the invention, the antigen binding protein can be administered once every 49 to 60 day, for example every 56 days.

In an embodiment of all aspects of the ion, the antigen binding protein has a 2 fold, or 4 fold, or 6 fold, or 8 fold or greater affinity for human FcRn at pH 6 as assessed by ProteOn XPR36 protein interaction array system at 25°C wherein the antibodies are immobilised on the chip. Preferably, the antigen binding protein has an affinity for human FcRn between about 100 to about 500 KD(nM), such as between about 130 to about 360 KD(nM) or between about 140 to about 250KD(nM) or between about 140 to about 210KD(nM).

In one embodiment, the clearance of the antigen g protein is about 2 to about 10 ml/hr, preferably about 2 to about 5m|/hr or 2 to 4m|/hr or 2 to 3m|/hr, such as about 2, about 2.5, 3, 4 or 5 ml/hr. In one ment the n binding protein of the invention shows a clearance rate which is 2 fold, 3 fold, 4 fold or 5 fold lower than adalimumab. In one embodiment, clearance for an antigen binding protein according to the ion is in the ranges specified above or 2 fold, 3 fold, 4 fold or 5 fold lower than adalimumab at a human dose of about 40 mg.

In one aspect, the antigen g protein of the invention is a variant of adalimumab (lgG comprising the light chain sequence of SEQ ID No. 2 and the heavy chain sequence of SEQ ID No.12), the variant comprising one or more substitutions in the FcRn binding portion of the lgG constant domain to increase the half-life of the variant compared with adalimumab, wherein when the variant is administered to patients at a single dose of 40 mg at a four to eight weekly interval, preferably eight weekly al, the mean -state trough antibody concentration in the t population does not fall below 5 pg /m|. In one embodiment the mean steady-state trough antibody concentration in the patient tion does not fall below 6 pg /m|, between dosing intervals.

In a further embodiment, the antigen binding n comprises at least one amino acid modification in the Fc region of said n binding protein, wherein said modification is at one or more of positions 250, 252, 254, 256, 257, 259, 308, 428 or 434 of the Fc region as compared to same position in the adalimumab sequence, wherein the numbering of the amino acids in the Fc region is that of the EU index in Kabat.

The wild type human lgG1 has amino acid residues Val-Leu-His-Gln-Asp-Trp-Leu at positions 308- 314, amino acid residues Leu-Met- lle-Ser-Arg-Thr at positions 251-256, amino acid residues Met- His-Glu-Ala-Leu-His-Asn-HisTyr at ons 428-436, and amino acid residues Gly—Gln-Pro-Glu-Asn at positions 385-389. Residue numbering may differ for lgG2-4.

Described are embodiments where the antigen binding protein of the invention comprises one or more amino acid tution relative to the human IgG1 constant domain sing the sequence of SEQ ID No. 13.

Described are embodiments where the one or more amino acid substitution in the FcRn g portion of the human IgG1 heavy chain constant domain is at amino acid residues 252, 254 and 256 numbered according to EU index of Kabat and the aa substitution at residue 252 is a substitution of met with tyr, phe, tryp or thr; the aa substitution at residue 254 is a substitution of ser with thr; and the aa substitution at residue 256 is a substitution of thr with ser, arg, glu, asp or thr. In the embodiments of the invention, the aa substitution at residue 252 is a substitution with tyr; the aa substitution at residue 254 is a substitution with thr and the substitution at residue 256 is a substitution with glu. Preferably, the IgG1 constant domain is as shown in SEQ ID No: 7.

In one embodiment, the one or more amino acid substitutions in the FcRn binding portion of the human IgG1 constant domain is at amino acid residues 250 and 428 numbered ing to EU index of Kabat and the aa substitution at residue 250 is a substitution of thr with glu or gln; the aa substitution at residue 428 is a substitution of met with leu or phe. Preferably, the aa substitution at residue 250 is a tution with glu and the aa substitution at residue 428 is a substitution with leu. Preferably, the IgG1 nt domain is as shown in SEQ ID No: 16.

In one embodiment, the one or more amino acid substitution in the FcRn binding portion of the human IgG1 constant domain is at amino acid es 428 and/ or 434 numbered according to EU index of Kabat. ably, the aa substitution at residue 428 is a substitution of met with leu and the aa tution at residue 434 is a substitution of asn with ser. Preferably, the IgG1 constant domain is as shown in SEQ ID No: 10.

In one embodiment, the one or more amino acid substitution in the FcRn binding portion of the human IgG1 constant domain is at amino acid residues 259 or 308 numbered according to EU index of Kabat.

Preferably, the substitution at residue 259 is a substitution of val with ile and the aa substitution at residue 308 is a tution of val with phe. Preferably, the IgG1 constant domain is as shown in SEQ ID No: 19 or SEQ ID No: 22.

In one embodiment, the one or more amino acid substitution in the FcRn binding portion of the human IgG1 heavy chain constant domain is at amino acid residues 257 and 434 numbered according to EU index of Kabat as shown in SEQ ID No: 25.

In one embodiment, the one or more amino acid tution in the FcRn binding portion of the human IgG1 heavy chain constant domain is at amino acid residues 433 and 434 numbered according to EU index of Kabat for example the residues are H433K and N434F Preferably, the IgG1 constant domain 40 is as shown in SEQ ID No: 165 or SEQ ID No: 167.

WO 11076 In one embodiment, the n binding protein comprises any of the IgG1 constant domain modifications listed in Table A.

In one embodiment, the antigen binding protein is an antibody.

In one embodiment, the antigen binding protein comprises a variable domain of SEQ ID NO: 6 and/or SEQ ID NO: 3 or a variant f which contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, insertions or deletions and/or shares at least 90% identity across the length of SEQ ID NO: 6 or SEQ ID NO: 3.

In one embodiment, the antigen g protein comprises the heavy chain sequence as shown in SEQ ID No 5, 9 or 15 optionally with a light chain sequence as shown in SEQ ID No: 2.

In one embodiment, the antigen binding protein comprises a variable heavy domain sequence as shown in SEQ ID NO: 78 or 80.

In one embodiment, the antigen binding protein comprises a heavy chain sequence as shown in SEQ ID NO: 145 or SEQ ID NO: 146 optionally with a light chain variant as shown in SEQ ID Nos. 148, 150 or 152.

In one embodiment, the antigen binding protein comprises the heavy chain sequence as as shown in SEQ ID No 18 or 21 optionally with a light chain sequence as shown in SEQ ID No: 2.

In one embodiment the antigen g n according to the invention comprises any of the variable domains specified in Table A. In one embodiment, the antigen binding protein according to the invention comprises the variable heavy domain having the sequence of VH, -VH, cb1VH, cb1VH, cb2VH, cb2VH, cb2VH, cb2-28—VH, cb2-38—VH, cb2VH, cb1- 8—VL or cb1VL as shown in Table A.

In one embodiment, the antigen binding n according to the invention ses the variable light domain having the sequence of cb1VL, cb1VL, cb1VL, cb1VL, cb1VL, cb1VL, cb1VL, cb1VL, cb1VL, cb1VL, -VL, cb1VL, cb1VL, VL, cb2 VL, cb2VL, cb2VL, cb2-28—VL, cb2VL, cb1VL, cb2VL or cb2VL as shown in Table A.

For example, the antigen binding protein according to the invention comprises a variable domain having the sequence of cb1-3VH, cb2-44VH or cb2-6VL as shown in Table A.

In one embodiment the antigen binding protein according to the invention comprises any of the variable domains specified in Table A. In one embodiment, the antigen binding n according to the invention comprises the variable heavy domain having a sequence selected from SEQ ID NO: 170 or SEQ ID NO: 174 or SEQ ID NO:178 In one embodiment, the antigen binding protein according to the invention comprises the variable Iight domain having a sequence selected from SEQ ID NO: 171 or SEQ ID NO: 175 or SEQ ID NO:179 In a further embodiment the antigen binding protein comprises any of the IgG1 constant domain modifications listed in Table A.

Variants of all the above mentioned variable domains or heavy chain sequences or light chain sequences which contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, insertions or deletions and/or share at least 90% identity across the length of any of these ces are also within the scope of the ion.

In one embodiment, the antigen binding protein of the invention comprises a variant of CDRH3 (SEQ ID No: 29) which variant has 1, 2, 3 or 4 amino acid substitutions as compared to SEQ ID No: 29. In one embodiment, the variant CDRH3 may have the sequence as shown in any one of SEQ ID Nos. 40 to 49.

In one ment, the n binding protein of the invention comprises a variant of CDRH1 (SEQ ID No: 27) which variant has 1 or 2 amino acid substitutions as compared to SEQ ID No: 27. In one embodiment, the variant CDRH1 may have the sequence as shown in any one of SEQ ID Nos. 33 to In one ment, the antigen binding protein of the invention ses a variant of CDRL1 (SEQ ID No: 30) which variant has 1, 2 or 3 amino acid substitutions as ed to SEQ ID No: 30. In one embodiment, the variant CDRL1 may have the sequence as shown in any one of SEQ ID Nos. 50 to In one embodiment, the antigen binding n of the invention comprises a t of CDRL2 (SEQ ID No: 31) which variant has 1, 2 or 3 amino acid substitutions as compared to SEQ ID No: 31. In one ment, the variant CDRL2 may have the sequence as shown in any one of SEQ ID Nos. 62 to In one embodiment, the antigen binding protein of the invention comprises a variant of CDRL3 (SEQ ID No: 32) which variant has 1, 2 or 3 amino acid substitutions as compared to SEQ ID No: 32. In one embodiment, the variant CDRL3 may have the sequence as shown in any one of SEQ ID Nos. 73 to In one embodiment, the invention relates to an antigen g protein which specifically binds to TNF-alpha comprising one or more or all CDRs selected from: CDRH1 (SEQ ID NO: 27), CDRH2 (SEQ ID NO: 28), CDRH3 (SEQ ID No: 29), CDRL1 (SEQ ID NO: 30), CDRL2 (SEQ ID NO: 31), and CDRL3 (SEQ ID NO: 32); wherein any of the CDRs could be a variant CDR which contains 1, 2, 3 or 4 amino acid tutions, ions or deletions as compared to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3. In one aspect, the antigen binding protein of the invention comprises CDRH1, CDRH3, CDRL1, CDRL2 and CDRL3 wherein any of the CDRs could be a variant CDR which contains 1, 2, 3 or 4 amino acid substitutions, insertions or deletions ed to CDRH1, CDRH3, CDRL1, CDRL2, or CDRL3. In one aspect, the antigen binding protein of the invention comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 wherein any of the CDRs could be a variant CDR which contains 1, 2, 3 or 4 amino acid substitutions, insertions or ons compared to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3 In one aspect, the ion s to a method of treating a human patient with a disease, the method comprising administering an antigen binding protein according to the invention.

The invention also relates to an antigen binding protein as disclosed herein for the treatment of disease in a human.

The invention also relates to use of an n binding protein as disclosed herein in the manufacture of a medicament for the treatment of disease, and an antigen binding protein as disclosed herein for use in treatment of disease.

In one embodiment, the disease to be treated by the antigen binding protein of the invention is toid arthritis, polyarticularjuvenile idiopathic arthritis, psoriatic arthritis, ankylosing litis, Ulcerative colitis, spondyloarthropathy, Crohn's disease or Psoriasis.

In one embodiment, the antigen binding protein of the invention is to be administered with methotrexate. The methotrexate can be delivered before, after or at the same time, or ntially the same time, as the antigen binding protein. In a preferred embodiment the n binding protein of the ion is to be administered with methotrexate to a patient suffering from rheumatoid arthritis. In one ment, methotrexate is administered to patients receiving an antigen binding protein of the invention to reduce the genic effect of the antigen binding protein. In one embodiment, the antigen binding protein of the invention is administered to patients already receiving methotrexate. Methotrexate may be substituted by another acceptable compound which reduced the immune se to the antigen binding protein, for example corticosteroids.

In one aspect, the invention relates to a method of treating a patient with a disease, the method comprising administering an antigen binding protein of the invention. In one embodiment, the method ses administering an antigen binding protein to the patient as a single 20, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 mg dose no more than once every four weeks, preferably once every 5, 6, 7, or 8 weeks and most preferably once every 8 weeks. Preferably, the dose is 40 to 80 mg, for example 40mg.

The invention also es a polynucleotide sequence encoding any amino acid sequence sed , including a heavy chain of any of the antigen binding constructs described herein, and a polynucleotide encoding a light chain of any of the antigen binding constructs described herein. Such polynucleotides represent the coding ce which corresponds to the lent polypeptide sequences, however it will be understood that such polynucleotide sequences could be cloned into an expression vector along with a start codon, an appropriate signal sequence and a stop codon. The polynucleotide may be DNA or RNA.

The invention also provides a host cell, for example a recombinant, transformed or ected cell, comprising one or more polynucleotides ng a heavy chain and/or a light chain of any of the antigen binding constructs described herein.

The invention further provides a pharmaceutical composition comprising an antigen binding construct as described herein a pharmaceutically acceptable carrier.

The invention further provides a method for the production of any of the antigen binding constructs described herein which method comprises the step of culturing a host cell comprising a first and second vector, said first vector comprising a polynucleotide encoding a heavy chain of any of the antigen binding constructs described herein and said second vector comprising a polynucleotide encoding a light chain of any of the antigen binding constructs described herein, in a serum- free / chemically defined / animal d component free culture media. atively a method may comprise culturing a host cell comprising a vector comprising a polynucleotide encoding a heavy chain of any of the antigen binding constructs described herein and a cleotide encoding a light chain of any of the antigen binding constructs described herein, suitably in a serum- free / chemically defined / animal derived ent free culture media.

In another embodiment, the ion es a method of increasing the half-life of an antibody by modifying an Fc according to the cations described herein.

In another embodiment, the invention includes an antigen binding protein as described herein with enhanced FcRn binding and having one or more onal substitutions, deletions or insertions that modulate another property of the effector function.

Once expressed by the desired method, the antigen g protein of the invention is then examined for in vitro activity by use of an appropriate assay. Presently conventional ELISA and Biacore assay formats are employed to assess qualitative and quantitative binding of the antigen binding construct to its target. Additionally, other in vitro assays may also be used to verify neutralizing efficacy prior to subsequent human clinical studies performed to evaluate the persistence of the antigen binding protein in the body despite the usual clearance mechanisms.

The dose and duration of treatment relates to the relative duration of the molecules of the present invention in the human circulation, and can be adjusted by one of skill in the art depending upon the condition being treated and the general health of the patient based on the information ed herein. It is envisaged that repeated dosing (e.g. once every 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeks) over an extended time period (e.g. four to six months) maybe required to achieve maximal therapeutic efficacy.

The mode of administration of the eutic agent of the ion may be any suitable route which delivers the agent to the host. The antigen binding proteins, and pharmaceutical compositions of the invention are particularly useful for eral administration, i.e., subcutaneously (s.c.), intrathecally, intraperitoneally, intramuscularly (i.m.), intravenously (i.v.), or intranasally. In one embodiment the n binding proteins and pharmaceutical compositions of the invention are administered via a subcutaneous auto injector pen or a subcutaneous pre-filled syringe.

Antigen binding proteins of the invention may be prepared as pharmaceutical compositions containing an effective amount of the antigen binding n of the invention as an active ingredient in a pharmaceutically acceptable carrier. In the prophylactic agent of the ion, an aqueous suspension or on containing the antigen binding construct, preferably buffered at logical pH, in a form ready for injection is preferred. The compositions for eral administration will commonly comprise a solution of the antigen binding uct of the invention or a cocktail thereof dissolved in a pharmaceutically acceptable r, preferably an aqueous carrier. A variety of aqueous carriers may be employed, e.g., 0.9% , 0.3% e, and the like. These solutions may be made sterile and generally free of particulate . These solutions may be sterilized by conventional, well known ization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, etc. The concentration of the antigen binding protein of the invention in such pharmaceutical formulation can vary widely, i.e., from less than about 0.5%, usually at or at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc., according to the particular mode of stration selected.

It has been reported that adalimumab is difficult to formulate at high trations. W02004016286 describes an adalimumab formulation comprising a e-phosphate buffer and other components including a polyol and a ent. The oral presentation “Humira® - from Development to Commercial Scale Production” presented on 25 October 2005 at the PDA Conference reports formulations comprising (i) e-phosphate buffer; (ii) acetate-phosphate buffer; and (iii) ate buffer. The acetate-phosphate buffer tested displayed the worst ising effect upon adalimumab.

Curtis et al. (2008) Current Medical Research and Opinion, Volume 27, p71-78, report the incidence of injection-site burning and stinging in patients with rheumatoid arthritis using injectable adalimumab.

The burning and stinging has been partly attributed to citrate -based formulations (Basic and al Pharmacology & Toxicology, Volume 98, p218—221, 2006; and Journal of Pharmaceutical Sciences, Volume 97, p3051-3066, 2008). However, WO20100129469 describes a high adalimumab concentration ation that still comprises a citrate-phosphate buffer and other components including a polyol with no sodium chloride. The more recent WO2012065072 describes an adalimumab formulation comprising a surfactant and a polyol with no buffer, thus potentially avoiding any citrate buffer effects upon injection.

In one ment there is provided a liquid formulation comprising a TNF-alpha antigen binding n and an acetate buffer. In a further embodiment the TNF-alpha binding protein comprises a CDRH1 selected from SEQ ID NO:27 or SEQ ID NO:’s 33-38 and/or a CDRH2 of SEQ ID NO:28 and/or a CDRH3 selected from SEQ ID NO:29 or SEQ ID NO:’s 40-49 and/or a CDRL1 selected from SEQ ID NO:30 or SEQ ID NO:’s 50-61 and/or a CDRL2 selected from SEQ ID NO:31 or SEQ ID NO:’s 62-72 and/or a CDRL3 of SEQ ID NO:32 or SEQ ID NO:’s73-76. For example the TNF-alpha antigen binding protein comprises CDRH1 of SEQ ID N027 and CDRH2 of SEQ ID N028 and CDRH3 of SEQ ID N029 and CDRL1 of SEQ ID NO:30 and CDRL2 ed from SEQ ID NO:31 and a CDRL3 of SEQ ID NO:32 or variants f.

The TNF-alpha antigen binding n may be adalimumab. The TNF-alpha antigen binding protein may be BPC1494. The TNF-alpha antigen binding protein may be BPC 1496.

The TNF-alpha antigen binding proteins described herein are formulated in an acetate buffer. The formulation may be in liquid form. The formulation may further comprise one or more, a ation, or all of: a surfactant; a chelator; a salt; and an amino acid. The pha antigen binding proteins are formulated at high concentrations, for example at 50 mg/mL. In one embodiment, the formulation does not comprise a polyol. In another embodiment, the formulation does not comprise a further buffer component, for e citrate. Therefore, the formulations described herein solve the problem of providing TNF-alpha antigen binding proteins, in particular the TNF-alpha n binding proteins as described in Table A, at high concentrations in a stable formulation, and avoid the burning and stinging effects of citrate-based buffers.

In one embodiment, the acetate buffer ation further comprises a surfactant and a chelator. In r embodiment, the acetate buffer formulation further comprises a surfactant and a salt. In another embodiment, the e buffer ation further comprises a surfactant and an amino acid.

In another embodiment, the acetate buffer formulation further comprises a chelator and a salt. In another ment, the acetate buffer formulation further comprises a chelator and an amino acid.

In another embodiment, the acetate buffer formulation further comprises a salt and an amino acid.

In one ment, the acetate buffer formulation further comprises a surfactant, a chelator, and a salt. In another embodiment, the acetate buffer formulation further comprises a surfactant, a chelator, and an amino acid. In another embodiment, the acetate buffer formulation further comprises a surfactant, a salt, and an amino acid. In another embodiment, the acetate buffer ation further comprises a chelator, a salt, and an amino acid.

In one embodiment, the buffer is sodium acetate trihydrate. This may be at a concentration of 10 to 100 mM sodium acetate trihydrate (1.361 to g/mL). Sodium acetate trihydrate may be present in an amount of 20 to 80 mM, 30 to 70 mM, 40 to 60 mM, or about 40mM, about 45mM, about 50mM, about 55mM, or about 60mM. In one embodiment, sodium acetate trihydrate is at a concentration of about 50mM (6.80mg/mL).

The acetate buffer may be the sole . In other words, the formulation may not comprise another buffer component, such as phosphate or citrate buffer. Citrate buffer may be detrimental to the formulation for a number of reasons: (i) it may not be a good buffer because the values of the three dissociation constants are too close to permit ction of the three proton receptor ; (ii) citrate may act as a metal or and thus nce metal ion balance: (iii) citrate is a metabolite of the citric acid cycle and has the potential to influence cellular lism.

Suitable surfactants (also known as detergents) may include, e.g., polysorbates (for example, polysorbate 20 or 80), polyoxyethylene alkyl ethers such as Brij 35.RTM., poloxamers (for example poloxamer 188, Poloxamer 407), Tween 20, Tween 80, Cremophor A25, Sympatens ALM/230, and Mirj. In one embodiment, the surfactant is polysorbate 80. The formulation may comprise a concentration of 0.01 to 0.1 % polysorbate 80 (0.1 to 1mg/mL). Polysorbate 80 may be t in an amount of 0.01 to 0.05%, or 0.01 to 0.03%; or about 0.015%, about 0.02%, or about 0.025%. In one embodiment, polysorbate 80 is at a concentration of about 0.02% w/v (0.2mg/mL). A high tration of polysorbate 80, for example more than 0.1%, may be detrimental to the formulation because this surfactant may contain high levels of oxidants which may increase levels of oxidation upon storage of the formulation and therefore reduce shelf life. 2012/064129 Suitable chelating agents may e EDTA and metal complexes (e.g. Zn-protein complexes). In one embodiment, the chelating agents is EDTA. The formulation may comprise a concentration of 0.02 to 0.2 mM EDTA (0.00748 to 0.0748mg/mL). EDTA may be present in an amount of 0.02 to 0.15 mM, 0.02 to 0.1 mM, 0.03 to 0.08 mM, or 0.04 to 0.06 mM; or about 0.03 mM, about 0.04 mM, about 0.05 mM, or about 0.06 mM. In one embodiment, EDTA is at a concentration of about 0.05mM (0.018mg/mL).

Suitable salts may include any orming counterions, such as sodium. For example, sodium chloride may be used, or c acetate instead of chloride as a counterion in a sodium salt may be used. In one embodiment, the salt is sodium de. The formulation may comprise a concentration of 25 to 100 mM sodium chloride (1.461 to 5.84mg/mL). Sodium chloride may be present in an amount of 35 to 90 mM, 45 to 80 mM, 25 to 70 mM, or 45 to 60mM; or 45mM, 46mM, 47mM, 48mM, 49mM, 50mM, 51mM, 52mM, 53mM, 54mM, 55mM. In one embodiment, sodium chloride is at a concentration of about 51mM (2.98mg/mL).

Suitable amino acids may e ne. The formulation may comprise a concentration of 0.5 to % arginine free base (5 to 50mg/mL). Arginine free base may be present in an amount of In other embodiments, the arginine free base may be between 0.5 to 4.0%, 0.5 to 3.5%, 0.5 to 3.0%, 0.5 to 2.5%, or about 0.5%, about 0.75%, about 1%, about 1.5%, about 2%, or about 3%. In one embodiment, arginine is at a concentration of about 1% (10mg/mL).

A polyol is a substance with multiple hydroxyl groups, and includes sugars (reducing and non- reducing sugars), sugar alcohols and sugar acids. Examples of polyols include fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose, glucose, sucrose, trehalose, sorbose, melezitose, raffinose, mannitol, l, erythritol, threitol, sorbitol, glycerol, L-gluconate and metallic salts f. In one embodiment, the formulation of the invention does not comprise a polyol.

In one embodiment, the acetate buffer formulation further comprises one or more, a combination, or all of: polysorbate 80, EDTA, sodium de, and arginine free base.

The pH of the formulation may be adjusted to pH 5.0 to 7.0. In one embodiment, acetic acid is present (about 100 mM acetic acid) to adjust the formulation to about pH 5.5. In other embodiments, the pH may be ed to pH 5.0, 5.5, 6.0, 6.5 or 7.0. In yet other embodiments of the invention, NaOH or HCl is used to adjust the pH to 5.0, 5.5, 6.0, 6.5 or 7.0.

The TNF-alpha antigen binding proteins described herein may be formulated in the tration range of 20 to 300 mg/mL. For example, the antigen binding n is present in a concentration of -200 mg/mL or 50-100 mg/mL; or about 40 mg/mL or about 45 mg/mL or about 50 mg/mL or about 55 mg/mL or about 60 mg/mL or about 70 mg/mL or about 80 mg/mL or about 90 mg/mL, or about 100mg/mL. In one embodiment, the TNF-alpha antigen binding protein is at a concentration of about 50 mg/mL.

The TNF-alpha antigen g protein may be ada|imumab. The TNF-alpha n binding protein may be BPC1494. The TNF-alpha antigen binding protein may be BPC 1496.

In one embodiment, the formulation is stable for at least 1 year, at least 18 months, or at least 2 years. For example, the formulation is stable at a temperature of about 5°C for at least 1 year, at least 18 months, or at least 2 years. In another embodiment, the formulation is stable at room ature (about 25°C). For example, the formulation is stable at a ature of about 25°C for at least 14 weeks, at least 2 weeks, at least 1 week, at least 6 days, at least 5 days, at least 4 days, at least 3 days, at least 2 days or at least 1 day. In another embodiment, the formulation is stable at a ature of about 40°C. For example, the formulation is stable at a temperature of about 40°C for at least 9 weeks or at least 4 weeks.

As shown by Examples 25 and 26 below, the formulations are stable at room temperature (about °C). Therefore, there is l risk of aggregates or low molecular weight fragments forming in pre-filled devices for injection that may be left at room temperature for more than the recommended time. Aggregates are potentially immunogenic (see The AAPS Journal 2006; 8 (3) Article 59 Themed lssue: Proceedings of the 2005 AAPS Biotec Open Forum on Aggregation of Protein Therapeutics, Guest Editor - Steve Shire, Effects of Protein Aggregates: An Immunologic Perspective) and low molecular weight fragments may illicit pre-existing autoantibodies (see J l 2008; 83- 3192; Human Anti-lgG1 Hinge Autoantibodies Reconstitute the Effector Functions of Proteolytically lnactivated lgGs1).

The stability of a pha antigen binding protein in a liquid formulation may be assessed by any one or a combination of: appearance by visual observation, protein concentration (A280nm), size exclusion chromatography (SEC), Capillary lso-Electric Focussing (c-lEF), and by a onal binding assay ). For example, the percentage of monomer, aggregate, or fragment, or combinations thereof, can be used to determine stability. In one embodiment, a stable liquid formulation is a formulation having less than about 10%, or less than about 5% of the TNF-alpha antigen binding protein being present as aggregate in the formulation. The formulation may have a monomer content of at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%. The formulation may have a r content of at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% at room temperature (about 25°C) after about 2 weeks. The formulation may have a monomer content of at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% at 2012/064129 room temperature (about 25°C) after about 1 week. The ation may have a monomer content of at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% at room temperature (about 25°C) after about 1 day.

Thus, a pharmaceutical composition of the ion for injection could be prepared to n 1 mL sterile buffered water, and between about 1 mg to about 100 mg, e.g. about 30 mg to about 100 mg or more preferably, about 35 mg to about 80mg, such as 40, 50, 80 or 90 mg of an antigen binding construct of the invention. Actual methods for preparing parenterally strable compositions are well known or will be apparent to those skilled in the art and are described in more detail in, for example, Remington's ceutical Science, 15th ed., Mack Publishing Company, Easton, Pennsylvania. For the preparation of intravenously administrable antigen binding construct formulations of the invention see Lasmar U and Parkins D “The formulation of Biopharmaceutical products”, Pharma. Sci.Tech.today, page 129-137, Vol.3 (3rd April 2000), Wang, W “Instability, stabilisation and formulation of liquid protein pharmaceuticals”, Int. J. Pharm 185 (1999) 129-188, Stability of Protein Pharmaceuticals Part A and B ed Ahern T.J., Manning M.C., New York, NY: Plenum Press (1992), Akers,M.J. ient-Drug interactions in Parenteral Formulations”, J.Pharm Sci 91 (2002) 300, lmamura, K et al “Effects of types of sugar on ization of Protein in the dried state”, J Pharm Sci 92 (2003) 266-274,lzutsu, Kkojima, S. “Excipient crystalinity and its protein- structure-stabilizing effect during freeze-drying”, J Pharm. Pharmacol, 54 (2002) 1033-1039, Johnson, R, “Mannitol-sucrose mixtures-versatile formulations for protein lyophilization”, J. Pharm. Sci, 91 (2002) 914-922. ably, the antigen binding protein of the invention is provided or administered at a dose of about 40 mg. Preferably the antigen binding protein is suitable for subcutaneous delivery and is delivered subcutaneously. Other dosing or administration routes may also be used, as disclosed herein.

In one emboduiment the antigen binding proteins according to any aspect of the invention shows increased Mean Residence Time as compared to an lgG comprising the light chain sequence of SEQ ID No.2 and heavy chain sequence of SEQ ID No.12.

The binding ability of modified lgGs and molecules comprising an lgG constant domain or FcRn binding portion thereof can be characterized by various in vitro assays. PCT publication WO 97/34631 by Ward discloses various methods in detail. For e, in order to compare the ability of the modified lgG or fragments f to bind to FcRn with that of the wild type lgG, the modified lgG or fragments f and the wild type lgG can be radio-labeled and reacted with FcRn-expressing cells in vitro. The radioactivity of the cell-bound fractions can be then counted and compared. The cells sing FcRn to be used for this assay are may be endothelial cell lines including mouse pulmonary capillary endothelial cells (B10, D2.PCE) d from lungs of B10.DBA/2 mice and SV40 transformed endothelial cells (SVEC) (Kim et al., J lmmunol., 40: 457-465,1994) derived from C3H/HeJ mice. However, other types of cells which express ient number of FcRn, including mammalian cells which express recombinant FcRn of a species of , can be also used.

Alternatively, after counting the radioactivity of the bound fraction of modified lgG or that of fied lgG, the bound molecules can be then extracted with the detergent, and the percent release per unit number of cells can be calculated and compared.

Affinity of antigen g ns of the inventions for FcRn can be measured by surface plasmon resonance (SPR) measurement using, for example, a BlAcore 2000 (BlAcore Inc.) as described previously (Popov et al., Mol. lmmunol., 33: 493-502,1996; Karlsson et al., J lmmunol. Methods, 145: 229-240,1991, both of which are incorporated by reference in their entireties). In this method, FcRn molecules are coupled to a e sensor chip (e. g., CM5 chip by Pharmacia) and the binding of modified lgG to the immobilized FcRn is measured at a certain flow rate to obtain sensorgrams using BIA evaluation 2.1 software, based on which on-and off-rates of the modified lgG, constant domains, or nts thereof, to FcRn can be calculated. ve affinities of antigen g proteins of the invention and unmodified lgG for FcRn can be also measured by a simple competition binding assay. rmore, affinities of modified lgGs or fragments thereof, and the wild type lgG for FcRn can be also measured by a saturation study and the Scatchard analysis.

Transfer of modified lgG or fragments f across the cell by FcRn can be measured by in vitro transfer assay using radiolabeled lgG or fragments thereof and FcRn- expressing cells and comparing the ctivity of the one side of the cell monolayer with that of the other side. Alternatively, such er can be measured in vivo by feeding 10-to 14-day old suckling mice with radiolabeled, modified lgG and periodically counting the radioactivity in blood samples which indicates the transfer of the lgG through the intestine to the circulation (or any other target , e. g., the lungs). To test the dose-dependent inhibition of the lgG transfer through the gut, a mixture of radiolabeled and unlabeled lgG at certain ratio is given to the mice and the radioactivity of the plasma can be ically measured (Kim et al., Eur. R lmmunol., 24: 2429-2434,1994).

The half-life of antigen binding proteins can be measured by cokinetic studies according to the method described by Kim et al. (Eur. J. of lmmuno. 24: 542,1994), which is incorporated by reference herein in its entirety. According to this method, abeled antigen binding protein is injected intravenously into mice and its plasma concentration is periodically measured as a function of time, for example, at 3 minutes to 72 hours after the injection. The clearance curve thus obtained should be biphasic. For the determination of the in vivo half-life of the modified lgGs or fragments thereof, the clearance rate in B-phase is calculated and compared with that of the unmodified lgG.

Antigen binding ns of the invention may be d for the ability to immunospecifically bind to an antigen. Such an assay may be performed in solution (e. g., Houghten, BiolTechniques, 13: 412- 421,1992), on beads (Lam, Nature, 354: 82-84,1991, on chips (Fodor, Nature, 364: 6,1993), on bacteria (U. S. Patent No. 5,223,409), on spores (U. S. Patent Nos. 698; 5,403,484; and ,223,409), on plasmids (Cull et al., Proc. Natl. Acad. Sci. USA, 89: 1865-1869,1992) or on phage (Scott and Smith, Science, 249: 386-390,1990; Devlin, Science, 249: 404-406,1990; Cwirla et al., Proc. Natl. Acad. Sci. USA, 87: 6378-6382, 1990; and Felici, J : Mol. Biol., 222: 301-310, 1991) (each of these references is incorporated herein in its entirety by reference). Antibodies that have been identified to immunospecifically bind to an antigen or a fragment thereof can then be assayed for their specificity affinity for the antigen.

The antigen binding proteins of the invention may be d for immunospecific g to an antigen and cross-reactivity with other antigens by any method known in the art. lmmunoassays which can be used to e immunospecific binding and cross-reactivity include, but are not limited to, competitive and non-competitive assay s using ques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay),"sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, scent immunoassays, protein A immunoassays, to name but a few. Such assays are e and well known in the art (see, e. g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc, New York, which is incorporated by nce herein in its entirety). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).

In a preferred embodiment, BlAcore kinetic analysis is used to determine the binding on and off rates of antibodies to an antigen. BlAcore kinetic analysis ses analyzing the binding and dissociation of an antigen from chips with immobilized antibodies on their surface.

Antigen binding protein: The term “antigen binding protein” as used herein includes reference to antibodies, antibody fragments and other n constructs, which are capable of binding to TNF- alpha.

Antibody: The term “antibody” is used herein in the broadest sense and includes reference to molecules with an immunoglobulin-like domain and includes monoclonal, recombinant, polyclonal, chimeric, humanised, bispecific and heteroconjugate antibodies.

Human lgG1 heavy chain constant domain: refers to human amino acid ce for the lgG1 heavy chain constant domain that is found in nature, including allelic variations.

“Half-life (t1/2)” refers to the time required for the concentration of the n binding polypeptide to reach half of its original value. The serum half-life of proteins can be measured by pharmacokinetic studies according to the method described by Kim et al. (Eur. J. of lmmuno. 24: 542, 1994). According to this method, radiolabeled protein is injected intravenously into mice and its plasma concentration is periodically measured as a function of time, for example, at about 3 s to about 72 hours after the injection. Other methods for pharmacokinetic analysis and determination of the half-life of a molecule will be familiar to those skilled in the art. Details may be found in Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and in Peters et al, Pharmacokinetic analysis: A Practical Approach (1996). Reference is also made to “Pharmacokinetics”, M Gibaldi & D , published by Marcel Dekker, 2nd Rev. ex edition (1982), which describes pharmacokinetic parameters such as t alpha and t beta half lives and area under the curve (AUC), and “Clinical Pharmacokinetics: Concepts and Applications”, Rowland and Tozer, Third Edition (1995).

“Clearance (CL)” refers to the volume of plasma irreversibly cleared of a protein per unit time.

Clearance is ated as the Dose/AUC (AUC : is the Area Under Curve or Area under the plasma drug concentration time curve). Clearance can also be calculated by the rate of drug ation divided by the plasma tration of the drug (rate of elimination = CL*concentration) “Mean nce Time (MRT)” is the average time that the antigen binding polypeptides reside in the body before being irreversibly eliminated. Calculated as MRT= AUMC/AUC.

“Steady state concentration” (Css) is the concentration reached when the drug elimination rate becomes equal to drug administration rate as a result of ued drug administration. Css fluctuates between peak and trough levels and is measured in microgram/ml. “Mean steady-state trough concentration” refers to the mean of the trough level across the patient tion at a given time.

“Comparable mean steady-state trough concentration” refers to mean steady-state trough concentration which is the same or within about 10% to 30% of the stated value. Comparable mean steady-state trough concentration for the antigen binding polypeptides of the ion may be considered to be those mean steady-state trough concentrations that are 0.8 to 1.25 times the mean steady-state trough concentration achieved with an lgG comprising the light chain sequence of SEQ ID No.2 and the heavy chain ce of SEQ ID No. 12.

Half lives and AUC can be determined from a curve of serum concentration of drug (for example the n binding ptide of the present invention) against time. Half life may be determined through tmental or non-compartmental analysis. The WINNONLINTM analysis package (available from Pharsight Corp., Mountain View, CA94040, USA) can be used, for example, to model the curve. In one embodiment, “half life” refers to the terminal half life. 2012/064129 Specifically binds: The term “specifically binds” as used throughout the t specification in relation to antigen binding proteins means that the antigen binding protein binds to pha with no or insignificant binding to other unrelated proteins. The term however does not exclude the fact that the antigen binding proteins may also be cross-reactive with closely related molecules. The n g proteins described herein may bind to TNF-alpha with at least 2, at least 5, at least 10, at least 50, at least 100, or at least 1000 fold greater affinity than they bind to closely related molecules.

CDRs: “CDRs” are defined as the complementarity determining region amino acid sequences of an antigen binding protein. These are the hypervariable regions of immunoglobulin heavy and light chains. There are three heavy chain and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Thus, "CDRs" as used herein refers to all three heavy chain CDRs, all three light chain CDRs, all heavy and light chain CDRs, or at least two CDRs.

Throughout this specification, amino acid residues in variable domain sequences and full length dy sequences are ed according to the Kabat numbering convention. Similarly, the terms “CDR”, “CDRL1”, “CDRL2”, “CDRL3”, “CDRH1”, “CDRH2”, “CDRH3” used in the Examples follow the Kabat numbering convention. For further information, see Kabat et al., Sequences of Proteins of Immunological lnterest, 4th Ed., US. Department of Health and Human Services, National Institutes of Health (1987). % identity of variants : The term “identical" or “sequence identity” indicates the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with riate insertions or deletions. The variants described herein may have 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to the native CDR or variable domain sequences at the amino acid level.

It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine study, numerous equivalents to the specific procedures described herein. Such lents are ered to be within the scope of this ion and are covered by the claims. All ations and patent ations mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains.

All publications and patent applications are herein orated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be orated by reference. The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more, at least one," and "one or more than one." The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and r." Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not e additional, unrecited elements or method steps. In one aspect such open ended terms also comprise within their scope a restricted or closed definition, for e such as "consisting essentially of", or "consisting of".

The term "or combinations thereof" as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, sly included are ations that contain s of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, , and so forth. The skilled artisan will understand that lly there is no limit on the number of items or terms in any combination, unless otherwise nt from the t.

All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

All documents referred to herein are incorporated by nce to the fullest extent permissible.

Any element of a sure is explicitly contemplated in combination with any other element of a disclosure, unless otherwise apparent from the context of the application.

The present ion is further described by reference to the following examples, not limiting upon the t invention.

Examples Example 1: Cloning of antibody expression vectors The DNA expression constructs encoding the variable heavy (VH) and variable light (VL) domains of an NFor antibody were previously prepared de novo and included ction sites for cloning into mammalian expression vectors. Both heavy and light chain variable domain sequences were sequence sed for expression in mammalian cells (for methodology see W02009024567 and Kotsopoulou et al, J Biotechnol (2010) 146: 186-193). Information bing the heavy and light chain variable region sequences can be found in US patent U86090382. To generate the constructs used in this study, the variable heavy domain (VH) sequences were amplified using PCR. The PCR primers contained Hind/ll and Spel restriction sites to frame the VH domain containing the signal sequence for cloning into a pTT mammalian expression vectors ning the human y1 constant region. Similarly the VL domain sequence was amplified by PCR using primers ning Hind/ll and Bsin restriction sites to facilitate cloning into a pTT mammalian expression vector containing the human kappa constant region. The heavy chain expression plasmid was given the code SJC322 and the light chain expression d was given the plasmid code SJC321.

DNA expression constructs encoding ative le heavy and light chain regions of anti-TNFor dies with modifications in the CDR regions (as bed in Rajpal et al. PNAS (2005) 102(24): pg 8466-8471) were prepared de novo by build up of overlapping oligonucleotides and similar molecular biology techniques to those described above. The resulting plasmids encoding the heavy and light chains of variants cb1-3, cb2-6 and cb2-44 are described in Table 1.

Example 2: Engineering of the Fc region Forward and reverse priming primers were used to introduce cations (M252Y/S254T/T256E and M428L) into the human y1 constant region of the plasmid encoding the heavy chain of pascolizumab lL-4 antibody) using the ange protocol (Promega).

As described in Example 1 above, a PCR fragment encoding the VH domain of an anti-TNFor antibody was generated using a previously constructed, codon optimised vector as a template. The resulting fragment was cloned using Hind/ll and Spel into a pTT expression vector containing the modified human y1 constant region described in the preceding paragraph. The plasmid encoding the heavy chain of the anti-TNFor antibody with the M252Y/S254T/T256E modification was designated SJC324. The plasmid encoding the heavy chain with the T25OQ/M428L modification was designated SJC323.

Forward and reverse priming primers were used to introduce cations into the human y1 constant region of NFor heavy chain expression plasmid SJC322 using the Quikchange protocol (Promega). Plasmid SJC326 encodes the anti-TNFor heavy chain containing the M428L/N434S modification in the human y1 constant region. Plasmid SJC328 encodes the anti-TNFor heavy chain containing the V308F modification in the human y1 constant region.

Example 3: Expression of antibodies in HEK2936E cells using pTT5 episomal vectors Expression plasmids encoding the heavy and light chains described above were transiently co- transfected into HEK 293 BE cells. Expressed antibody was purified from the supernatant by affinity chromatography using a 1m| HiTrap Protein A column (GE Healthcare). Table 1 below shows the list of antibodies produced.

Some antibodies were also expressed in CHO cells using a different set of expression vectors. See Examples 13, 14 and 15 for a description of the molecular y, expression and purification.

Table 1: List of expressed antibodies BBC cadej ,cDR'variant _: ‘ , Heay‘y :sea Light Inof aha-n expressiom heavy, expression chain] Vader, ,gchain" BPC1492 None Wild-type SJC322 12 4 None M252Y/8254T/T256 SJC324 5 SJC321 BPC1496 None M428L/N434S SJC326 SJC321 Example 4: Binding of antibodies to tumour necrosis factor alpha in a direct binding ELISA A binding ELISA was carried out to test the g of the expressed antibodies purified using protein A to recombinant tumour necrosis factor alpha . ELISA plates were coated with recombinant human TNFd at 0.1ug/ml and blocked with blocking solution (4% BSA. Various ons of the ed antibody were added (diluted in 4% BSA in T Tris-buffered saline at pH8.0 containing 0.05% Tween ) and the plate was incubated for 1 hour at room temperature before g in deionised water.

Binding was detected by the addition of a peroxidase labelled anti human kappa light chain antibody (Sigma A7164) in blocking solution. The plate was incubated for 1 hour at room temperature before washing in deionised water. The plate was developed by addition of CPD substrate (Sigma P9187) and colour development d by addition of 2M HCI. ance was measured at 490nm with a plate reader and the mean absorbance plotted against concentration. The results are shown in Figure 1 and confirm that all the antibodies have a similar profile.

Example 5: Analysis of antibodies in an L929 in vitro neutralisation assay This assay was used to test the neutralising ability of the antibodies to neutralise TNF-d and inhibit cell death. Briefly, L929 cells were seeded in a 96-well flat-bottomed plate at /well in 100p| RPMI 1640 (w/o phenol red) and incubated overnight at 37°C, 5% C02. Cells were sensitised with 1.25pg/ml actinomycin D for 1 hour. For the neutralising study, 0.001-60pg/ml (0.0067- 400 nM) anti- TNF-d mAb was pre-incubated with approx. 2ng/ml (approximately 0.05nM) TNF-d in a 1:1 ratio for 1 hour at room temperature. For control group, RPMI was used in place of the antibody. Following the 1 h cubation with mycin D, 20 pl of antibody-antigen complex was added per well. 10u| media alone was added to wells as a negative l. Plates were incubated at 18 hour at 37°C, 5% C02. Following this treatment period, cell viability was determined by a cell titer-Glo Luminescent assay kit according to manufacturer’s instructions ga, Madison USA). For L929 assay, the percentage cell viability of the unknowns was expressed as a percentage of the untreated group (taken as a 100%) and |C50 values were determined by Graphpad prism. Differences in |C50 values of antibodies was assessed by one-way ANOVA (Newman—Keuls post hoc test) and considered significant at es of less than 0.05. Data is represented as mean i SEM, of n=4 experiments measured in duplicate. |C50 values for each antibody were determined and are listed in Table 2 below. The results show that the potency of all the antibodies tested are able.

Table 2: |C5o values for various anti-TNFa antibodies in an L929 neutralisation assay BPC1492 1.19:0.10 BPC1494 1.20 i 0.13 BPC1496 1.18:0.10 Adalimumab 1.09 i 0.07 WO 11076 Table 3 shows the |C50 values derived from the experiment. The results indicate that the improved anti-TNFor antibodies (BPC1499, 0, BPC1501) show increased y in this assay compared to BPC1492 and ada|imumab.

Table 3: |C5o values for improved anti-TNFa antibodies in an L929 neutralisation assay BPC1499 0.21 i 0.04 BPC1500 0.13 i 0.02 BPC1501 0.21 i 0.03 Adalimumab 1.09 i 0.07 Example 6: Effect of antibodies on in vitro |L-6 e The neutralising ability of antibodies was determined by measuring their effect on inhibiting TNF-d mediated lL-6 release from whole blood cells. Briefly, 130 pL of whole blood was added to each well and plates were ted at 37°C in a humidified 5% C02 incubator for 1 hour. For the neutralising study, 0.001-30ug/ml 7- 200 nM) TNF-d mAb was pre-incubated with 10ng/ml (approx. 0.4 nM) pha in a 1:1 ratio for 1 hour at 4°C. For control group, RPMI was used in place of the antibody.

Following this pre-treatment, 20 pl of antigen-antibody x or RPMI (negative control) was added per well and plates were incubated for 24 hour at 37°C, 5% C02. 100 pL PBS (w/o MgCl2 or CaClz) added to each well and placed on plate shaker for 10 mins at 500 rpm. Plates were then spun at 2000 rpm for 5 mins. 120 pL supernatant was carefully removed and transferred to fresh 96-well round bottomed plate and lL-6 release was determined using an MSD based assay kit (Meso Scale Diagnostics, Maryland USA). For the whole blood assay, the MSD signal for each sample was read using a MSD SECTOR® lmager 2400 and lL-6 release from the cells was quantified using a standard data analysis package in PRISM 4.00 software (GraphPad. San Diego, USA). The percentage of lL-6 inhibition by each antibody was expressed as a percentage of the TNF-d alone treated group. Hence, dose response curves were ed for each antibody and |C50 values were determined. Using the log of the |C50 values, the difference in potency of the antibodies was determined by one-way ANOVA (Newman—Keuls post hoc test) and considered significant at P-values of less than 0.05 for each donor (n=3). Data is represented as mean i SEM of three donors, measured in duplicate.

Table 4 below shows the |C50 values derived from these data. These s suggest that there is no significant difference in potency between the antibodies tested.

Table 4: |C5o values for various anti-TNF antibodies in a nduced |L-6 release assay BPC1492 0.72 i 0.32 BPC1494 0.62 i 0.11 BPC1496 0.64 i 0.13 Adalimumab 0.47 i 0.09 The |C50 values are shown in Table 5. The results te that the improved anti-TNFor antibodies (BPC1499, BPC1500, BPC1501) show increased potency in this assay.

Table 5: |C5o values for various improved NF antibodies in a TNFa—induced |L-6 release assay BPC1492 0.72 i 0.32 4 0.62 i 0.12 BPC1499 0.14 i 0.02 BPC1500 0.11 i 0.05 BPC1501 0.15 i 0.03 Adalimumab 0.47 i 0.09 Example 7: Accelerated stressor studies Prior to the study, antibodies to be tested were fied on a spectrophotometer at OD280nm and diluted to 1.1mg ml in PBS (pH7.4). An t was removed and 10%v/v of 500mM sodium acetate was added to give a final concentration of1mg/ml at pH5.5 and the sample inspected for itation.

The remaining sample in PBS had 10% PBS v/v added to a final concentration of 1mg/ml at pH7.4 and an aliquot of this sample was removed to provide a baseline aggregation level (as monitored by size exclusion chromatography). The s were then ted at 37°C for two weeks in an incubator, after which the samples were re-quantified on a spectrophotometer at OD280nm and assessed (by size exclusion chromatography) for aggregation. The samples were tested for human TNFq binding in a direct binding ELISA. The results are shown in Figure 2 and confirm that the binding activity of all antibodies tested is comparable following the accelerated stressor study.

Example 8: Stability study in 25% human serum Prior to the study, antibodies to be tested were quantified on a spectrophotometer at OD280nm and diluted to 1.25 mg/ml in PBS (pH7.4). An aliquot was removed and 25%v/v of human serum was added to give a final concentration of 1mg/ml. The remaining sample in PBS had 25% PBS v/v added to a final concentration of 1mg/ml and an aliquot of this sample was removed to e a baseline level. The samples were then incubated at 37°C for two weeks in an incubator, after which the samples were tested for human TNFd binding in a direct binding ELISA. The results are shown in Figure 3 and confirm that the binding activity of all antibodies tested is comparable following incubation in 25% human serum for two weeks.

Example 9: Analysis of g to human TNFor following freeze-thaw Antibody samples were diluted to 1mg/ml in a buffer containing 50mM Acetate and 150mM NaCl (pH6.0), snap-frozen in dry ice and then thawed at 4°C overnight. g of the antibodies to human TNFd was tested in comparison to an dy which had not been snap-frozen.To assess the binding activity following freeze-thaw, ELISA plates were coated with recombinant human TNFd at 1ug/ml and blocked with blocking solution (4% BSA in Tris ed saline). Various concentrations were added to the coated plates and incubated for 1 hour at room temperature before washing in deionised water. Binding was detected by the addition of a peroxidase labelled anti human kappa light chain antibody (Sigma A7164) in blocking solution. The plate was incubated for 1 hour at room temperature before washing in deionised water. The plate was developed by addition of CPD substrate (Sigma P9187) and colour development stopped by addition of 2M HCL. Absorbance was ed at 490nm with a plate reader and the mean ance plotted against concentration. The results are shown in Figure 4 and confirm that the binding ty of all antibodies tested is able following freeze-thaw.

Example 10: Analysis of binding of NFa antibodies to Felella ELISA plates were coated with recombinant human FClella (V158 and F158 variants) at 1pg/ml and blocked with ng solution (4% BSA in Tris buffered saline). Various concentrations were added to the coated plates and incubated for 1 hour at room temperature before washing in deionised water.

Binding was detected by the addition of a peroxidase labelled anti human kappa light chain antibody (Sigma A7164) in blocking solution. The plate was incubated for 1 hour at room temperature before washing in deionised water. The plate was developed by on of CPD substrate (Sigma P9187) and colour pment stopped by addition of 2M HCl. Absorbance was measured at 490nm with a plate reader and the mean ance plotted against concentration. The s are shown in Figure 5a and 5b and confirms that BPC1494 has reduced capacity to bind FClella (V158 and F158 variants) compared to BPC1492 and BPC1496.

Example 11: ProteOn Analysis: FcRn Binding Antibodies for testing were immobilised to similar levels on a GLC biosensor chip (BioRad 176-5011) by primary amine ng. Recombinant human and cynomolgus FcRn were used as analytes at 2048nM, 512nM, 128nM, 32nM, and 8nM, an injection of buffer alone (i.e. OnM) was used to double reference the binding curves. Regeneration of the antibody surface following FcRn ion used HBS—N at pH9.0, the assay was run on the n XPR36 Protein Interaction Array System at 25°C and run in HBS—N pH7.4 and HBS—N pH6.0 with the FcRn diluted in appropriate buffer. Affinities were calculated using Equilibrium model, inherent to the ProteOn analysis re, using a “Global R- max” for binding at pH6.0 and the R-max from binding at pH6.0 for affinity calculation at pH7.4. Since the binding curves did not reach tion at pH7.4, the values obtained are unlikely to be true ties however they can be used to rank constructs. The results are shown in Table 6 and confirm that BPC1494 and BPC1496 have an improved affinity for human and cyno FcRn at pH6.0 when compared to BPC1492.

Table 6 Affinities of Anti-TNF alpha constructs binding to Human and Cyno FcRn BPC1492 BPC1496 BPC1497 BPC1498 BPC1493 Example 12: PK studies in human FcRn transgenic mice.

In a single dose pharmacokinetic study BPC1494 and BPC1492, were stered intravenously (IV) at 1 mg/kg to two different strains of FcRn humanised mice and one strain deficient in FcRn (Petkova et al. Int. Immunol (2010) 18(12): 1759-1769). Plasma samples were analyzed for BPC1494 or BPC1492, as appropriate, using a validated b fluorescent immunoassay.

The methods used biotinylated human TNF alpha as the capture antigen and an Alexa labelled anti- human lgG (Fc specific) antibody as the detection antibody. Using an aliquot of mouse plasma diluted 1:10 with assay buffer, the lower limit of quantification (LLQ) was 100 ng/mL and the higher limit of quantification (HLQ) was100,000 ng/mL. Plasma concentrations below the lowest standards were considered to be not quantifiable. QC samples prepared at three different concentrations and stored with the study samples, were ed with each batch of samples against separately 2012/064129 prepared calibration standards. For the analyses to be acceptable, at least one QC at each concentration must not deviate from nominal concentration by more than 20%. The QC results from this study met these acceptance criteria.

PK analysis was performed by non-compartmental pharmacokinetic analysis using WinNonLin, version 6.1. All computations utilised the nominal blood sampling times. The ic exposure to BPC1494 and BPC1492 was determined by calculating the area under the plasma concentration time curve (AUC) from the start of dosing until the last quantifiable time point (AUC0_t) using the linear log trapezoidal calculation method. Further PK parameters could not be derived from the data due discrepancies in sample labelling.

Table 7 - Summary pharmacokinetic parameters for BPC1494 and BPC1492 following a single intravenous administration (bolus))at a target dose of 1 mg/kg to transgenic mice Cmax AUC Compound Strain (ug/mL) (hr*ug/mL) Strain 1 = chRn-l- hFcRn (32) Tg/Tg Strain 2 = chRn-l- hFcRn (276) Tg/Tg Rag1-/- Strain 3 = chRn g1-/- Similar Cmax concentrations were obtained for all groups. In both human FcRn in mouse strains BPC1494 had a higher exposure (AUC0_t) than 2, although this ence was not notable (1.3 fold). In the e of both human and mouse FcRn BPC1492 had a higher exposure than BPC1494.

Example 13: Cloning of antibody expression vectors into pEF vectors In some cases, the DNA encoding the sion cassettes for the heavy and light chains were excised from the s described in Example 3 using Hind/ll and EcoRI and cloned into pEF vectors, where expression occurs from the hEF1a promoter, using standard molecular biology ques (for description of vectors see Kotsopoulou et al J. Biotechnol (2010) 146: 186-193).

Table 8 BPC1494 M252Y/8254T/T256E SJC331 SJC329 6 M428L/N434S SJC332 SJC329 _— Example 14: Expression of dies in CHO cells using pEF expression vectors Expression plasmids encoding heavy and light chains were co-transfected into CHO DG44 cells and expressed at scale to produce antibody. For the generation of BPC1492 plasmids SJC329 and SJC330 were used. For the expression of BPC1494 plasmids SJC329 and SJC331 were used. For 6 plasmids SJC329 and SJC332 were used.

Briefly, 30ug DNA (15ug heavy chain and 15ug light chain) was linearised overnight with Not1 restriction enzyme. The resultant cted DNA was then ethanol precipitated and re-dissolved in TE buffer. From culture, 6X106 CHO DG44 cells were obtained and washed in 10ml of PBS. The cell pellet was then re-suspended in 300m of Amaxa solution V. 100p| of the entioned cell suspension was then added into to each of three Amaxa cuvettes, which also contained 3ug of the ised DNA. The cuvettes were inserted into an Amaxa nucleofector || device and electroporated with pre-set programme U-023. The contents of the three es (300m) of electroporated cells were added to 10ml of warmed MR14 medium (including nucleosides and BSA) and incubated in a T75 flask for 48 hours. Following this period, the medium was changed to nucleoside-free-MR14 (MR14 containing only BSA)). Every 3-4 days, conditioned medium was removed and replaced with fresh ion medium. Once cells had undergone recovery, the medium was substituted to 2X MR14 and lgG expression was confirmed by nephlometry. 2L shake-flasks were seeded with 1L of the lgG-expressing cells at 0.6X106/ml and grown for 7 days. Cells were separated from supernatant by centrifugation and the supernatant was used for protein purification. 1 litre cell e atants were purified using a 2-step automated s on an AKTA Xpress system. The antibody was captured on a 5m| MabSelectSure column and then washed prior to n. The eluted antibody was then loaded onto a 440ml Superdex 200 gel filtration column and 2m| fractions collected in a 96-well block. Fractions of purified antibody were pooled and 0.2pm filtered and then concentrated to ~5mg/ml using Amicon spin concentrators. The final material was again 0.2pm filtered and then dispensed into sterile tubes for delivery. The final material was subject to analytical SEC to determine aggregation, an endotoxin assay, LC-MS for accurate mass determination (included PNGaseF and untreated material to determine glycosylation), SDS PAGE electrophoresis, PMF for sequence confirmation and A280 for concentration determination.

WO 11076 Example 15: Alternative method for expression of antibodies in CHO cells using pEF expression vectors DHFR-null CHO DG44 cells were obtained from Dr. Chasin of Columbia University. These cells were subsequently adapted to a chemically defined medium. These adapted host cells were designated DG44-c and are cultured in proprietary chemically defined medium supplemented with Glutamax and HT-supplement.

Generation of the polyclonal pool: For more s on protocols see W02009024567 and Kotsopoulou et al, J. hnol (2010) 164(4): 186-193. Briefly, DG44-c cells were transfected with plasmids encoding the heavy and light chains and DHFR and neoR respectively by electroporation (using the Amaxa fector system). At 48 hours post transfection, selection was initiated by addition of G418 (at a final concentration of 400ug/ml) and l of HT. When viability and cell counts sed sufficiently (in this case 2 months post transfection) methotrexate (MTX) was added at a final concentration of 5nM. Cells were scaled up and production curves were initiated 9-16 days after on of MTX. For these production curves cells were seeded at 0.6-0.8X106 cells/ml in chemically defined media and were fed on days 6, 9 or 10, 12 or 13 and/or 16. Supernatant was collected when viability dropped to approximately 50% and the cells were removed by centrifugation at 40009 for 30 mins followed by filtration through a sartobran capsule.

Antibodies were purified at room temperature using a two step chromatographic procedure: Initial capture was performed using a 50ml ect SuRe column (GE Healthcare) followed by Size Exclusion Chromatography (SEC) with a 1.5L ex 200 pg SEC (GE Healthcare). The conditioned media was loaded onto a pre-equilibrated MabSelect SuRe column at a flow rate of 9cm/h. Following washing to base line with equilibration buffer (50mm Tris pH 8.0, 2M NaCl) the column was washed with a low salt buffer buffer (50mM NaCl Tris pH 8.0, 150mM NaCl) until conductivity was stable. The column was then eluted with elution buffer (25mM Citrate pH 2.5).

Fractions corresponding to peak protein elution were immediately neutralized with 1/10 vol. 1.0M Tris pH 8.0 which were then pooled and filtered through a 0.2pm bottletop filter. The recovered sample was loaded at 21cm/h onto the SEC column pre-equilibrated with SEC buffer (50mM Na Acetate, 150mM NaCl). The fractions containing the main (monomeric) protein peak were pooled and filter sterilized.

Antibodies ed by this method were used for analytical comparability studies summarised in the following example.

Examples 16: Analytical comparability on stressed and control s Size ion chromatography was carried out to determine the aggregation levels of the protein.

The optimised method involved injection of the sample onto a TOSOH TSK WXL column which had been equilibrated in 100 mM sodium phosphate, 400 mM NaCl, pH 6.8. Absorbance was 2012/064129 measured at both 280nm and 214nm. Reverse-phase HPLC separates proteins and their isoforms based on hydrophobicity. Protein was ed onto a PLRP-S 1000 °A 8pm column and eluted using a gradient produced by 50%Formic acid, and 95% Acetonitrile. Absorbance was measured at 280nm. The purity of the molecule is reported as a percentage of the main peak area relative to the total peak area. ent isoforms of the mAb were separated on the basis of their pl values using capillary ctric focussing (clEF). lEF separation was performed on a 10cm, UV280 transparent cartridge capillary. The optimised method involved a solution containing 5% pH 3-10 ampholytes, 10mM NaOH, protein of interest and al pl markers (7.05 and 9.5) which was loaded into the capillary by re injection.

The specific activity of antibodies (adalimumab, 4, BPC1496) was determined using MSD. In brief, 96-well plates were coated with 50uL per well TNFd diluted to 1 pg/mL in PBS. The plate was incubated on the bench top at ambient temperature t shaking for 2 hours. The coating solution was removed and the plate was blocked with 50uL per well of 1% BSA in PBS, with 0.05% Polysorbate 20. The plate was incubated for 1 hour at 24°C with shaking at 400 rpm and then washed 4 times with wash buffer. The antibodies were diluted in 0.1% BSA in PBS with 0.05% Polysorbate 20 and 30p| of each sample was added to the plate. The plate was incubated for 1 hour at 24°C with shaking at 400 rpm. The plate was then washed 4 times with wash buffer. Anti-human lgG sulfotag was diluted 1 in 5000 in assay buffer. 30uL was added to each well of the plate and then incubated for 1.5 hour at 24°C, with shaking at 400 rpm. The plate was then washed 4 times with wash buffer. The 4x MSD Read Buffer concentrate was diluted to 1x using deionised water. 100uL was then added per well of the plate. The plate was then read using the MSD Sector lmager instrument. From the signals obtained from the assay, specific activities of the molecules were calculated.

Deamidation analysis Deamidation is a common post-translational modification that can occur to asparagine and glutamine residues, but is most commonly observed with asparagine residues, ularly when nt to a glycine residue. In order to examine how susceptible these residues are and to determine the effects of ation on potency, adalimumab, 4 and BPC1496 were exposed to a stress study.

The stress was carried out by incubation in 1% ammonium bicarbonate at pH 9.0, for 48 hrs, conditions which have previously been shown to cause deamidation. The stressed samples were incubated alongside a control (in PBS) and were compared to this as well as an unstressed reference and analysed using c-lEF, SEC and Binding ELISA. Forced deamidation was also done on all samples in the presence and absence of EDTA. It has been shown previously that forced deamidation conditions cause fragmentation in on to deamidation. EDTA prevents and or minimizes the fragmentation.

Oxidation analysis 2012/064129 Oxidation of various residues can occur throughout the processing and storage of proteins; r the most commonly oxidised residue is methionine, which was the focus of this screen. Oxidation susceptibility of these residues was examined h exposure to stress conditions by incubation in 5mM and 50 mM H202 for 30minutes and evaluated using RP-HPLC, SEC and ELISA.

Summary of results Both BPC1494 and BPC1496 behave very favourably compared to adalimumab as shown by analytical comparability on both stressed and control samples. For all antibodies tested, no significant degradation was observed under forced ion conditions as shown by all analytical techniques employed. Significant ation as measured by c-IEF was observed at pH 9.0 as expected for all antibodies tested. In addition we saw significant fragmentation for all antibodies tested as shown by SEC at pH 9.0 in samples without EDTA, this is also as expected. There is a ion in the pi value, ximately 0.2) of BPC1494 when compared to adalimumab. This is attributed to the presence of an additional ic acid residue in the heavy chain sequence of the BPC1494 thus making it more acidic. Forced deamidation and oxidation had minimal impact on binding and this was observed for BPC1494, BPC1496 and adalimumab.

Example 17: Analysis of binding of improved antibodies by ELISA Antibodies BPC1499, 1500 and 1501 were assessed for g activity by ELISA as described in Example 4. Using two different antigen coating trations (0.1 and 1.0 pg/ml), the antibodies did not show any ence in their binding profile when ed with BPC1492. Under the conditions tested, it appears that the ELISA does not discriminate between antibodies with different reported binding activities. The same antibodies were assessed using methodologies described in Examples 18, 5 and 6 which are considered more sensitive assays. In these assays, antibodies BPC1499, 1500 and 1501 show improved binding affinity and improved potency when compared with BPC1492.

Example 18: Biacore Analysis of TNF alpha binding using a Capture surface n A and anti-human IgG (GE Healthcare BR-1008—39) were coupled on separate flow cells on a CM3 biosensor chip. These surfaces were used to capture the antibodies for binding is.

Recombinant human and lgus TNF alpha were used as analytes at 64nM, M, 7.11nM, , 0.79nM, an injection of buffer alone (i.e. OnM) used to double reference the binding curves.

Regeneration of the capture surface was carried out using 100mM phosphoric acid and 3M MgCIZ.

The run was carried out on the Biacore T100 machine at 37°C using HBS-EP as running buffer. The constructs BPC1494 and BPC1496 showed reduced binding to Protein A and the uman lgG surface making these surfaces unsuitable for generating kinetics for those molecules.

Table 9 Kinetic Analysis of Human and Cyno TNF alpha Binding to Captured Anti-TNF alpha Antibodies.

BPC1492, human TNFOt L Protein A 061 1.10E-04 0.05196 BPC1494 human TNFoc Protein A Analysable BPC1496 human TNFOt Protein A Analysable BPC1500 human TNFOt Protein A 2.68E+06 4.19E-05 0.01561 BPC1492 human TNFOt anti-human lgG 6.78E+06 1.73E-04 0.02554 BPC1494 human TNFOt anti-human lgG Analysable BPC1496 human TNFOt anti-human lgG able BPC1500 human TNFOt anti-human lgG 4.51E+06 05 0.01568 BPC1492 Cyno TNFOt Protein A 1.10E+06 1.11E-04 0.101 BPC1494 Cyno TNFOt Protein A Analysable BPC1496 Cyno TNFOt Protein A Analysable BPC1500 Cyno TNFOt Protein A 2.34E+06 3.51 E05 0.01503 BPC1492 Cyno TNFOt anti-human lgG 1.96E+06 04 0.1911 BPC1494 Cyno TNFOt anti-human lgG Analysable BPC1496 Cyno TNFOt anti-human lgG not analysable BPC1500 Cyno TNFOt anti-human lgG 06 2.09E-04 0.04667 Example 19: ProteOn Reverse Assay Binding is ylated TNF alpha was mixed with biotinylated BSA at a 1:49 ratio, at a final total protein concentration of 20ug/ml (i.e. 0.4ug ylated TNF alpha and 19.6ug biotinylated BSA). This mixture was captured on a NLC sor chip (a single flowcell) d 176-5021). The chip surface was conditioned with 10mM glycine pH3.0 till a stable signal was achieved. The antibodies to be tested were used as analytes at 256nM, 64nM, 16nM, 4nM and 1nM and OnM. The binding curves were referenced against a flowcell coated with biotinylated BSA alone. Regeneration was achieved using 10mM glycine pH3.0. Data was fitted to the 1:1 model inherent to the ProteOn analysis softwa re.

WO 11076 Table 10 Apparent Kinetics of Anti-TNF alpha antibodies binding to Neutravidin Captured TNF alpha 9 2.27E+06 1.72E-05 BPC1500 2.06E+06 05 BPC1501 1.17E+06 6.97E-05 BPC1496 6.33E+05 4.04E-04 4 7.23E+05 3.50E-04 BPC1492 05 3.21E-04 This data is one set of two experiments which were carried out (second set not shown). The KD ranking of the data is representative of both data sets.

Example 20: Construction of alternative antibodies which bind to human TNFa The DNA expression constructs encoding additional variable heavy s with modifications in the CDR regions (as described in Rajpal et al. PNAS (2005) 102(24): pg 8466-8471) were prepared de novo by build up of overlapping oligonucleotides and similar molecular biology techniques to those described in Example 1. Examples of DNA sequences encoding the variable heavy domains of these variant antibodies are given in SED IQ NO: 81, 83, 85, 87, 89, 91, 93 and 95. The DNA sion ucts encoding additional variable light domain regions with modifications in the CDR regions (as described in Rajpal et al. PNAS (2005) 102(24): pg 8466-8471) were prepared de novo by build up of overlapping oligonucleotides and similar lar biology techniques to those described in e 1. Examples of DNA sequences encoding the variable light domains of these t antibodies are given in SED IQ NO:97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127, 129, 131, 133, 135 and 137. Once constructed, the expression plasmids encoding the heavy and light chains were transiently co-transfected into HEK 293 BE cells. Expressed antibody were purified from the supernatant and assessed for activity using the methods similar to those described in Example 6..

Example 21: Construction of expression vectors for BPC2604 (Pascolizumab-YTE) The pTT-based DNA expression constructs encoding the heavy chain of pascolizumab was engineered to include the following changes M252Y/S254T/T256E (EU index numbering) using the Quikchange protocol (Promega).

Example 22: Expression/purification of Pasco and Pasco-YTE vectors Expression plasmids encoding the heavy and light chains of BPC2604 were ently co-transfected into HEK 293 BE cells. Expressed antibody was purified from the bulk supernatant using a two step purification carried out by ty tography and SEC using a 5m| MabSelectSure column and Superdex 200 column on an AKTA Xpress.

Example 23: BlAcore is of Pasco vs. Pasco YTE for FcRn binding Antibodies were immobilised on a GLM chip (20ug/ml in acetate pH4.5) by y amine coupling.

Human, cynomolgus, rat and mouse FcRn receptors used at 2048, 512, 128, 32 and 8nM. OnM used for double referencing. Assay were carried out in HBS—EP pH7.4 and HBS—EP pH6.0 (FcRn receptor diluted in appropriate running buffer for each pH. The surface was regenerated for FcRn binding with 200mM Tris pH9.0. Data was fitted to an equilibrium model, with R-max set to highest R-max obtained of any construct. The results are shown in Table 11 below and confirm that the YTE- ed pascolizumab (BPC2604) shows improved binding to FcRn at pH6.0 compared to pascolizumab.

Table 11: Affinities of anti-lL-4 antibody constructs for Human and Cyno FcRn (n.a.b. is no analysable binding) KD (nM) at pH6.0: R-max = KD (nM) at pH7.4: R-max = 1020 1020 Antibody Fc cation Cyno Mouse Rat Human Cyno Mouse Rat FcRn FcRn FcRn FcRn FcRn FcRn BPC2604 M252Y/8254T/T256E 98 W 11600 11100 2160 Example 24: PK studies with Pasco vs. Pasco-YTE Figure 6 shows the e dose normalised plasma concentrations of izumab-YTE (BPC2604)) in female cynomolgus monkeys and pascolizumab in male cynomolgus monkeys following a single intravenous (1 hr infusion) administration at a target dose of 1 mg/kg. The data for BPC2604 and pascolizumab were generated in separate studies. Plasma antibody concentrations for pascolizumab and BPC2604 were assessed by chemi-luminescence ELISA using lL-4 as the capture reagent and anti-human lgG (Fc specific)-HRP conjugate as the detection reagent. The validated range for the assay was 50-5000 ng/mL. The results are shown in Figure 6. Both compounds had similar Cmax but BPC2604 had a 3-fold lower plasma clearance resulting in 3-fold increase in AUC and 2-fold increase in half-life (T1/2).

Example 25: Formulation studies at 5mg/ml The ity of adalimumab and the TNF-alpha variant BPC1494 in two formulations was ed.

Formulation ‘A’ (citrate-phosphate buffer) is the marketed umab formulation made up of 6.16 mg/ml Sodium de + 0.30 mg/ml Sodium e monobasic + 1.30 mg/mL Citric acid monohydrate + 12 mg/ml ol + 0.86mg/mL Monobasic sodium phosphate dihydrate + 1.53mg/mL Dibasic sodium phosphate dihydrate + 1.0 mg/ml P880 at pH 5.2.

Formulation ‘B’ (acetate buffer) is composed of 6.81mg/mL (50mM) Sodium Acetate trihydrate + 10mg/mL (1% w/v) Arginine + 0.0186mg/mL (0.05mM) EDTA + 2.98mg/mL (51mM) Sodium de + 0.2mg/mL (0.02% w/v) Polysorbate 80, adjusted to pH 5.5 using HCl or NaOH.

The TNF-alpha variant BPC1494 material used in this study was made in a Chinese Hamster Ovary (CHO DG44) cell line and purified using a two step process involving mAb Select Sure followed by Superdex column 200pg. Adalimumab (Product code NDC 007402, Lot number 91073LX40) manufactured by Abbott Laboratories was used.

Adalimumab was re-formulated into Formulation ‘B’ by overnight dialysis at 5°C using a 10KDa Slide — A — Lyzer cassette (Product Number 66830, Lot Number LJ150514); produced by Thermo Scientific (Rockford, IL ;USA). This experiment was carried out at a different time point to the other three formulations.Both Adalimumab in Formulations ‘A’ and ‘B’ were diluted to 5mg/mL using their respective formulation buffers. The TNF-alpha variant BPC1494 le was also formulated in Formulations A and B at ~5mg/mL. A total of 4 samples were filtered through a MilleXGV 0.22um filter under a clean laminar flow condition before being transferred into labelled erilized glass vials and ted at 5°C, 25°C and 40°C for up to 14 weeks. Samples were taken at selected time points and analysed using SEC-HPLC (Table 12), clEF (Table 13). Other assays as described below were also d out to assess the stability of the antibodies.

Appearance by visual observation s were inspected for y under daylight conditions. Both antibodies in each formulation remained ged (clear colourless solution) after 14 weeks storage at 5°C, 25°C and 40°C.

Protein concentration (A280nm) Measurement Protein concentration was measured using a nanodrop spectrometer, which is indicative of protein stability. The extinction cient for adalimumab is 1.46 and for TNF-alpha variant BPC1494 is 1.48.

There was no significant ence in the results after 14 weeks storage at 5°C, 25°C and 40°C. pH was measured for all samples stored under different storage conditions to determine whether any significant pH drifts had ed. All results remained within assay variability after 14 weeks storage at 5°C, 25°C and 40°C.

Size exclusion chromatography (SEC) This method separates soluble protein molecules in the solution based on size and not molecular . In theory, small molecules will penetrate every small pore of the stationary phase and hence will elute later. The chromatogram obtained s the determination of percentage area of aggregates, monomer and low molecular weight (MW) nts. The ce of ates and/or low molecular weight s is indicative of protein degradation. lncreased ity corresponds to a high percentage of monomeric species (Mono) together with a low percentage of Total Aggregates (TA) and Total Low lar weight Fragments ).

SEC-HPLC data (Table 12) shows that the TNF-alpha variant BPC1494 was relatively more stable in formulation ‘B’ compared to formulation A after storage at 25°C and 40°C for 14 weeks. Furthermore, TNF-alpha variant BPC1494 was relatively more stable, or at least as stable as adalimumab in formulation A. The results for adalimumab in formulation B are all within 5% TA and/or TLMWF.

Therefore, formulation B has advantages over formulation A for both TNF-alpha variant BPC1494 and adalimumab.

For example, Table 12 shows that after storage at 25°C for 8 weeks, TNF-alpha variant BPC1494 in formulation A has 2.3% TLMWF while formulation B produced only 1.5%. Furthermore, TNF-alpha variant BPC1494 in formulation B was relatively more stable than adalimumab in formulation A (1.8% TLMWF). Similarly, at the 14 week time point at 25°C, 3.15% TLMWF was observed for TNF-alpha variant BPC1494 in formulation ‘A’ compared to 2.3% TLMWF in formulation ‘B’. Furthermore, TNF- alpha variant BPC1494 in ‘B’ was relatively more stable than adalimumab in formulation A (3.4% TLMWF). A similar trend for TLMWF was observed for both molecules on tion at 40°C for 4 weeks (adalimumab in ‘A’: 3.6%; TNF-alpha variant BPC1494 in ‘A’: 4.1%; TNF-alpha variant BPC1494 in ‘B’: 2.6%).

Also, results for Total Aggregate (TA) show that at 14 weeks at 25°C, the TNF-alpha t BPC1494 was relatively more stable in ‘B’ (0.3%) than in ‘A’ (0.5%); and relatively more stable than adalimumab in formulation A (0.4%).

Capillary lso-Electric Focusing (c-IEF) This technique is used for determining the charge profile of molecules. A broad pl range reflects greater charge heterogeneity of the Product and in addition a broad pl range may be indicative of degradation. Typically the number of peaks will se with sed degradation. The C-IEF data of Table 12 supports the SEC gs in Table 13.

The % area of main isoform (%AMI) was comparable between adalimumab in formulation A and TNF- alpha variant BPC1494 in formulation B at Weeks 8 and 14 at 25°C (56.0-57.7 and 53.2 respectively).

At these time points and temperature, formulation B shows a slight advantage over ‘A’ for TNF-alpha variant BPC1494.

Similarly, adalimumab is relatively more stable in formulation ‘B’ than in formulation ‘A’ (see Week 4 data). For example, increased changes in charge heterogeneity (i.e. increase in number of peaks) were observed for adalimumab incubated for up to 4 weeks at 40°C in formulation ‘A’ compared to formulation ‘B’ (8 peaks and 6 peaks respectively). TNF-alpha variant BPC1494 showed a more consistent charge geneity of 5 peaks at all timepoints and atures.

Functional binding assay The binding activity of adalimumab and TNF-alpha variant BPC1494 in the two formulations was assessed by e. Over a 14 week period of storage at 5°C, 25°C and 40°C, the samples showed similar %binding within assay variability.

Hence, it can be ded that formulation ‘B’ can serve as an alternative to formulation ‘A’ in a clinical setting without compromising the stability of the protein and ially eliminating the pain ated with the marketed adalimumab formulation (A).

Importantly, this data shows that not only does the acetate formulation (B) improve the stability of the TNF-alpha variant BPC1494 compared to the e-phosphate formulation (A); but the acetate formulation is comparable or slightly better than the citrate-phosphate formulation when stabilising adalimumab.

Table 12: SEC-HPLC of adalimumab and TNF-alpha t BPC1494 in Formulation ‘A’ and ‘B’ at 5°C, 25°C and 40°C. TLMWF: Total Low Molecular Weight Fragment; Mono: Monomer; TA: Total Aggregate. N = 2 %%%E%% Egaaagaafi agaaggfigifiéa __ _______ aéfiaéa _<. .m _mco=m_:E._oh_ .< .m _ . 83259 ”5:352:09. 53335.8 E E E E vmiomm agfi vmgumm Eagaagagfi nmEzE=mUm nmEzE=mvm Egg Emzm> £235 agaagaaéaaég 29m- -n_z._. _, agaaaglaiaég m; mfo .8 Nimm mg E. 80 vmd m2 HEEHEEI- @9wa 5528 0° pm 9mm 92 00m 00mm 0% 9mm o m ia0|2 HOZII ._.Z 2012/064129 .003. 00mm .Uom sass—35:0“— wmvvomm u:m_._m> m;Q_m-u_z._. 39:35.25 wNm Dom Em N Em a u_m=-m_0 %%%%%E.x. -Nm.m wmfi -wa Nod ”9‘ 2558 00: 00m 00mm Ooov 00m 0mm Ooov win...

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Example 26: Formulation studies at | As shown in the previous example 25, adalimumab and TNF-alpha variant BPC1494 at 5mg/mL in formulation ‘B’ can serve as an alternative to formulation ‘A’. This example is focused on comparing the stability of adalimumab in its marketed formulation ‘A’ compared to formulation ‘B’ and other TNF-alpha variants at 50mg/ml.

Two samples of TNF-alpha t BPC1494 were ed, one expressed in CHO DG44 cells and one expressed in CHOK1 cells. A second TNF-alpha variant BPC1496 was made in a CHO-DG44 cell line. All three samples were expressed and purified using mAb Select Sure. In contrast to Example 25, no Superdex column step was carried out. Adalimumab (Product code N 00515-01, Lot number H12) manufactured by Abbott Laboratories, as in Example 25.

Adalimumab was formulated in formulations ‘A’ (as purchased) and ‘B’ (by buffer exchange) as described above in e 25, and the TNF-alpha variants (BPC1494 and 1496) were formulated in ‘B’, all at mL (total of 5 samples). The samples were ed with GV 0.22um filter under clean laminar flow conditions before being transferred into labelled pre- sterilized glass vials and incubated at 5°C and 40°C for up to 9 weeks. At selected time-points, samples were taken and analysed using SEC-HPLC (Table 14), clEF (Table 15). Other assays as described below were also carried out.

Appearance by visual observation.

Samples were observed for clarity under daylight conditions. Both antibodies in both formulations ed unchanged (clear colourless solution) after 9 weeks storage at 5°C and 40°C.

Protein concentration (A280nm) Measurement Protein concentration was measured using a nanodrop spectrometer, which is indicative of protein stability. There was no significant difference in the results after 9 weeks storage at 5°C and 40°C.

Size ion chromatography (SEC) SEC-HPLC data (Table 13) showed that umab at 50mg/ml was relatively more stable in formulation ‘B’ compared to formulation ‘A’ after storage at 40°C for 9 weeks. Also, the TNF-alpha variants 94 and 1496) were relatively as stable or more stable in ‘B’ as adalimumab in ‘B’.

No comparison between the variants in ‘A’ and ‘B’ was carried out.

Note that the Initial TA levels for the TNF-alpha variants were relatively higher than for umab. Therefore, the results include a % change column at the right hand side to compare the changes from l to Week 9 at 40°C. For example, table 13 shows that after 9 week storage, the percentage change in total low molecular weight fragment (TLMWF) in formulation ‘B’ was between 3.82-4.96% compared to 6.08% in formulation ‘A’. Similarly, the r percentage change in formulation ‘A’ was greater for adalimumab than for ‘B’ (7.54 and 4.52% respectively). The TNF-alpha variants in ‘B’ were all vely at least as stable or more stable as adalimumab in formulation ‘A’ (% change at Week 9). The s at week 4 for all samples are within the 5% TA and/or TLMWF allowance for a commercial product. Therefore, ‘B’ has advantages over ‘A’ for both TNF-alpha variants and adalimumab at 50 mg/ml.

In particular, the TNF-alpha variant BCP1496 showed a low TLMWF value of 3.86 at Week 9 at 40°C.

Capillary lso-Electric ng (c-IEF) C-IEF data (Table 15) ts the findings in Table 14.

Formulation B shows a reduced % change of %AMI at week 9 for adalimumab as compared to Formulation A ( 23.53 and 27.57 respectively).

The TNF-alpha variants in ‘B’ are more stable in terms of charge heterogeneity (i.e. increase in number of peaks) than adalimumab (in both ‘A’ and ‘B’). For example, at Week 9 there were 5 and 6 peaks for each of the variants; and 6 and 9 peaks for adalimumab, at 5°C and 40°C respectively.

In particular, the TNF-alpha variant BCP1496 and adalimumab, both in ‘B’, showed a low % change in %AMI at week 9 of 25.83 and 23.53 respectively. The relatively higher % change in %AMI at week 9 for the pha variant BCP1496 (CHO DG44) of 38.13 may be due to the relatively high initial %AMI of 75.03.

Functional binding assay (ELISA) The biological activity of adalimumab and the TNF-alpha variants in the two formulations was assessed by Biacore. Over the 9 week period of storage at 5°C and 40°C, the samples showed the same %binding within assay variability.

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Plasma samples were analyzed for BPC1494 using a qualified analytical method based on sample dilution followed by immunoassay analysis Plasma samples were analyzed for BPC1494 or BPC1492. The method used 10 pg/ml biotinylated inant human TNF-alpha as the capture antigen and a 1:100 dilution of luor 647-labelled anti-human lgG (Fc specific) antibody as the detection antibody 45). The lower limit of quantification (LLQ) for BPC1494 was 1 pg/mL using a 50 pL aliquot of 100-fold diluted monkey plasma with a higher limit of quantification (HLQ) of 100 pg/mL. The computer systems that were used on this study to acquire and quantify data included Gyrolab Workstation n 5.2.0, Gyrolab Companion version 1.0 and SMSZOOO version 2.3. PK analysis was performed by mpartmental pharmacokinetic analysis using WinNonlin Enterprise Pheonix version 6.1.

Pharmacokinetic data is presented in Table 16 with parameters determined from last dose received on Week 4 to the time point (t) 840 hours post dosing for 30 mg/kg/week dose group (2) and last dose received on Week 3 to the time point (t)1008 hours post dosing for 30 & 100 mg/kg/biweekly dose groups (3 and 4).

Table 16: Individual and Mean cokinetic Parameters for BPC1494 in the Male Cynomolgus Monkey Following Subcutaneous Dosing of BPC1494 at mglkglweek or 30 and 100 mglkglbiweekly over a 4-Week Investigative Study Dose Animal cokinetic ters b (mg/kg/waeekly) Number_ Estimated Estimated WM272------O125 WM273 --_0-141 - P12M- (568) (0.977) (4)82 (292 (1.09) (404) P12M275---_-°“5 PMM27“ ----141 P12M- III-II.-Mean 641 1.70 36 309 363 0.128 54.9 —-------- -—-------P12M'2802080---123-244° ---118Group2animals received 30 mg/kg weekly for 4 weeks b) Pharmacokinetic parameters determined from last dose received on Week 4 to the time point (t) 840 hours post dosing for 30 mglkglweek and last dose received on Week 3 to the time point (t)1008 hours post dosing for 30 & 100 biweekly c) C|_F and Vz_F are estimates due to elimination phase following multiple doses and steady state not yet achieved. Parameter estimates have been calculated from i) using AUCO- 168 or 336, ii) extrapolation of data from week 1 based on half-life and iii) using total dose over the defined sampling with AUCO-inf d) Animal 274 and 277 excluded from mean pharmacokinetic ations based on scientific judgment that these animals are likely to be exhibiting an anti-drug antibody response.

Mean data shown in parentheses are inclusive of these animals.

Example 28: SPR binding analysis of FcRn to Protein L captured anti-TNFd mAbs The study was carried out using the ProteOnTM XPR36 (BioRadTM) biosensor machine, a surface plasmon based machine designed for label free c/affinity measurements. n L was immobilised on a GLM chip (BioRad, Cat No: 12) by primary amine ng. This surface was then used to capture the humanised antibodies, human and cyno FcRn (both in- house materials) was then used as analytes at 2048nM, 512nM, 128nM, 32nM, and 8nM, an injection of buffer alone (i.e. OnM) used to double nce the g curves. Regeneration of the protein L surface was carried out using e-HCI pH1.5. The assay was run at 25°C and run in HBS—EP pH7.4 and HBS—EP pH6.0 with human or cynomolgus FcRn diluted in appropriate buffer. Affinities were calculated using the Equilibrium model, inherent to the ProteOn analysis software, using a l R-max” for binding at pH6.0 and the R-max from binding at pH6.0 for affinity ation at pH7.4. Since the binding curves did not reach saturation at pH7.4, the values obtained are unlikely to be true affinities however were used to rank the binding of the antibodies tested.

The binding affinity of different batches of BPC1492, BPC1494 and BPC1496 for human FcRn was compared using antibodies captures by Protein L. Table 17shows the results from a series of experiments using this format. The data confirms that BPC1494 and BPC1496 have an improved affinity for recombinant human FcRn compared to BPC1492 at both pH6.0 and pH7.4. The fold improvement in binding ty of BPC1494 for FcRn compared to BPC1492 differs from experiment to experiment due to changes in the Protein L activity on the capture. However, in the experiments shown in Table 17, the fold improvement in binding affinity at pH6.0 ranges between 3.5-fold and 16.3-fold. It was not possible to determine the fold improvement in binding affinity at pH7.4 due to the weak binding ty of human lgG for FcRn at neutral pH.

The binding affinity of different batches of BPC1492, BPC1494 and BPC1496 for cynomolgus FcRn was also compared using antibodies captured with Protein L. Table 18 shows the results from the experiment using this format. The data confirms that BPC1494 has an ed affinity for recombinant cynomolgus FcRn compared to BPC1492 at both pH6.0 and pH7.4. The fold improvement in g affinity of BPC1494 (range 41 .8—46.8nM) for cynomolgus FcRn compared to BPC1492 (range 394-398nM) is approximately 9-fold at pH6. It was not possible to determine the fold improvement in binding affinity at pH7.4 due to the weak binding activity of BPC1492 for FcRn.

Table 17 Recombinant human FcRn g affinities using the Protein L e method """AffinityKD(nM) '- BPC1494 BPC1496 I‘VHEKVITV, HEKL WGRITSWHEKHEKGRITS’ 00 .

(JO A .0" O NO) N A :5 (JO A F” (D A .0"A Z >w Z>w 11700 8980 9830 9670 01o co 019‘ oo 01 01 01 3.60 2.33 N A Z>01 Z>01 Z>w N550 A CA A 3'o A F” CO A .‘l 00 —\'\1 0&9 Oix) 1.810 A 0our0 Z>m 1} 10900 7830 8050 8460 .O .O N04>A c3 '_\ A N (O A 9° (D «>00 01 .0 co ‘1 co 00(3) \l O.546 A O (DO NAB zAB NAB 2010 9190 9330 9480 9550 (JOA CO 01 Z ZU N A 22 CU >w I!22 z Z>w I!22 22 NO A O ZU - although data points have been reported, the values should be treated with caution because these data are not consistent with the data obtained for the other batches of the same molecule during this experiment NAB = no analysable g ND = not tested in this experiment ## = high affinity binding — beyond the sensitivity of the machine Table 18 Recombinant cynomolgus FcRn binding affinities using the Protein L capture method __ mpH6'g pH74 , 7 Watch-number " . CONStrUCt :’ * r - : *e: s: BPC1494 H GRITS44463 ' ' ‘ 46.8 14800 MCB16Marc2012 BPC1494 41.8 13300 GRITS42954 BPC1494 43.2 13700 Clinical grade BPC1492 394 No binding 4348 2 398 No binding Table A —Description Sequence fier (SEQ ID NO) cleotide Amino acid anti-TNF antibody heavy chain plus 4; U'IOON M252Y/8254T/T256E modification Anti-TNF antibody heavy variable domain (VH) lgG1 nt domain plus \l M252Y/8254T/T256E modification Anti-TNF antibody heavy chain plus M428L/N434S modification lgG1 constant domain plus M428L/N434S I _\ O modification Anti-TNF antibody heavy chain (wild-type lgG1) _\_\ N lgG1 constant domain (wild-type) (JO Anti-TNF antibody heavy chain plus A 4; A 01 T25OQ/M428L modification lgG1 constant domain plus T25OQ/M428L _\ CD modification Anti-TNF antibody heavy chain plus V308F —\ 00 cation lgG1 constant domain plus V308F modification —\ Anti-TNF antibody heavy chain plus V259| N —\<9 modification lgG1 nt domain plus V259| modification Anti-TNF antibody heavy chain plus P257L and MN AN lgG1”constant domain plus P257L and N434Y N01 NNNN (JOOOQJ AOCDCO‘IO) 0O (D WO 11076 (DCDCDCOCOCOCOCOV #NOCOCD-RNOCO (9 CD (D CO Anti-TNF antibody heavy chain variant cb1VH plus M252Y/S254T/T256E modification Anti TNF antibody heavy chain variant cb2 44- VH plus M252Y/S254T/T256E modification Anti-TNF antibody light chain variant cb1 VL Anti-TNF antibody light chain variant cb2VL Anti-TNF antibody light chain t cb2VL Anti-TNF antibody heavy chain variant cb1 VH Anti-TNF antibody heavy chain variant cb2 Pascolizumab heavy chain containing M252Y/S254T/T256E modifications Pascolizumab light chain Pascolizumab heavy chain Alternative NF antibody heavy chain plus M428L/N434S modification Alternative lgG1 constant domain plus M428L/N434S modification Anti-TNF antibody heavy chain plus N434F modification lgG1 constant domain plus H433K/N434F modification ative anti TNF antibody heavy chain plus H433K/N434F modification ative lgG1 constant domain plus H433K/N434F modification Alternative anti TNF antibody heavy chain plus M428L/N434S modification Alternative lgG1/2 constant domain plus N434S modification WO 11076 Seguence g SEQ ID NO: 1Polynucleotide sequence of the anti-TNF antibody light chain GATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGAIAGAGTGACCA TCACCTGCCGGGCCAGCCAGGGCATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTG GCAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCACCCTGCAGAGCGGCGTGCCCAGCA GATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCG AGGACGTGGCCACCTACTACTGCCAGCGGTACAACAGAGCCCCTTACACCTTCGGCCAGGG CACCAAGGTGGAGATCAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGC GATGAGCAGCTCAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCC GGGAGGCCAAAGTGCAGTGGAAAGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGA GCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGA GCAAGGCCGACTACGAGAAGCACAAAGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGT CCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC SEQ ID NO: 2 Protein sequence of the anti-TNF antibody light chain DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGS TLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 3 Protein sequence of the anti-TNF antibody variable domain (VL) DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGS GSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRT SEQ ID NO: 4 Polynucleotide sequence of the anti-TNF dy heavy chain plus M252Y/SZ54T/T256E modification GAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACT GAGCTGTGCCGCCAGCGGCTTCACCTTCGACGACTACGCCATGCACTGGGTGAGGCAGGC CCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTA CGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTA CCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGTCC TACCTGAGCACCGCCAGCAGCCTGGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCC AGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGC GGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTG TCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGC AGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAG ACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGC CCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAG GCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGTACATCACCAGAGAGCC CGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTG GTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAA CAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAA GGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGC AAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAG CTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCG CCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGC TGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCA GCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAG AAGAGCCTGAGCCTGTCCCCTGGCAAG SEQ ID NO: 5 Protein sequence of the anti-TNF antibody heavy chain plus M252Y/SZ54T/T256E cation SGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYAD SVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL YITREPEVTCWVDVSHEDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK SEQ ID NO: 6 Protein sequence of the anti-TNF antibody heavy variable domain (VH) SGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYAD SVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSS SEQ ID NO: 7 Protein ce of the IgG1 constant domain plus M252Y/SZ54T/T256E modification ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLYITREPEVTCVVVDVSHEDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRWSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ SEQ ID NO: 8 Polynucleotide sequence of the anti-TNF antibody heavy chain plus M428L/N434S modification GAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACT GAGCTGTGCCGCCAGCGGCTTCACCTTCGACGACTACGCCATGCACTGGGTGAGGCAGGC CCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTA CGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTA CCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGTCC TACCTGAGCACCGCCAGCAGCCTGGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCC AGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGC GGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTG TCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGC AGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAG ACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGC CCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAG GCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCC CGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTG GTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAA CAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAA GGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGC AAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAG CTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCG CCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGC TGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCA GCAGGGCAACGTGTTCAGCTGCTCCGTGCTGCACGAGGCCCTGCACAGCCACTACACCCA GAAGAGCCTGAGCCTGTCCCCTGGCAAG SEQ ID NO: 9 Protein sequence of the anti-TNF antibody heavy chain plus M428L/N434S modification EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYAD SVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCWVDVSHEDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSL SPGK SEQ ID NO: 10 n sequence of the IgG1 nt domain plus M428L/N434S modification ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLM|SRTPEVTCVWDVSHEDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQ KSLSLSPGK SEQ ID NO: 11 Polynucleotide sequence of the anti-TNF dy heavy chain (wild-type IgG1) GAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACT GAGCTGTGCCGCCAGCGGCTTCACCTTCGACGACTACGCCATGCACTGGGTGAGGCAGGC 2012/064129 CCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTA CGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTA CCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGTCC TACCTGAGCACCGCCAGCAGCCTGGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCC AGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGC ACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTG TCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGC AGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAG ACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGC CCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAG GCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCC CGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTG GTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAA CAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAA GGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGC AAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAG CTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCG CCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGC GCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCA GCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAG AAGAGCCTGAGCCTGTCCCCTGGCAAG SEQ ID NO: 12 Protein sequence of the anti-TNF antibody heavy chain (wild-type IgG1) EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYAD SVEGRFHSRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSUTHNGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSVWVSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTWCNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL hMSRTPEVTCVVVDVSHEDPEVKFhNVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDMH.

NGKEYKCKVSNKALPAMEKWSKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDMNE VVESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWKMQGNVFSCSVMHEALHNHYTQKSLSL SPGK SEQ ID NO: 13 Protein sequence of the IgG1 constant domain (wild-type) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWHVSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTWCNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK PKDTUWSRTPEVTCVVVDVSHEDPEVKHmNYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDMANGKEYKCKVSNKALPAMEKWSKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQ ID NO: 14 Polynucleotide sequence of the anti-TNF antibody heavy chain plus M428Lrnodeafion GAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACT GAGCTGTGCCGCCAGCGGCTTCACCTTCGACGACTACGCCATGCACTGGGTGAGGCAGGC CCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTA CGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTA CCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGTCC TACCTGAGCACCGCCAGCAGCCTGGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCC AGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGC GGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTG TCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGC AGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAG ACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGC CCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAG GCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACmmCTGATGATCAGCAGAACCCCC GAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGT ACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACA GCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGG AGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAA GGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCT GACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCC GTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTG GACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGC AGGGCAACGTGTTCAGCTGCTCCGTGWGCACGAGGCCCTGCACAATCACTACACCCAGAA GAGCCTGAGCCTGTCCCCTGGCAAG SEQ ID NO: 15 Protein sequence of the anti-TNF antibody heavy chain plus T250Q/M428L modflbafion SGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYAD SVEGRFHSRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSUTHNGQGTLVTVSSASTKG APSSKSTSGGTAALGCLVKDYFPEPVTVSVWVSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTWCNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDQL EVTCVVVDVSHEDPEVKFhNVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDMH.

NGKEYKCKVSNKALPAMEKWSKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDMNE VVESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWKMQGNVFSCSVLHEALHNHYTQKSLSL SPGK SEQ ID NO: 16 Protein ce of the IgG1 constant domain plus T250Q/M428L modification ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWHVSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTWCNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK PKDQLN“SRTPEVTCVVVDVSHEDPEVKHmNYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH 2012/064129 QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQ KSLSLSPGK SECIID NCk 17 denudeofide sequence ofthe anfiJTNF anfibody heavy cham pMs V308F modflbafion GAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACT TGCCGCCAGCGGCTTCACCTTCGACGACTACGCCATGCACTGGGTGAGGCAGGC CCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTA CGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTA CCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGTCC TACCTGAGCACCGCCAGCAGCCTGGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCC AGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGC GGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTG TCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGC AGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAG ACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGC CCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAG GCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCC CGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTG GTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAA CAGCACCTACCGGGTGGTGTCCGTGCTGACCflbCTGCACCAGGATTGGCTGAACGGCAAG GAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCA AGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGC TGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGC CGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCT GGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAG CAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGA AGAGCCTGAGCCTGTCCCCTGGCAAG SEQ ID NO: 18 Protein sequence of the anti-TNF antibody heavy chain plus V308F modification SGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYAD SVEGRFHSRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSUTHNGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSVWVSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTWCNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL hMSRTPEVTCVVVDVSHEDPEVKFhNVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTFLHQDMH.

NGKEYKCKVSNKALPAMEKWSKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDMNE VVESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWKMQGNVFSCSVMHEALHNHYTQKSLSL SPGK SEQ ID NO: 19 Protein ce of the IgG1 constant domains plus V308F modification ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLM|SRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTFLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQ ID NO: 20 cleotide sequence of the anti-TNF antibody heavy chain plus V259| modification GAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACT GAGCTGTGCCGCCAGCGGCTTCACCTTCGACGACTACGCCATGCACTGGGTGAGGCAGGC CCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTA CGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTA CCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGTCC TACCTGAGCACCGCCAGCAGCCTGGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCC AGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGC GGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTG TCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGC AGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAG ACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGC CCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAG GCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCC CGAGATCACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAAGTGAAGTTCAACTGG TACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAAC AGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAG AAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCA AGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGC AGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGC CGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCT GGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAG CAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGA AGAGCCTGAGCCTGTCCCCTGGCAAG SEQ ID NO: 21 Protein sequence of the anti-TNF antibody heavy chain plus V259| modification EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYAD SVEGRFHSRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSUTHNGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSVWVSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTWCNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEITCWVDVSHEDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WO 11076 WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK SEQ ID NO: 22 Protein sequence of the lgG1 nt domains plus V259| modification ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWHVSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTWCNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLNHSRTPHTCVVVDVSHEDPEVKFhNVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDMANGKEYKCKVSNKALPAMEKWSKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQ ID NO: 23 Polynucleotide sequence of the NF antibody heavy chain plus P257L and N434Y variant CAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACT GAGCTGTGCCGCCAGCGGCTTCACCTTCGACGACTACGCCATGCACTGGGTGAGGCAGGC CCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTA CGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTA CCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGTCC TACCTGAGCACCGCCAGCAGCCTGGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCC AGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGC GGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTG TCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGC AGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAG ACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGC CCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAG GCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCT GGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTG GTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAA CAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAA GGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGC AAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAG CTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCG CCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGC TGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCA GCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACTATCACTACACCCAG AAGAGCCTGAGCCTGTCCCCTGGCAAG SEQ ID NO: 24 Protein sequence of the anti-TNF antibody heavy chain plus P257L and N434Y modflbafion EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYAD SVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHYHYTQKSLSL SPGK SEQ ID NO: 25 Protein sequence of the IgG1 constant domains plus P257L and N434Y modification ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLM|SRTLEVTCVVVDVSHEDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRWSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHYHYTQ KSLSLSPGK SEQ ID NO: 26 Signal peptide sequence MGWSCIILFLVATATGVHS SEQ ID NO: 27 anti-TNF antibody CDRH1 DYAMH SEQ ID NO: 28 anti-TNF antibody CDRH2 AITWNSGHIDYADSVEG SEQ ID NO: 29 anti-TNF antibody CDRH3 VSYLSTASSLDY SEQ ID NO: 30 NF antibody CDRL1 RASQGIRNYLA SEQ ID NO: 31 anti-TNF antibody CDRL2 SEQ ID NO: 32 anti-TNF antibody CDRL3 QRYNRAPYT SEQ ID NO: 33 NF antibody CDRH1 variant QYAMH SEQ ID NO: 34 anti-TNF antibody CDRH1 variant HYALH SEQ ID NO: 35 anti-TNF antibody CDRH1 variant HYAMH SEQ ID NO: 36 NF antibody CDRH1 variant QHALH SEQ ID NO: 37 anti-TNF dy CDRH1 variant QHAMH SEQ ID NO: 38 anti-TNF antibody CDRH1 variant DHALH SEQ ID NO: 39 Cimzia (certolizumab) LC (VL + Ck) DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKALIYSASFLYSGVPYRFSG SGSGTDFTLTISSLQPEDFATYYCQQYNIYPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 40 anti-TNF antibody CDRH3 variant VHYLSTASQLHH SEQ ID NO: 41 anti-TNF antibody CDRH3 variant VQYLSTASSLQS SEQ ID NO: 42 anti-TNF antibody CDRH3 variant VKYLSTASSLHY SEQ ID NO: 43 anti-TNF antibody CDRH3 variant VKYLSTASNLES SEQ ID NO: 44 anti-TNF antibody CDRH3 variant VHYLSTASSLDY SEQ ID NO: 45 NF antibody CDRH3 variant VSYLSTASSLQS SEQ ID NO: 46 anti-TNF dy CDRH3 variant VRYLSTASNLQH SEQ ID NO: 47 anti-TNF antibody CDRH3 variant VQYLSTASQLHS SEQ ID NO: 48 NF antibody CDRH3 variant VRYLSTASQLDY SEQ ID NO: 49 anti-TNF antibody CDRH3 variant VRYLSTASSLDY SEQ ID NO: 50 anti-TNF antibody CDRL1 variant RNYLA SEQ ID NO: 51 anti-TNF antibody CDRL1 variant HASRKLRNYLA SEQ ID NO: 52 anti-TNF antibody CDRL1 variant HASRRLRNYLA SEQ ID NO: 53 anti-TNF antibody CDRL1 variant HASKRIRNYLA SEQ ID NO: 54 anti-TNF antibody CDRL1 variant HASRKIRNYLA SEQ ID NO: 55 anti-TNF antibody CDRL1 t HASRRIRNYLA SEQ ID NO: 56 anti-TNF antibody CDRL1 variant HASREIRNYLA SEQ ID NO: 57 anti-TNF antibody CDRL1 variant HASQGIRNYLA SEQ ID NO: 58 anti-TNF dy CDRL1 variant RNYLA SEQ ID NO: 59 anti-TNF antibody CDRL1 variant RASRGLRNYLA SEQ ID NO: 60 anti-TNF dy CDRL1 variant HASQRIRNYLA SEQ ID NO: 61 anti-TNF antibody CDRL1 variant RASRRIRNYLA SEQ ID NO: 62 anti-TNF antibody CDRL2 variant AASSLLR SEQ ID NO: 63 anti-TNF antibody CDRL2 variant AASSLLK SEQ ID NO: 64 anti-TNF antibody CDRL2 variant AASSLLP SEQ ID NO: 65 NF antibody CDRL2 variant AASSLQP SEQ ID NO: 66 anti-TNF dy CDRL2 variant AASSLLH SEQ ID NO: 67 anti-TNF antibody CDRL2 variant AASSFLP SEQ ID NO: 68 NF antibody CDRL2 variant AASSLLQ SEQ ID NO: 69 anti-TNF antibody CDRL2 variant AASSLQQ SEQ ID NO: 70 anti-TNF antibody CDRL2 variant AASTLLK SEQ ID NO: 71 anti-TNF antibody CDRL2 variant AASSLQN SEQ ID NO: 72 anti-TNF antibody CDRL2 variant SEQ ID NO: 73 anti-TNF antibody CDRL3 variant QRYDRPPYT SEQ ID NO: 74 anti-TNF antibody CDRL3 t QRYDKPPYT SEQ ID NO: 75 anti-TNF antibody CDRL3 variant QRYNRPPYT SEQ ID NO: 76 anti-TNF dy CDRL3 variant QRYNKPPYT SEQ ID NO: 77 Polynucleotide sequence of anti-TNF antibody variable heavy domain variant cb1VH (aka cb2VH) GAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACT GAGCTGTGCCGCCAGCGGCTTCACCTTCGACCAGTACGCCATGCACTGGGTGAGGCAGGC CCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTA CGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTA CCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGTCC TACCTGAGCACCGCCAGCAGCCTGGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCC SEQ ID NO: 78 n sequence of NF antibody variabIe heavy domain variant cb1VH (aka cb2VH) EVQLVESGGGLVQPGRSLRLSCAASGFTFDQYAMHWVRQAPGKGLEWVSAITWNSGHIDYAD SVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSS SEQ ID NO: 79 Polynucleotide sequence of anti-TNF antibody variabIe heavy domain t cb2VH GAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACT GAGCTGTGCCGCCAGCGGCTTCACCTTCGACGACCACGCCCTGCACTGGGTGAGGCAGGC CCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTA CGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTA CCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGAG GAGCACCGCCAGCAGCCTGGACTACTGGGGCCAGGGCACACTAGTGACCGTGTC CAGC SEQ ID NO: 80 Protein sequence of anti-TNF antibody variabIe heavy domain variant cb2VH EVQLVESGGGLVQPGRSLRLSCAASGFTFDDHALHWVRQAPGKGLEWVSAITWNSGHIDYADS VEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVRYLSTASSLDYWGQGTLVTVSS SEQ ID NO: 81 Polynucleotide sequence of anti-TNF antibody variabIe heavy domain variant cb1VH GAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACT GAGCTGTGCCGCCAGCGGCTTCACCTTCGACCACTACGCCCTGCACTGGGTGAGGCAGGC CAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTA CGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTA CCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGTCC TACCTGAGCACCGCCAGCAGCCTGGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCC SEQ ID NO: 82 n sequence of anti-TNF antibody variable heavy domain variant cb1VH EVQLVESGGGLVQPGRSLRLSCAASGFTFDHYALHWVRQAPGKGLEWVSAITWNSGHIDYADS VEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSS SEQ ID NO: 83 Polynucleotide ce of anti-TNF antibody variable heavy domain variant cb1VH GAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACT GAGCTGTGCCGCCAGCGGCTTCACCTTCGACGACTACGCCATGCACTGGGTGAGGCAGGC CCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTA CGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTA CCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGCAC TACCTGAGCACCGCCAGCCAACTGCACCACTGGGGCCAGGGCACACTAGTGACCGTGTCC SEQ ID NO: 84 Protein sequence of NF antibody variabIe heavy domain variant cb1VH EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYAD SVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVHYLSTASQLHHWGQGTLVTVSS SEQ ID NO: 85 Polynucleotide sequence of anti-TNF antibody variabIe heavy domain variant cb2VH GAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACT TGCCGCCAGCGGCTTCACCTTCGACCACTACGCCATGCACTGGGTGAGGCAGGC CCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTA CGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTA GATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGCA GTACCTGAGCACCGCCAGCAGCCTGCAGAGCTGGGGCCAGGGCACACTAGTGACCGTGTC CAGC SEQ ID NO: 86 Protein sequence of anti-TNF antibody variabIe heavy domain variant cb2VH EVQLVESGGGLVQPGRSLRLSCAASGFTFDHYAMHWVRQAPGKGLEWVSAITWNSGHIDYAD SVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVQYLSTASSLQSWGQGTLVTVSS SEQ ID NO: 87 PonnucIeotide sequence of anti-TNF antibody variable heavy domain variant cb2VH GAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACT GAGCTGTGCCGCCAGCGGCTTCACCTTCGACCAGTACGCCATGCACTGGGTGAGGCAGGC CCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTA CGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTA CCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGAAG TACCTGAGCACCGCCAGCAGCCTGCACTACTGGGGCCAGGGCACACTAGTGACCGTGTCC SEQ ID NO: 88 Protein sequence of NF antibody variable heavy domain variant cb2VH EVQLVESGGGLVQPGRSLRLSCAASGFTFDQYAMHWVRQAPGKGLEWVSAITWNSGHIDYAD SVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVKYLSTASSLHYWGQGTLVTVSS SEQ ID NO: 89 PonnucIeotide sequence of anti-TNF antibody variabIe heavy domain variant GAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACT GAGCTGTGCCGCCAGCGGCTTCACCTTCGACCAGCACGCCCTGCACTGGGTGAGGCAGGC CCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTA CGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTA CCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGCAC TACCTGAGCACCGCCAGCAGCCTGGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCC SEQ ID NO: 90 n sequence of anti-TNF antibody variabIe heavy domain variant cb2VH EVQLVESGGGLVQPGRSLRLSCAASGFTFDQHALHWVRQAPGKGLEWVSAITWNSGHIDYADS VEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVHYLSTASSLDYWGQGTLVTVSS SEQ ID NO: 91 PonnucIeotide sequence of anti-TNF dy variabIe heavy domain variant cb2VH GAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACT TGCCGCCAGCGGCTTCACCTTCGACCAGTACGCCATGCACTGGGTGAGGCAGGC CCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTA CGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTA CCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGCAC AGCACCGCCAGCCAGCTGCACCACTGGGGCCAGGGCACACTAGTGACCGTGTCC SEQ ID NO: 92 Protein sequence of anti-TNF antibody variabIe heavy domain variant cb2-28—VH EVQLVESGGGLVQPGRSLRLSCAASGFTFDQYAMHWVRQAPGKGLEWVSAITWNSGHIDYAD SVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVHYLSTASQLHHWGQGTLVTVSS SEQ ID NO: 93 Polynucleotide sequence of anti-TNF dy variable heavy domain variant cb2-38—VH GAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACT GAGCTGTGCCGCCAGCGGCTTCACCTTCGACCAGCACGCCATGCACTGGGTGAGGCAGGC CCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTA CGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTA CCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGTCC TACCTGAGCACCGCCAGCAGCCTGGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCC SEQ ID NO: 94 Protein sequence of anti-TNF antibody variable heavy domain variant cb2-38—VH EVQLVESGGGLVQPGRSLRLSCAASGFTFDQHAMHWVRQAPGKGLEWVSAITWNSGH|DYAD SVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSS SEQ ID NO: 95 Polynucleotide sequence of anti-TNF antibody variable heavy domain t cb2VH GAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACT GAGCTGTGCCGCCAGCGGCTTCACCTTCGACCAGTACGCCATGCACTGGGTGAGGCAGGC CCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTA CGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTA CCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGAAG AGCACCGCCAGCAACCTGGAGAGCTGGGGCCAGGGCACACTAGTGACCGTGTCC SEQ ID NO: 96 Protein sequence of anti-TNF antibody variable heavy domain variant cb2VH SGGGLVQPGRSLRLSCAASGFTFDQYAMHWVRQAPGKGLEWVSAITWNSGHIDYAD SVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVKYLSTASNLESWGQGTLVTVSS SEQ ID NO: 97 Polynucleotide sequence of anti-TNF antibody variable light domain t cb1- 8—VL GATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCA GCCACGCCAGCAAGAAGATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGG CAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGAGGGGCGTGCCCAGCAG ATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGA GGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGG CACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 98 Protein sequence of anti-TNF antibody variable light domain variant cb1VL DIQMTQSPSSLSASVGDRVTITCHASKKIRNYLAWYQQKPGKAPKLLIYAASSLLRGVPSRFSGS GSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 99 Polynucleotide sequence of anti-TNF antibody variable light domain variant cb1- 43-VL GATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCA TCACCTGCCACGCCAGCAGGAAGCTGAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGG CAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGAAGGGCGTGCCCAGCAG CGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGA GGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGG CACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 100 Protein sequence of anti-TNF antibody variable light domain variant cb1VL DIQMTQSPSSLSASVGDRVTITCHASRKLRNYLAWYQQKPGKAPKLLIYAASSLLKGVPSRFSGS GSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 101 Polynucleotide sequence of anti-TNF antibody variable light domain t cb1- 45-VL GATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCA GCCACGCCAGCAGGAGGCTGAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGG CAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGCCCGGCGTGCCCAGCAG ATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGA GGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGG CACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 102 Protein sequence of anti-TNF antibody variable light domain variant cb1VL DIQMTQSPSSLSASVGDRVTITCHASRRLRNYLAWYQQKPGKAPKLLIYAASSLLPGVPSRFSGS GSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 103 Polynucleotide sequence of anti-TNF dy variable light domain variant cb1- 4-VL GATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCA TCACCTGCCACGCCAGCAGGAAGCTGAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGG CAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGAGGGGCGTGCCCAGCAG ATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGA GGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGG CACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 104 Protein sequence of anti-TNF dy le light domain variant cb1VL DIQMTQSPSSLSASVGDRVTITCHASRKLRNYLAWYQQKPGKAPKLLIYAASSLLRGVPSRFSGS GSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 105 Polynucleotide sequence of NF antibody variable light domain variant cb1- 41-VL GATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCA TCACCTGCCACGCCAGCAAGAGGATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGG CAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGAAGGGCGTGCCCAGCAG ATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGA GGACGTGGCCACCTACTACTGCCAGCGGTACGACAAGCCCCCTTACACCTTCGGCCAGGG CACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 106 Protein sequence of anti-TNF antibody variable light domain variant cb1VL SPSSLSASVGDRVTITCHASKRIRNYLAWYQQKPGKAPKLLIYAASSLLKGVPSRFSGS GSGTDFTLTISSLQPEDVATYYCQRYDKPPYTFGQGTKVEIKRT SEQ ID NO: 107 Polynucleotide sequence of anti-TNF antibody variable light domain variant cb1- 37-VL GATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCA TCACCTGCCACGCCAGCAGGAAGCTGAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGG CCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGAGGGGCGTGCCCAGCAG ATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGA GGACGTGGCCACCTACTACTGCCAGCGGTACAACAGACCCCCTTACACCTTCGGCCAGGGC ACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 108 Protein sequence of anti-TNF antibody variable light domain variant cb1VL DIQMTQSPSSLSASVGDRVTITCHASRKLRNYLAWYQQKPGKAPKLLIYAASSLLRGVPSRFSGS GSGTDFTLTISSLQPEDVATYYCQRYNRPPYTFGQGTKVEIKRT SEQ ID NO: 109 Polynucleotide ce of anti-TNF antibody variable light domain variant cb1- 39-VL GATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCA TCACCTGCCACGCCAGCAGGAAGATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGG CAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGCCCGGCGTGCCCAGCAG ATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGA GGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGG CACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 110 Protein ce of anti-TNF antibody variable light domain variant -VL DIQMTQSPSSLSASVGDRVTITCHASRKIRNYLAWYQQKPGKAPKLLIYAASSLQPGVPSRFSGS GSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 111 Polynucleotide sequence of anti-TNF antibody variable light domain variant cb1- 33-VL GATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGAIAGAGTGACCA TCACCTGCCACGCCAGCAGGAGGATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGG CAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGCACGGCGTGCCCAGCAG ATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGA GGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGG GGTGGAGATCAAGCGTACG SEQ ID NO: 112 Protein sequence of anti-TNF antibody variable light domain variant cb1VL DIQMTQSPSSLSASVGDRVTITCHASRRIRNYLAWYQQKPGKAPKLLIYAASSLLHGVPSRFSGS GSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 113 Polynucleotide sequence of anti-TNF antibody variable light domain variant cb1- -VL GATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGAIAGAGTGACCA TCACCTGCCACGCCAGCAGGAGGCTGAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGG CAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGCCCGGCGTGCCCAGCAG ATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGA GGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGG GGTGGAGATCAAGCGTACG SEQ ID NO: 114 Protein sequence of anti-TNF antibody variable light domain variant cb1VL DIQMTQSPSSLSASVGDRVTITCHASRRLRNYLAWYQQKPGKAPKLLIYAASSLQPGVPSRFSG SGSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 115 Polynucleotide sequence of anti-TNF dy variable light domain variant cb1- 31-VL GATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCA TCACCTGCCACGCCAGCAGGAGGCTGAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGG CAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGAAGGGCGTGCCCAGCAG ATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGA GGACGTGGCCACCTACTACTGCCAGCGGTACAACAAGCCCCCTTACACCTTCGGCCAGGGC ACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 116 Protein sequence of anti-TNF antibody variable light domain variant cb1VL DIQMTQSPSSLSASVGDRVTITCHASRRLRNYLAWYQQKPGKAPKLLIYAASSLLKGVPSRFSGS GSGTDFTLTISSLQPEDVATYYCQRYNKPPYTFGQGTKVEIKRT SEQ ID NO: 117 Polynucleotide sequence of anti-TNF antibody le light domain variant cb1- 29-VL CAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGAIAGAGTGACCA TCACCTGCCACGCCAGCAGGAAGATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGG CCCTAAGCTGCTGATCTACGCCGCCAGCAGCTTCCTGCCCGGCGTGCCCAGCAG ATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGA GGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGG CACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 118 n sequence of anti-TNF antibody variable light domain variant cb1VL DIQMTQSPSSLSASVGDRVTITCHASRKIRNYLAWYQQKPGKAPKLLIYAASSFLPGVPSRFSGS GSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 119 Polynucleotide sequence of anti-TNF antibody variable light domain variant cb1- 22-VL GATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCA TCACCTGCCACGCCAGCAAGAAGATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGG CAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGCCCGGCGTGCCCAGCAG ATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGA GGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGG CACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 120 Protein sequence of anti-TNF antibody variable light domain variant cb1VL DIQMTQSPSSLSASVGDRVTITCHASKKIRNYLAWYQQKPGKAPKLLIYAASSLQPGVPSRFSGS GSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 121 Polynucleotide sequence of anti-TNF antibody variable light domain variant cb1- 23-VL CAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCA TCACCTGCCACGCCAGCAGGAGGATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGG CAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGCAGGGCGTGCCCAGCAG ATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGA GGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGG CACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 122 Protein sequence of anti-TNF antibody variable light domain variant cb1VL DIQMTQSPSSLSASVGDRVTITCHASRRIRNYLAWYQQKPGKAPKLLIYAASSLLQGVPSRFSGS TLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 123 Polynucleotide sequence of anti-TNF antibody variable light domain variant cb1- 12-VL GATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCA TCACCTGCCACGCCAGCAGGAAGCTGAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGG CAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGCAGGGCGTGCCCAGCAG ATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGA GGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGG CACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 124 Protein ce of anti-TNF antibody variable light domain variant cb1VL SPSSLSASVGDRVTITCHASRKLRNYLAWYQQKPGKAPKLLIYAASSLQQGVPSRFSG SGSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 125 Polynucleotide sequence of NF antibody variable light domain variant cb1- -VL CAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCA TCACCTGCCACGCCAGCAGGAAGCTGAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGG CAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGCCCGGCGTGCCCAGCAG ATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGA GGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGG CACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 126 Protein sequence of anti-TNF antibody variable light domain variant cb1VL DIQMTQSPSSLSASVGDRVTITCHASRKLRNYLAWYQQKPGKAPKLLIYAASSLLPGVPSRFSGS GSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 127 Polynucleotide sequence of anti-TNF antibody variable light domain variant cb2- 1-VL GATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCA TCACCTGCCACGCCAGCAGGGAGATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGG CAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGCCCGGCGTGCCCAGCAG ATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGA GGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGG CACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 128 Protein sequence of anti-TNF antibody variable light domain t VL DIQMTQSPSSLSASVGDRVTITCHASRE|RNYLAWYQQKPGKAPKLLIYAASSLLPGVPSRFSGS GSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 129 Polynucleotide sequence of anti-TNF antibody variable light domain variant cb2- 11-VL GATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCA TCACCTGCCACGCCAGCCAGGGCATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGG CAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCACCCTGCTGAAGGGCGTGCCCAGCAG ATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGA GGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGG CACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 130 Protein sequence of anti-TNF dy variable light domain variant cb2VL DIQMTQSPSSLSASVGDRVTITCHASQGIRNYLAWYQQKPGKAPKLLIYAASTLLKGVPSRFSGS GSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 131 Polynucleotide sequence of anti-TNF antibody variable light domain t cb2- 40-VL GATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCA TCACCTGCCACGCCAGCCAGAAGATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGG CAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGCAGGGCGTGCCCAGCAG ATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGA GGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGG CACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 132 Protein sequence of anti-TNF antibody variable light domain variant cb2VL DIQMTQSPSSLSASVGDRVTITCHASQKIRNYLAWYQQKPGKAPKLLIYAASSLQQGVPSRFSGS GSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 133 Polynucleotide sequence of anti-TNF antibody le light domain variant cb2- -VL GATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCA TCACCTGCCACGCCAGCAGGAGGCTGAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGG CAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGCACGGCGTGCCCAGCAG ATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGA GGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGG CACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 134 Protein sequence of anti-TNF antibody variable light domain variant cb2VL DIQMTQSPSSLSASVGDRVTITCHASRRLRNYLAWYQQKPGKAPKLLIYAASSLLHGVPSRFSGS GSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 135 Polynucleotide sequence of anti-TNF antibody variable light domain variant cb2- 28—VL GATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCA TCACCTGCCACGCCAGCAGGAGGCTGAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGG CCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGAAGGGCGTGCCCAGCAG ATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGA WO 11076 GGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGG CACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 136 Protein sequence of NF antibody le light domain variant cb2-28—VL DIQMTQSPSSLSASVGDRVTITCHASRRLRNYLAWYQQKPGKAPKLLIYAASSLLKGVPSRFSGS GSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 137 Polynucleotide sequence of anti-TNF antibody le light domain variant cb2- -VL GATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCA TCACCTGCCACGCCAGCAAGAGGATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGG CAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGAGGGGCGTGCCCAGCAG ATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGA GGACGTGGCCACCTACTACTGCCAGCGGTACAACAAGCCCCCTTACACCTTCGGCCAGGGC ACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 138 Protein sequence of anti-TNF antibody variable light domain variant cb2VL DIQMTQSPSSLSASVGDRVTITCHASKRIRNYLAWYQQKPGKAPKLLIYAASSLLRGVPSRFSGS GSGTDFTLTISSLQPEDVATYYCQRYNKPPYTFGQGTKVEIKRT SEQ ID NO: 139 Polynucleotide sequence of anti-TNF dy variable light domain variant cb1- 3-VL GATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCA TCACCTGCCACGCCAGCAGGAAGATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGG CAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGAGGGGCGTGCCCAGCAG ATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGA GGACGTGGCCACCTACTACTGCCAGCGGTACGACAAGCCCCCTTACACCTTCGGCCAGGG CACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 140 Protein sequence of anti-TNF antibody variable light domain variant cb1VL DIQMTQSPSSLSASVGDRVTITCHASRKIRNYLAWYQQKPGKAPKLLIYAASSLLRGVPSRFSGS GSGTDFTLTISSLQPEDVATYYCQRYDKPPYTFGQGTKVEIKRT SEQ ID NO: 141 Polynucleotide sequence of anti-TNF antibody variable light domain variant cb2- 6-VL GATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCA TCACCTGCCACGCCAGCAAGAGGATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGG CAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGAAGGGCGTGCCCAGCAG ATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGA GGACGTGGCCACCTACTACTGCCAGCGGTACAACAAGCCCCCTTACACCTTCGGCCAGGGC ACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 142 Protein sequence of NF antibody variable light domain variant cb2VL DIQMTQSPSSLSASVGDRVTITCHASKRIRNYLAWYQQKPGKAPKLLIYAASSLLKGVPSRFSGS TLTISSLQPEDVATYYCQRYNKPPYTFGQGTKVEIKRT SEQ ID NO: 143 cleotide sequence of anti-TNF antibody variable light domain variant cb2- 44-VL GATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCA TCACCTGCCACGCCAGCAGGAAGATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGG CAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGCCCGGCGTGCCCAGCAG ATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGA GGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGG CACCAAGGTGGAGATCAAGCGTACG SEQ ID NO: 144 Protein sequence of anti-TNF antibody variable light domain variant cb2VL DIQMTQSPSSLSASVGDRVTITCHASRKIRNYLAWYQQKPGKAPKLLIYAASSLLPGVPSRFSGS GSGTDFTLTISSLQPEDVATYYCQRYDRPPYTFGQGTKVEIKRT SEQ ID NO: 145 n sequence of anti-TNF antibody heavy chain variant cb1VH plus SZ54T/T256E modification EVQLVESGGGLVQPGRSLRLSCAASGFTFDQYAMHWVRQAPGKGLEWVSAITWNSGHIDYAD SVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL YITREPEVTCWVDVSHEDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK SEQ ID NO: 146 Protein sequence of anti-TNF antibody heavy chain variant cb2VH plus M252Y/SZ54T/T256E modification EVQLVESGGGLVQPGRSLRLSCAASGFTFDDHALHWVRQAPGKGLEWVSAITWNSGHIDYADS VEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVRYLSTASSLDYWGQGTLVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLY ITREPEVTCVWDVSHEDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK SEQ ID NO: 147 Polynucleotide sequence of anti-TNF antibody light chain variant cb1VL WO 11076 GATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGAIAGAGTGACCA TCACCTGCCACGCCAGCAGGAAGATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGG CAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGAGGGGCGTGCCCAGCAG ATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGA GGACGTGGCCACCTACTACTGCCAGCGGTACGACAAGCCCCCTTACACCTTCGGCCAGGG GGTGGAGATCAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGC GATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCC GGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGA GCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGA GCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGT CCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC SEQ ID NO: 148 Protein sequence of anti-TNF antibody light chain variant cb1VL DIQMTQSPSSLSASVGDRVTITCHASRKIRNYLAWYQQKPGKAPKLLIYAASSLLRGVPSRFSGS GSGTDFTLTISSLQPEDVATYYCQRYDKPPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 149 Polynucleotide sequence of anti-TNF antibody light chain variant cb2VL GATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCA TCACCTGCCACGCCAGCAAGAGGATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGG CAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGAAGGGCGTGCCCAGCAG ATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGA GGACGTGGCCACCTACTACTGCCAGCGGTACAACAAGCCCCCTTACACCTTCGGCCAGGGC ACCAAGGTGGAGATCAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGC GATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCC GGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGA GCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGA GCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGT CCGTGACCAAGAGCTTCAACCGGGGCGAGTGC SEQ ID NO: 150 Protein sequence of anti-TNF antibody light chain variant cb2VL DIQMTQSPSSLSASVGDRVTITCHASKRIRNYLAWYQQKPGKAPKLLIYAASSLLKGVPSRFSGS GSGTDFTLTISSLQPEDVATYYCQRYNKPPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 151 cleotide sequence of anti-TNF antibody light chain variant cb2 GATATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCTCTGTGGGCGATAGAGTGACCA TCACCTGCCACGCCAGCAGGAAGATCAGAAACTACCTGGCCTGGTATCAGCAGAAGCCTGG CAAGGCCCCTAAGCTGCTGATCTACGCCGCCAGCAGCCTGCTGCCCGGCGTGCCCAGCAG ATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGA GGACGTGGCCACCTACTACTGCCAGCGGTACGACAGACCCCCTTACACCTTCGGCCAGGG CACCAAGGTGGAGATCAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGC GATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCC GGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGA GCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGA GCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGT CCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC SEQ ID NO: 152 Protein sequence of anti-TNF antibody light chain variant -VL DIQMTQSPSSLSASVGDRVTITCHASRKIRNYLAWYQQKPGKAPKLLIYAASSLLPGVPSRFSGS GSGTDFTEHSSLQPEDVATYYCQRYDRPPYTFGQGTKVHKRTVAAPSVFFPPSDEQLKSGTA SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 153 Polynucleotide sequence of anti-TNF antibody heavy chain variant cb1- 3-VH GAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACT GAGCTGTGCCGCCAGCGGCTTCACCTTCGACCAGTACGCCATGCACTGGGTGAGGCAGGC CAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTA CGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTA CCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGTCC TACCTGAGCACCGCCAGCAGCCTGGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCC AGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGC GGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTG TCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGC AGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAG ACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGC CCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAG GCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCC CGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTG GTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAA CAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAA GGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGC AAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAG AAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCG CCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGC TGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCA GCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAG AAGAGCCTGAGCCTGTCCCCTGGCAAG SEQ ID NO: 154 Protein sequence of anti-TNF antibody heavy chain variant cb1VH EVQLVESGGGLVQPGRSLRLSCAASGFTFDQYAMHWVRQAPGKGLEWVSAITWNSGHIDYAD HSRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSUTHNGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSVWVSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTWCNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL hMSRTPEVTCVVVDVSHEDPEVKFhNVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDMH.

NGKEYKCKVSNKALPAMEKWSKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDMNE QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWKMQGNVFSCSVMHEALHNHYTQKSLSL SPGK SEQ ID NO: 155 Polynucleotide sequence of anti-TNF antibody heavy chain variant cb2- 44-VH GAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGCAGAAGCCTGAGACT GAGCTGTGCCGCCAGCGGCTTCACCTTCGACGACCACGCCCTGCACTGGGTGAGGCAGGC CCCTGGCAAGGGCCTGGAGTGGGTGTCCGCCATCACCTGGAATAGCGGCCACATCGACTA CGCCGACAGCGTGGAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTA CCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGGTGAG GAGCACCGCCAGCAGCCTGGACTACTGGGGCCAGGGCACACTAGTGACCGTGTC CAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAG CGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGT GTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAG CAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCA GACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAG CCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGA GGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCC CCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACT GGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACA ACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCA AGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAG CAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGA GCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATC GAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTG CTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGC AGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCA GAAGAGCCTGAGCCTGTCCCCTGGCAAG SEQ ID NO: 156 Protein sequence of anti-TNF antibody heavy chain t cb2VH EVQLVESGGGLVQPGRSLRLSCAASGFTFDDHALHWVRQAPGKGLEWVSAITWNSGHIDYADS VEGRFHSRDNAKNSLYLQMNSLRAEDTAVYYCAKVRYLSTASSUTflNGQGTLVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK SEQ ID NO: 157 Polynucleotide sequence of pascolizumab heavy chain containing the M252Y/8254T/T256E modifications CAGGTGACCCTGAGGGAGAGCGGCCCCGCCCTGGTGAAGCCCACCCAGACCCTGACCCTG ACCTTCAGCGGCTTTAGCCTCAGCACCTCCGGCATGGGCGTGAGCTGGATCAGGC AGCCACCCGGCAAAGGCCTGGAGTGGCTGGCCCACATCTACTGGGACGACGACAAGAGGT ACAACCCCAGCCTGAAGAGCCGGCTGACCATCAGCAAGGATACCAGCAGGAACCAGGTGG TGCTGACCATGACCAACATGGACCCCGTGGACACCGCTACCTACTACTGCGCCAGGAGGGA GACCGTCTTCTACTGGTACTTCGACGTGTGGGGAAGGGGCACACTAGTGACCGTGTCCAGC GCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGC GGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCC TGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGC GGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACC TACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCA AGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCC CCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGchHkmCAGAgmCCCGAGG TGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGT GGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCAC CTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTA CAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCC AAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACC AAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGG AGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACA GCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGG GCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAG CCTGAGCCTGTCCCCTGGCAAG SEQ ID NO:158 Protein sequence of pascolizumab heavy chain containing the M252Y/8254T/T256E cations QVTLRESGPALVKPTQTEHICTFSGFSLSTSGMGVSWHRQPPGKGLEMAAHPHNDDDKRYNPS LKSRLHSKDTSRNQVVLTMTNMDPVDTATYYCARRETVFWNYFDVMKEKNIVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSVWVSGALTSGVHTFPAVLQSSGLYSLSSVVTV TQTWCNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYI TREPEVTCVVVDVSHEDPEVKHmNYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWKNG KEYKCKVSNKALPAMEKHSKAKGQPREPQVYTLPPSRDELTKNQVSJILVKGFYPSDMNEM/ ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS SEQIDIVO:159 eofidesequenceofpasmfluunmblbhtcham GACATCGTGCTGACCCAGAGCCCCTCTTCCCTGAGCGCAAGCGTGGGCGATAGGGTGACC ATCACCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACATGAACTGGTACC AGCAGAAGCCCGGCAAGGCCCCCAAACTGCTGATCTACGCCGCCAGCAACCTCGAGTCAG GCATTCCCAGCAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTCACCTTCACAATCAGCAG CCTGCAGCCCGAGGACATCGCCACCTACTACTGCCAGCAGAGCAACGAGGACCCTCCCAC CTTCGGACAGGGCACCAAGGTCGAGATCAAGCGTACGGTGGCCGCCCCCAGCGTGTTCAT CTTCCCCCCCAGCGATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAA CAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGG CCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAG CACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGAC CCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC SEQ ID NO: 160 Protein sequence of pascolizumab light chain DIVLTQSPSSLSASVGDRVTITCKASQSVDYDGDSYMNWYQQKPGKAPKLLIYAASNLESGIPSR FSGSGSGTDFTFTISSLQPEDIATYYCQQSNEDPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKS GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 161 Protein sequence of pascolizumab heavy chain QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYNPS LKSRLTISKDTSRNQWLTMTNMDPVDTATYYCARRETVFYWYFDVWGRGTLVTVSSASTKGPS SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS SEQ ID NO: 162 Alternative protein sequence of the anti-TNF antibody heavy chain plus N434S modification EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYAD SVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCWVDVSHEDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV 2012/064129 EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLS LSPGK SEQ ID NO:163 Alternative protein sequence of the lgG1 constant domain plus M428L/N434S modification ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLM|SRTPEVTCVWDVSHEDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYT QKSLSLSPGK SEQ ID NO:164 Protein sequence of the anti-TNF antibody heavy chain plus H433K/N434F modification EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYAD TISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCWVDVSHEDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALKFHYTQKSLSL SPGK SEQ ID NO: 165 n sequence of the lgG1 nt domain plus H433K/N434F modification ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLM|SRTPEVTCVWDVSHEDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALKFHYTQ KSLSLSPGK SEQ ID NO: 166 Alternative protein sequence of the anti-TNF antibody heavy chain plus H433K/N434F modification EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYAD SVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCWVDVSHEDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALKFHYTQKSLS LSPGK SEQ ID NO:167 Alternative protein ce of the lgG1 constant domain plus N434F modification ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLM|SRTPEVTCVWDVSHEDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALKFHYT QKSLSLSPGK SEQ ID NO: 168 Alternative protein sequence of the anti-TNF antibody heavy chain plus M428L/N434S modification EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYAD SVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTL MISRTPEVTCWVDVSHEDPEVQFNWWDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWL NGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLS LSPGK SEQ ID NO:169 Alternative protein sequence of the IgG1/2 constant domain plus M428L/N434S modification ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPPVAGPSVFLFPPKP KDTLM|SRTPEVTCVVVDVSHEDPEVQFNWWDGVEVHNAKTKPREEQFNSTFRWSVLTVVHQ DWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQ KSLSLSPGK SEQ ID NO: 170 Golimumab_VH QVQLVESGGGVVQPGRSLRLSCAASGFIFSSYAMHWVRQAPGNGLEWVAFMSYDGSNKKYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGIAAGGNYYYYGMDVWGQGTTVTVS SEQ ID NO: 171 mab_VL EIVLTQSPATLSLSPGERATLSCRASQSVYSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGS GSGTDFTLTISSLEPEDFAVYYCQQRSNWPPFTFGPGTKVDIKRT SEQ ID NO: 172 Golimumab_HC WO 11076 QVQLVESGGGVVQPGRSLRLSCAASGFIFSSYAMHWVRQAPGNGLEWVAFMSYDGSNKKYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGIAAGGNYYYYGMDVWGQGTTVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLYITREPEVTCVWDVSHEDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRVVSVLTVL GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK SEQ ID NO: 173 Golimumab_LC EIVLTQSPATLSLSPGERATLSCRASQSVYSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGS GSGTDFTLTISSLEPEDFAVYYCQQRSNWPPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGT ASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 174 Remicade_VH EVKLEESGGGLVQPGGSMKLSCVASGFIFSNHWMNWVRQSPEKGLEWVAEIRSKSINSATHYA ESVKGRFTISRDDSKSAVYLQMTDLRTEDTGWYCSRNYYGSTYDYWGQGTTLTVSS SEQ ID NO: 175 Remicade_VL DILLTQSPAILSVSPGERVSFSCRASQFVGSSIHWYQQRTNGSPRLLIKYASESMSGIPSRFSGS GSGTDFTLS|NTVESEDIADYYCQQSHSWPFTFGSGTNLEVKRT SEQ ID NO: 176 de _HC EVKLEESGGGLVQPGGSMKLSCVASGFIFSNHWMNWVRQSPEKGLEWVAEIRSKSINSATHYA ESVKGRFTISRDDSKSAVYLQMTDLRTEDTGWYCSRNYYGSTYDYWGQGTTLTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLY ITREPEVTCVWDVSHEDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK SEQ ID NO: 177 Remicade _LC DILLTQSPAILSVSPGERVSFSCRASQFVGSSIHWYQQRTNGSPRLLIKYASESMSGIPSRFSGS GSGTDFTLSINTVESEDIADYYCQQSHSWPFTFGSGTNLEVKRTVAAPSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 178 Cimzia (certolizumab) VH SGGGLVQPGGSLRLSCAASGWFTDYGMNWVRQAPGKGLEWMGWINTYIGEPIYAD SVKGRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARGYRSYAMDYWGQGTLVTVSS SEQ ID NO: 179 Cimzia (certolizumab) VL DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKALIYSASFLYSGVPYRFSG SGSGTDFTLTISSLQPEDFATYYCQQYNIYPLTFGQGTKVEIKRT SEQ ID NO: 180 Cimzia (certolizumab) HC (VH+CH1) SGGGLVQPGGSLRLSCAASGYVFTDYGMNWVRQAPGKGLEWMGWINTYIGEPIYAD SVKGRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARGYRSYAMDYWGQGTLVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCAA

Claims (38)

Claims

1. An antigen binding protein which specifically binds to pha comprising: (i) CDRH1 of SEQ ID NO: 27, CDRH2 of SEQ ID NO: 28, CDRH3 of SEQ ID No: 29, CDRL1 of SEQ ID NO: 30, CDRL2 of SEQ ID NO: 31, and CDRL3 of SEQ ID NO: 32; and (ii) a neonatal Fc receptor (FcRn) binding portion of a human IgG1 constant domain comprising amino acid substitutions relative to the human IgG1 constant domain wherein the amino acid substitutions are at amino acid residues 252, 254 and 256 numbered according to EU index of Kabat and the substitution at residue 252 is a substitution of met with tyr; residue 254 is a substitution of ser with thr and residue 256 is a substitution of thr with glu: and wherein the antigen binding protein has an increased FcRn binding affinity at pH 6 and/ or increased half-life as compared to an IgG sing the light chain sequence of SEQ ID No. 2 and the heavy chain sequence of SEQ ID No.12.

2. An antigen binding protein as claimed in claim 1 wherein the human IgG1 nt domain has the ce of SEQ ID No. 13 before amino acid substitutions are introduced.

3. An antigen binding n as claimed in any preceding claim for treatment of a disease wherein the antigen binding protein is to be administered to patients at a single dose between about 35 to about 45 mg at a four to eight weekly interval.

4. An antigen binding protein as claimed in claim 3 n the antigen binding protein is to be administered to patients subcutaneously as a single 40 mg dose no more than once every four weeks.

5. An antigen binding n as claimed in claim 3 or claim 4 wherein the antigen binding n is to be stered to patients subcutaneously as a single 40 mg dose no more than once every eight weeks.

6. An antigen binding protein as claimed in any one of claims 1 to 5 wherein the half-life of the antigen binding protein is increased 2 fold, 3 fold, 4 fold or 5 fold as compared to the native IgG.

7. An antigen binding protein as claimed in any preceding claim wherein administration of the antigen binding protein no more than once every four weeks in patients achieves the mean steadystate trough concentration in the patient population of n about 4 μg/ml to about 7 μg/ml.

8. An antigen binding protein as claimed in claim 7 wherein the mean steady-state trough concentration is between about 5 μg/ml to about 6 μg/ml.

9. An antigen binding protein as claimed in any one of claims 1 to 8 n the clearance of the antigen binding protein is about 2.ml/ hr to about 4ml/hr.

10. An antigen binding protein as claimed in any preceding claim sing a constant domain as shown in SEQ ID No: 7.

11. An antigen binding protein as claimed in any one of claims 1 to 10 wherein the n binding protein is an antibody.

12. An n binding protein as claimed in any one of claims 1 to 11 wherein the antigen binding protein is to be administered with methotrexate.

13. An n binding protein as claimed in any one of claims 1 to 11 wherein the antigen binding protein is to be stered for the treatment of rheumatoid arthritis.

14. An n binding protein as claimed in any one of claims 1 to 12 comprising the heavy chain sequence as shown in SEQ ID NO 5 optionally with a light chain sequence as shown in SEQ ID No:

15. Use of an antigen binding protein as claimed in any ing claim in the manufacture of a medicament for treating a disease.

16. Use according to claim 15, wherein the medicament is for administering to the patient subcutaneously as a single of dose about 35 to about 45 mg at a four to eight weekly interval.

17. Use of an antigen binding protein as d in any one of claims 1 to 14 in the manufacture of a medicament for the treatment of rheumatoid arthritis, polyarticular le thic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease or Psoriasis.

18. Use according to any one of claims 15 to 17, wherein the medicament is to be administered to patients at a single dose between about 35 to about 45 mg at a four to eight weekly interval.

19. Use according to claim 18, wherein the medicament is to be administered to patients subcutaneously as a single 40 mg dose of antigen binding protein no more than once every four weeks.

20. Use according to claim 18 or 19 n the medicament is to be administered to patients subcutaneously as a single 40 mg dose of antigen binding protein no more than once every eight weeks.

21. Use according to any one of claims 15 to 20 wherein the half-life of the antigen binding protein is increased 2 fold, 3 fold, 4 fold or 5 fold as compared to the native IgG.

22. Use ing to any one of claims 15 to 21 wherein administration of the medicament no more than once every four weeks in patients achieves the mean steady-state trough concentration in the patient population of between about 4 μg/ml to about 7 μg/ml.

23. Use according to in claim 22 wherein the mean steady-state trough concentration is between about 5 μg/ml to about 6 μg/ml.

24. Use according to any one of claims 15 to 23 wherein the clearance of the antigen binding protein is about 2.ml/ hr to about .

25. Use according to any one of claims 15 to 24 wherein the antigen binding protein comprises a constant domain as shown in SEQ ID No: 7.

26. Use according to any one of claims 15 to 25 wherein the antigen binding n is an antibody.

27. Use according to any one of claims 15 to 26 wherein the medicament is to be administered with methotrexate.

28. Use according to any one of claims 15 to 27, for the treatment of rheumatoid arthritis.

29. Use according to any one of claims 15 to 26, wherein the antigen binding n comprises the heavy chain sequence as shown in SEQ ID NO 5 optionally with a light chain sequence as shown in SEQ ID No: 2.

30. An n binding n as claimed in any one of claims 1 to 14 for use in the treatment of rheumatoid arthritis, polyarticular juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease or sis.

31. A nucleic acid comprising a nucleotide sequences encoding the antigen binding protein as d in any one of claims 1 to 14 or 30.

32. A host cell comprising the nucleic acid as claimed in claim 31, provided that the host cell is not within a human.

33. An antigen binding n according to claim 1, substantially as herein described or exemplified.

34. Use according to claim 15, substantially as herein described or exemplified.

35. Use according to claim 17, substantially as herein described or exemplified.

36. An antigen binding n according to claim 30, substantially as herein described or exemplified.

37. A nucleic acid according to claim 31, substantially as herein bed or exemplified.

38. A host cell according to claim 32, substantially as herein described or exemplified. WO 11076