This application claims priority to U.S. Provisional Application Serial No.

61/650,883, filed May 23, 2012 and U.S. Provisional Application Serial No. 61/720,102, filed October 30, 2012, both of which are herein incorporated by reference in their entireties.

BACKGROUND

Interleukin-6 (IL-6) is a major proinflammatory cytokine. It is responsible for the proliferation and differentiation of immunocompetent and hematopoietic cells. Human IL-6 is a single glycoprotein consisting of 212 amino acids with two N-liiiked glycosylation sites, and has a molecular we ght of about 26kDa. The structure of IL-6 comprises four a-helical domains with a motif of four cysteine residues which are necessary for its tertiary structure. IL-6 signalling is mediated by the binding of IL-6 to either soluble or surface bound IL-6 receptor alpha chain (IL-6Ra), enabling interaction of the complex with the cell surface transmembrane gpl30 subunit that mediates intracellular signalling.

IL-6 is implicated in the pathogenesis of inflammatory diseases, including

inflammatory autoimmune diseases such as rheumatoid arthritis (RA), spondy losing arthropathy, systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD) and Castlernan's disease. IL-6 is also implicated in the pathogenesis of cancers, including prostate cancer, diffuse large cell lymphoma, multiple myeloma, and renal cell cancer. A role for IL-6 in promoting cancer-related anorexia, oral mucositis and cachexia has also been reported.

Although IL-6 binding molecules derived from immunization of non-human animals are known in the art, these molecules have typically required extensive antibody engineering (e.g., CDR grafting and humanization) to reduce their immunogenicity. Moreover, the resulting humanized variants typically suffer from sub-optimal binding affinity to the IL-6 target and require extensive antibody engineering and affinity maturation in an attempt to restore IL-6 binding affinity. The end result is that most IL-6 antibodies exhibit sub-optimal binding affinity to the IL-6 target.

Therefore, given the importance of IL-6 in disease pathogenesis and the shortcomings of known IL-6 antibodies, there is clearly a need in the art for improved (e.g., minimally engineered) IL-6 agents that can inhibit the biological activity of IL-6, and hence treat diseases associated with IL-6 activity.

SUMMARY OF THE INVENTION

The present invention improves upon the state of the art by providing binding molecules (e.g., antibodies or antigen binding fragments thereof) with improved binding profiles that specifically bind to IL-6 (e.g., human and non-human primate IL-6) with high binding affinity (e.g., picomolar binding affinity) and potently inhibit its biological activity (e.g., binding to an IL-6 receptor). In certain exemplary embodiments, the IL-6 binding molecules of the invention are derived from the conventional antibody repertoire of a camelid species (e.g., llama) that has been subjected to active immunization with the IL-6 antigen. For example, the camelid-derived IL-6 binding molecules of the invention may comprise paired VH/VL domains or other alternative frameworks wherein one or more hypervariable loops (e.g., HI, H2, H3, LI, L2 and/or L3) of the VH or VL domains are derived from the camelid species. Moreover, in certain embodiments, at least one of the hypervariable loops adopt a canonical fold (or combination of canonical folds) that is identical or substantially identical to that of a human antibody. Such binding molecules exhibit high human homology (sequence and structure) and are therefore particularly useful for treating IL-6-associated diseases or disorders (e.g., inflammatory disease and cancer) due to their low

immunogenicity. Surprisingly, the IL-6 antibodies of the invention exhibit high binding affinity, manufacturability and thermal stability without the need for extensive and time- consuming antibody engineering and affinity maturation that is typically required of known IL-6 antibodies.

Accordingly, in one aspect, the invention provides, a binding molecule that specifically binds to IL-6, the binding molecule comprising at least one antibody CDR, wherein the CDR comprises at least one amino acid residue that is buried in the F229 cavity or the F279 cavity on IL-6 when the binding molecule is bound to IL-6, In certain embodiments, the binding molecule comprises a VH domain, the VH domain having an amino acid at position 98, according to abat, that is buried in the F229 cavity on IL-6 when the antibody or fragment is bound to IL-6. In one particular embodiment, the amino acid at position 98 is a tryptophan. In certain embodiments, the binding molecule comprises a VL domain, the VL domain having an amino acid at position 30, according to Kabat, that is buried in the F229 cavity on IL-6 when the antibody or fragment is bound to IL-6. In one particular embodiment, the amino acid at position 30 is a tyrosine, in certain embodiments, the binding molecule comprises a VH domain, the VH domain having an amino acid at position 99, according to Kabat, that is buried in the F279 cavity on IL-6 when the antibody or fragment is bound to IL-6, In one particular embodiment, the amino acid at position 99 is a valine.

In certain embodiments, the binding molecule comprises a VH domain and a VL domain, said VH domain comprising hypervariable loops HI, H2 and H3, wherein said VH domain polypeptide is paired with a VL domain comprising hypervariable loops LI, L2 and L3 wherein at least one of hypervariable loops H1-H3 and L1-L3 are obtained from a conventional antibody of a Lama species by active immunization of the Lama species with the IL-6 antigen. In one particular embodiment, at least one of the hypervariable loops HI, H2, LI, L2 and L3 exhibits a predicted or actual canonical fold structure which is identical or substantially identical to a corresponding canonical fold structure of a HI, H2, LI, L2 or L3 hypervariable loop which occurs in a human antibody. In one particular embodiment, at least one of the hypervariable loops HI and ί 12 each exhibit a predicted or actual canonical fold structure which is identical or substantially identical to the corresponding human canonical fold structure. In one particular embodiment, at least one of the hypervariable loops LI, L2 and L3 each exhibit a predicted or actual canonical fold structure which is identical or substantially identical to the corresponding human canonical fold structure. In one particular embodiment, at least one of the hypervariable loops HI and H2 form a combination of predicted or actual canonical fold structures which is identical or substantially identical to a corresponding combination of canonical fold structures known to occur in a human germline VH domain. In one particular embodiment, at least one of the hypervariable loops HI and H2 form a combination of canonical fold structures corresponding to a combination of human canonical fold structures selected from the group consisting of 1-1, 1- 2, 1-3, 1-4, 1-6, 2-1, 3-1 and 3-5. In one particular embodiment, at least one of the hypervariable loops LI and L2 form a combination of predicted or actual canonical fold structures which is identical or substantially identical to a corresponding combination of canonical fold structures known to occur in human germline VL domains. In one particular embodiment, at least one of the hypervariable loops LI and L2 form a combination of canonical fold structures corresponding to a combination of human canonical fold structures selected from the group consisting of 11-7, 13-7(A,B,C), 14-7 (A,B), 12-1 1 , 14-11, 12-12, 2- 1, 3-1, 4-1 and 6-1.

In certain embodiments, the binding molecule comprises a VH domain and a VL domain, wherein the VH domain and/or VI, domain of the binding molecule exhibits a sequence identity of 90% or greater, with one or more corresponding human VH or VL domains across framework regions FR 1, FR2, FR3 and FR4. In certain embodiments, the binding molecule comprises a VH domain and a V L domain and is a germlined variant of a parental binding molecule, wherein one or both of the VH domain and VL domain of the binding molecule comprise a total of between 1 and 10 amino acid substitutions across the framework regions as compared to the corresponding VH domain and VL domain of the parental non-human antibody. In one particular embodiment the parental binding molecule is a conventional camelid antibody. In certain embodiments, the binding molecule is an antibody or antigen binding fragment thereof.

In certain embodiments, the binding molecule comprises a VH domain, the VH domain comprising the HCDR3 amino acid sequence set forth in SEQ ID NO: 500

[X ₁ PDVVTGFHYDX ₂ ], or sequence variant thereof, wherein:

Xi is any amino acid, preferably D or Y;

X ₂ is any amino acid, preferably Y or N; and

wherein the sequence variant comprises one, two or three amino acid substitutions in the recited sequence. In one particular embodiment, the HCDR3 amino acid amino acid sequence is selected from the group consisting of SEQ ID NO: 497-499.

In certain embodiments, the VH domain further comprises the HCDR2 amino acid sequence set forth in SEQ ID NO: 507 [ VKi YX ₂ X ₃ DTYYSPSLX ₄ S] , or sequence variant thereof, wherein:

XI is any amino acid, preferably D, Y or N;

X2 is any amino acid, preferably D or E;

X3 is any amino acid, preferably A or G;

X4 is any amino acid, preferably E or K; and

wherein the sequence variant comprises one, two or three amino acid substitutions in the recited sequence. In one particular embodiment, the HCDR2 amino acid amino acid sequence is selected from the group consisting of SEQ ID NO: 501-506.

In certain embodiments, the VH domain further comprises the HCDR1 amino acid sequence set forth in SEQ ID NO: 512 j¾ X ₂ Y YX ₃ WX ₄ ] , or sequence variant thereof, wherein: XI is any amino acid, preferably T, S or P;

XI is any amino acid, preferably R or S;

X3 is any amino acid, preferably A or V;

X4 is any amino acid, preferably S or T; and

wherein the sequence variant comprises one, two or three amino acid substitutions in the recited sequence. In one particular embodiment, the HCDRl amino acid sequence is selected from the group consisting of SEQ ID NO: 508-51 1.

In certain embodiments, the binding molecule comprises a VII domain comprising the HCDR3, HCDR2 and HCDRl amino acid amino acid sequences set forth in SEQ ID NO: 497, 501 and 508, respectively.

In certain embodiments, the binding molecule further comprises a VL domain, wherein the VL domain comprises the LCDR3 amino acid sequence set forth in SEQ ID NO: 524 [ AS YXiXoXsX-sXsXe ?] ,_ r sequence variant thereof, wherein:

XI is any amino acid, preferably R or j

X2 is any amino acid, preferably N, H, R, S, D, T or Y:

X3 is any amino acid, preferably F, Y, T, 8 or R;

X4 is any amino acid, preferably N or I:

X5 is any amino acid, preferably N or D;

X6 is any amino acid, preferably V, N, G or A:

X7 is any amino acid, preferably V or I; and

wherein the sequence variant comprises one, two or three amino acid substitutions in the recited sequence. In one particular embodiment, the LCDR3 amino acid amino acid sequence is selected from the group consisting of SEQ ID NO: 513-523.

In certain embodiments, the VL domain further comprises the LCDR2 amino acid sequence set forth in SEQ ID NO: 535 or sequence variant thereof, wherein:

X I is any amino acid, preferably R, , D, A or E;

X2 is any amino acid, preferably S, N or T;

X3 is any amino acid, preferably T, K or Y;

X4 is any amino acid, preferably A, T or V; and

wherein the sequence variant comprises one, two or three amino acid substitutions in the recited sequence. In one particular embodiment, the LCDR2 amino acid amino acid sequence is selected from the group consisting of SEQ ID NO: 525-534. In certain embodiments, the VL domain further comprises the LCDRl amino acid sequence set forth in SEQ ID NO: 542 [AGX ₁ X ₂ X ₃ DX ₄ GX ₅ X ₆ X ₇ YVS], or sequence variant thereof, wherein

XI is any amino acid, preferably A or T;

X2 is any amino acid, preferably S or N;

X3 is any amino acid, preferably S, E or N;

X4 is any amino acid, preferably V or I;

X5 is any amino acid, preferably G, Y, T or F;

X6 is any amino acid, preferably G or Y;

X7 is any amino acid, preferably N, D or A; and

wherein the sequence variant comprises one, two or three amino acid substitutions in the recited sequence, in one particular embodiment, the LCDRl amino acid amino acid sequence is selected from the group consisting of SEQ ID NO: 538-541, In certain embodiments, the binding molecule comprises a VL domain comprising the comprising the LCDR3, LCDR2 and LCDRl amino acid amino acid sequences set forth in SEQ ID NO: 513, 525 and 536, respectively. In certain embodiments, the binding molecule comprises: a VH domain having the HCDR3, HCDR2 and HCDR1 amino acid amino acid sequences set forth in SEQ ID NO: 497, 501 and 508, respectively; and a VL domain having the LCDR3, LCDR2 and LCDRl amino acid amino acid sequences set forth in SEQ ID NO: 513, 525 and 536, respectively. In certain embodiments, the binding molecule comprises a VH domain with at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 152. In certain embodiments, the binding molecule comprises a VH domain amino acid sequence is selected from the group consisting of SEQ ID NO: 127-232 and 569-571. In certain embodiments, the binding molecule comprises a VH domain amino acid sequence is SEQ ID NO: 152. In certain embodiments, the binding molecule comprises a VL domain with at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 416. In certain embodiments, the binding molecule comprises a VL domain amino acid sequence is selected from the group consisting of SEQ ID NO: 391-496. In certain embodiments, the binding molecule comprises a VL domain amino acid sequence is SEQ ID NO: 416. In certain embodiments, the binding molecule comprises: a VH domain having the amino acid sequences set forth in SEQ ID NO: 152; and a VL domain having the amino acid sequence set forth in SEQ ID NO: 416. In certain embodiments, the binding molecule comprises the HI and H2 loops form a combination of canonical fold structures corresponding to the 3-1 combination of human canonical fold structures as found in a human 1 AC Y antibodv structure. In certain embodiments, the binding molecule comprises the LI and L2 loops form a combination of canonical fold structures corresponding to the 6λ- Ι combination of human canonical fold structures as found in a human 3MUG antibody structure. In certain embodiments, the binding molecule comprises the LI, L2 and L3 loops form a combination of canonical fold structures corresponding to the 6λ-1-5 combination of human canonical fold structures as found in the human SMUG antibody structure.

In certain embodiments, the binding molecule comprises a VI 1 domain, the VH domain comprising the HCDR3 amino acid sequence set forth in SEQ ID NO: 544

[ AGX ₁ GX ₂ G j, or sequence variant thereof, wherein:

X·. is any amino acid, preferably W;

X ₂ is any amino acid, preferably M, A, L, S or N: and

wherein the sequence variant comprises one, two or three amino acid substitutions in the recited sequence. In one particular embodiment, the HCDR3 amino acid amino acid sequence is selected from the group consisting of SEQ ID NO: 543, SEQ ID NO: 566, SEQ INO:567, and SEQ ID NO:568.

In certain embodiments, the VH domain further comprises the HCDR2 amino acid sequence set forth in SEQ ID NO: 554 [X ₁ ISX ₂ X ₃ GX ₄ SX ₅ X ₆ YX ₇ DSVKG], or sequence variant thereof, wherein:

XI is any amino acid, preferably A, P or R;

X2 is any amino acid, preferably A or S;

X3 is any amino acid, preferably S or G;

X4 is any amino acid, preferably G or V;

X5 is any amino acid, preferably A or T;

X6 is any amino acid, preferably Y, N or S;

X7 is any amino acid, preferably G, A or T; and

wherein the sequence variant comprises one, two or three amino acid substitutions in the recited sequence. In one particular embodiment, the HCDR2 amino acid amino acid sequence is selected from the group consisting of SEQ ID NO: 545-553.

In certain embodiments, the VH domain further comprises the HCDR1 amino acid sequence set forth in SEQ ID NO: 562 [X1X2X3X4 X5], or sequence variant thereof, wherein: XI is any amino acid, preferably S or T:

X2 is any amino acid, preferably H or Y;

X3 is any amino acid, preferably A or R:

X4 is any amino acid, preferably M or L;

X5 is any amino acid, preferably S or Y; and

wherein the sequence variant comprises one, two or three amino acid substitutions in the recited sequence. In one particular embodiment, the HCDR1 amino acid amino acid sequence is selected from the group consisting of SEQ ID NO: 555-561 ,

In certain embodiments, the VH domain comprises a HCDR3 having an amino acid amino acid sequence selected from the group consisting of SEQ ID NO:543, 566, 567, and 568, and the HCDR2 and HCDR1 amino acid amino acid sequences set forth in SEQ ID NO: 545 and 555, respectively. In certain embodiments, the binding molecule further comprises a VL domain, wherein the VL domain comprises the LCDR3 amino acid sequence set forth in SEQ ID NO: 563, or sequence variant thereof, wherein the sequence variant comprises one, two or three amino acid substitutions in the recited sequence. In certain embodiments, the VL domain further comprises the LCDR2 amino acid sequence set forth in SEQ ID NO: 564, or sequence variant thereof, wherein the sequence variant comprises one, two or three amino acid substitutions in the recited sequence, In certain embodiments, the VL, domain further comprises the LCDR1 amino acid sequence set forth in SEQ ID NO: 565, or sequence variant thereof, wherein the sequence variant comprises one, two or three amino acid substitutions in the recited sequence. In certain embodiments, the VL domain comprises the LCDR3, LCDR2 and LCDR1 amino acid amino acid sequences set forth in SEQ ID NO: 564, 564 and 565, respectively. In certain embodiments, the binding molecule: a VH domain having the HCDR3, HCDR2 and HCDR1 amino acid amino acid sequences set forth in SEQ ID NO: 544, 545 and 555, respectively; and a VL domain having the LCDR3, LCDR2 and LCDR1 amino acid amino acid sequences set forth in SEQ ID NO: 563, 564 and 565, respectively, In certain embodiments, the binding molecule comprises a VH domain with at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 86. In certain embodiments, the binding molecule comprises a VH domain having the amino acid sequence is selected from the group consisting of SEQ ID NO: 39- 126 and 569-571. In certain embodiments, the binding molecule comprises a VH domain having the amino acid sequence is selected from SEQ ID NO: 86, SEQ ID NO:569, SEQ ID NO:570 and SEQ ID NO:571. In certain embodiments, the binding molecule comprises a VL domain with at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 350. In certain embodiments, the binding molecule comprises a VL domain having the amino acid sequence is selected from the group consisting of SEQ ID NO: 303-390. In certain embodiments, the binding molecule comprises a VL domain having the amino acid sequence is SEQ ID NO: 350. In certain embodiments, the binding molecule comprises: a VH domain having the amino acid sequences set forth in SEQ ID NO: 86, SEQ ID NO:569, SEQ ID NO:570 or SEQ ID NO:571 ; and a VL domain having the amino acid sequences set forth in SEQ ID NO: 350.

In certain embodiments, the binding molecule comprises the HI and H2 loops form a combination of canonical fold structures corresponding to the 1-3 combination of human canonical fold structures as found in a human 1DFB antibody structure. In certain

embodiments, the binding molecule comprises the LI and L2 loops form a combination of canonical fold structures corresponding to the 7λ-1 combination of human canonical fold structures as found in a human IMF A antibody structure. In certain embodiments, the binding molecule comprises the LI, L2 and L3 loops form a combination of canonical fold structures corresponding to the 7λ-1-4 combination of human canonical fold structures as found in the human 3MUG antibody structure,

In certain embodiments, the binding molecule is a Fab fragment which binds to human IL-6 with an off-rate (k _Qff measured by surface Plasmon resonance) of less than 2 x lO ^"3 s ^"1 . In certain embodiments, the binding molecule binds to the human IL-6 antigen with sub-picomolar binding affinity. In certain embodiments, the binding molecule binds to the human IL-6 antibody with single digit femtomolar binding affinity. In certain embodiments, the binding molecule comprises the hypervariable loops are obtained from the conventional antibody of the Lama without subsequent affinity maturation. In certain embodiments, the binding molecule inhibits IL-6-induced proliferation of B9 hybrid oma cells with an IC50 of less than 0.1 pM.

In certain embodiments, the binding molecule exhibits a melting temperature (Tm) of greater than 65 °C. In certain embodiments, the binding molecule is a germlined variant of a parental camelid antibody, said germlined variant having a higher melting temperature than the parental camelid antibody. In certain embodiments, the binding molecule is expressed at the level of at least 20 mg/ml following transient expression in a HEK293 cell. In certain embodiments, the binding molecule is characterized by an EpiBase© score of less than about 10.0, e.g., less than about 6.0. In certain embodiments, the binding molecule inhibits binding of IL-6 to an IL-6 receptor. In certain embodiments, the binding molecule inhibits binding of gpl30 to an IL-6 receptor. In certain embodiments, the binding molecule binds specifically to human and cynomologus monkey IL-6. In certain embodiments, the binding molecule comprises at least one CDR from a camelid antibody that specifically binds to IL-6.

In another aspect, the invention provides, a pharmaceutical composition comprising the binding molecule of any of the preceding claims and one or more pharmaceutically acceptable carrier.

In another aspect, the invention provides, a method of treating an IL-6-associated di sense or disorder, comprising administering to a subject in need of treatment thereof an effective amount of the pharmaceutical composition of the invention.

In another aspect, the invention provides, an isolated nucleic acid encoding a binding molecule disclosed herein.

In another aspect, the invention provides, a recombinant expression vector comprising a nucleic acid molecule of the invention.

In another aspect, the invention provides a host cell comprising a recombinant expression vector of the invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 depicts the results of cell proliferation assays measuring the in vitro IL-6 neutralizing activity of antibodies of the invention.

Figure 2 depicts the results of epithelial ovarian cancer mouse tumor xenograph experiments measuring the in vivo efficacy of antibodies of the invention.

Figures 3A-B show that camelid-derived hypervariable loops (L1-L3, HI and H2) of the 61 H7 antibody of the invention adopts predicted canonical folds and canonical fold combinations of human antibodies.

Figures 4A-B show that camelid-derived hypervariable loops (L1 -L3, I II and H2) of the 68F2 antibody of the invention and its germlined variant (129D3) adopt predicted canonical folds and canonical fold combinations of human antibodies.

Figure 5 depicts a space-fill model of IL-6 overlaid with: (A) F229 of the IL-6 receptor; (B) F229 of the IL-6 receptor and W98 of the 61 H7 VH, (C) F229 of the IL-6 receptor and Y30 of the 68F2 VL; and (D) F229 of the IL-6 receptor, W98 of the 61 H7 VH and V99 of the 681 2 VH, according to Kabat numbering.

Figure 6 depicts a space-fil l model of the two surface binding cavities on IL-6 important for IL-6 receptor binding overlaid with residues F229 and F279 of the IL-6 receptor, and residues Y30 of the 68F2 VL and V99 of the 68F2 VH, according to Rabat numbering (Y32 and VI 04 in the structure),

Figures 7A-B depicts the thermal stability of 68F2 and its germlined variant 129D3 as measured in Biacore with immobilized glycosylated human IL-6 with respect to (A) other germlined variant IL-6 antibodies of the invention and (B) other reference antibodies, The upper part of each figure depicts the melting curves, while the lower part lists the Tm value for each antibody,

Figure 8 depicts the serum stability of antibody clones 68F2, 129D3 (a germlined variant of 68F2), and 103A1 (a variant of 61H7). Also included is the reference antibody GL 18.

Figure 9 depicts the low immunogenicity (Epibase) scores for IL-6 antibodies of the invention as compared to reference antibodies (shown in bold), including the fully human antibody adalimumab (Humira),

Figure 10A-B depicts an alignment of the VH and VL (A) 68F2 and (B) 61 H7 depicting the high level of sequence homology with the framework regions of their respective germlined variants 129D3 and 111A7. The minimal number of framework alterations introduced into each molecule (13 total) is also shown,

Figure 1 lA-B depicts an alignment of the VH and VK of (A) CNT0328 and (B) VH _. __rabbit (ALD518) depicting the high level of sequence homology with the framework regions of their respective germlined variants CNT0136 and VH_bum.an(ALD518). The minimal number of framework alterations introduced into each molecule (36 and 46 in total) is also shown.

Figure 12 depicts the pharmacokinetic profiles of 129D3 IgGl antibodies and variants thereof in cynomolgus monkeys.

Figure 13 depicts the results of serum amyloid A (SAA) mouse model experiments measuring the in vivo efficacy of antibodies of the invention.

Figure 14 depicts the results of mouse psoriasis xenograph experiments measuring the in vivo efficacy of antibodies of the invention.

Figure 15 depicts tumor growth data observed in experiments measuring the in vivo efficacy of antibodies of the invention in a renal cell cancer mouse tumor xenograph model.

Figure 16 depicts Kaplan-Meier plot of survival data observed in experiments measuring the in vivo efficacy of antibodies of the invention in a renal cell cancer mouse tumor xenograph model. Figure 17 depicts tumor growth data observed in experiments measuring the in vivo efficacy of antibodies of the invention in a renal cell cancer mouse tumor xenograph model with all agents dosed at 3 mg/kg.

Figure 18 depicts Kaplan-Meier plot of survival data observed in experiments measuring the in vivo efficacy of antibodies of the invention in a renal cell cancer mouse tumor xenograph model with all agents dosed at 3 mg/kg.

DESCRIPTION OF THE INVENTION L Definitions

In order that the present invention may be more readily understood, certain terms are first defined.

As used herein, the term "IL-6" refers to interleukin-6, IL-6 nucleotide and polypeptide sequences are well known in the art. An exemplary human IL-6 amino sequence is set forth in GenBank deposit GI: 10834984 and an exemplary mouse IL-6 amino sequence is set forth in GenBank deposit GI: 13624311.

As used herein, the term "antibody" refers to immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CHI , CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated VL) and a light chain constant region. The light chain constant region comprises one domain (CLl). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR).

As used herein, the term "antigen-binding fragment" of an antibody includes any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered

polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen- binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Non-limiting examples of antigen- binding portions include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR)). Other engineered molecules, such as diabodies, triabodies, tetrabodies and rainibodies, are also encompassed within the expression "antigen-binding portion."

As used herein, the terms "variable region" or "variable domain" refer to the fact that certain portions of the variable domains VH and VL differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its target antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called "hypervariable loops" in each of the VI, domain and the VH domain which form part of the antigen binding site. The first, second and third hypervariable loops of the VLambda light chain domain are referred to herein as Ι,1(λ), Τ2(λ) and L3( ) and may be defined as comprising residues 24-33 (L1 (X), consisting of 9, 10 or 1 1 amino acid residues), 49-53 (L2(X), consisting of 3 residues) and 90-96 (L3(X), consisting of 5 residues) in the VL domain (Morea et al,, Methods 20:267-279 (2000)). The first, second and third hypen'ariable loops of the VKappa light chain domain are referred to herein as LI (κ), L2( ) and L3( ) and may be defined as comprising residues 25-33 (L1 (K), consisting of 6, 7, 8, 11 , 12 or 13 residues), 49-53 (L2(K), consisting of 3 residues) and 90-97 (L3(K), consisting of 6 residues) in the VL domain (Morea et al., Methods 20:267-279 (2000)). The first, second and third hypervariable loops of the VH domain are referred to herein as HI, H2 and H3 and may be defined as comprising residues 25-33 (HI, consisting of 7, 8 or 9 residues), 52-56 i E 12. consisting of 3 or 4 residues) and 91 -105 (H3, highly variable in length) in the VH domain (Morea et al, Methods 20:267-279 (2000)).

Unless otherwise indicated, the terms LI, L2 and L3 respectively refer to the first, second and third hypervariable loops of a VL domain, and encompass hypervariable loops obtained from both Vkappa and Vlambda isotypes. The terms HI, H2 and H3 respectively refer to the first, second and third hypervariable loops of the VH domain, and encompass hypervariable loops obtained from any of the known heavy chain isotypes, including γ, ε, δ, α or μ.

The hypervariable loops LI, L2, L3, HI, H2 and H3 may each comprise part of a "complementarity determining region" or "CDR", as defined below. The terms

"hypervariable loop" and "complementarity determining region" are not strictly synonymous. since the hypervariable loops (HVs) are defined on the basis of structure, whereas

complementarity determining regions (CDRs) are defined based on sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD., 1983) and the limits of the HVs and the CDRs may be different, in some VH and VL domains.

The CDRs of the VL and VH domains can typically be defined as comprising the following amino acids: residues 24-34 (CDRL1), 50-56 (CDRL2) and 89-97 (CDRL3) in the light chain variable domain, and residues 31-35 or 31 -35b (CDRH1), 50-65 (CDRH2) and 95-102 (CDRH3) in the heavy chain variable domain: (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health,

Bethesda, MD. (1991)). Thus, the HVs may be comprised within the corresponding CDRs and references herein to the "hypervariable loops" of VH and VL domains should be interpreted as also encompassing the corresponding CDRs, and vice versa, unless otherwise indicated.

The more highly conserved portions of variable domains are called the framework region (FR), as defined below, The variable domains of native heavy and light chains each comprise four FRs (FR1, FR2, FR3 and FR4, respectively), largely adopting a β- sheet configuration, connected by the three hypervariable loops. The hypervariable loops in each chain are held together in close proximity by the FRs and, with the hypervariable loops from the other chain, contribute to the formation of the antigen-binding site of antibodies,

Structural analysis of antibodies revealed the relationship between the sequence and the shape of the binding site formed by the complementarity determining regions (Chothia et al., J. Mol. Biol. 227: 799-817 (1992)); Tramontane et al., J. Mol. Biol, 215: 175-182 (1990)).

Despite their high sequence variability, five of the six loops adop just a small repertoire of main-chain conformations, called "canonical structures". 'These conformations are first of all determined by the length of the loops and secondly by the presence of key residues at certain positions in the loops and in the framework regions that determine the conformation through their packing, hydrogen bonding or the ability to assume unusual main-chain conformations.

As used herein, the terms "complementarity determining region" or "CDR" refer to the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991), and by Chothia et al, J. Mol. Biol. 196:901-917 (1987) and by MacCallum et al., J. ol. Biol. 262:732-745 (1996) where the definitions include overlapping or subsets of amino acid residues when compared against each other. The amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth for comparison. Preferably, the term "CDR" is a CDR as defined by Kabat based on sequence comparisons.

Table 1: CDR definitions

'Residue numbering follows the nomenclature of Kabat et al., supra

"Residue numbering follows the nomenclature of Chothia et al., supra

'Residue numbering follows the nomenclature of MacCallum et al,, supra

As used herein he terms "framework region" or "FR region" include the amino acid residues that are part of the variable region, but are not part of the CDRs (e.g., using the Kabat definition of CDRs). Therefore, a variable region framework is between about 100- 120 amino acids in length but includes only those amino acids outside of the CDRs, F ^' or the specific example of a heavy chain variable region and for the CDRs as defined by Kabat et al., framework region 1 corresponds to the domain of the variable region encompassing amino acids 1 -30; framework region 2 corresponds to the domain of the variable region encompassing amino acids 36-49; framework region 3 corresponds to the domain of the variable region encompassing amino acids 66-94, and framework region 4 corresponds to the domain of the variable region from amino acids 103 to the end of the variable region. The framework regions for the light chain are similarly separated by each of the light claim variable region CDRs. Similarly, using the definition of CDRs by Chothia et al. or

McCallum et al, the framework region boundaries are separated by the respective CDR termini as described above. In preferred embodiments the CDRs are as defined by Kabat.

In naturally occurring antibodies, the six CDRs present on each monomelic antibody- are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen binding site as the antibody assumes its three dimensional configuration in an aqueous environment. The remainder of the heavy and light variable domains show less inter-molecular variabi lity in amino acid sequence and are termed the framework regions. The framework regions largely adopt a β- sheet conformation and the CDRs form loops which connect, and in some cases form part of, the β-sheet structure. Thus, these framework regions act to form a scaffold that provides for positioning the six CDRs in correct orientation by inter-chain, non-covalent interactions. The antigen binding site formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to the

immunoreactive antigen epitope. The position of CDRs can be readily identified by one of ordinary skill in the art.

As used herein, the term "F229 cavity" refers to the surface cavity of human IL-6 that is occupied by the phenylalanine 229 residue of the human IL-6 receptor in the IL-6/IL-6 receptor complex set forth in Boulanger et a!.., 2003, Science 27, 2101-2104, which is incorporated by reference herein in its entirety.

As used herein, the term "F279 cavity" refers to the surface cavity of human IL-6 that is occupied by the phenylalanine 279 residue of the human IL-6 receptor in the IL-6/IL-6 receptor complex set forth in Boulanger et al., 2003, Science 27, 2101-2104, which is incorporated by reference herein in its entirety.

As used herein, the term "camelid-derived" refers to antibody variable region amino acid sequences (e.g., framework or CDR sequences) naturally present in antibody molecules of a camelid (e.g., llama). Camelid-derived antibodies may be obtained from any camelid species, including, without limitation, llama, dromedary, alpaca, vicuna, guanaco or camel. In certain embodiments, the camelid (e.g., llama) has been actively immunised with IL-6 (e.g., human IL-6). in certain embodiments, the term "camelid-derived" is limited to antibody sequences that are derived from the conventional antibody repertoire of a camelid and specifically excludes antibody sequences derived from the heavy chain-only antibody (VHH) repertoire of the camelid.

As used herein, the term "conventional antibody" refers to antibodies of any isotype, including IgA, IgG, IgD, IgE or IgM. Native or naturally occurring "conventional" camelid antibodies are usually heterotetrameric glycoproteins, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end (N-terminal) a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain (VL) at one end (N-terminal) and a constant domain (CL) at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy- chain variable domains.

As used herein, the term "specifically binds to" refers to the ability of an antibody or antigen binding fragment thereof to bind to an IL-6 with an KD of at least about 1 x 10 ^" " (e.g, 1 x 10 ^"6 .VS . 1 x 10 ^"7 M, 1 x 10 ^"8 M, 1 x 10 ^"9 .VS . 1 x 10 ^"i0 M, 1 x 10 ^"11 M, 1 x 10 ¹² M, 1 x 10 ^" ° M, 1 x 10 ^~14 M, 1 x 10 ^"13 M or more), preferrably between 1 x 10 ^"32 M and 1 x 10 ^{"i 5} M or more and/or bind to IL-6 with an affinity that is at least two-fold greater than its affinity for a non-specific antigen. It shall be understood, however, that an antibody or antigen binding fragment thereof is capable of specifically binding to two or more antigens which are related in sequence. For example, the antibodies or antigen binding fragments thereof disclosed herein can specifically bind to both human and a non-human (e.g., mouse or non-human primate) IL-6,

As used herein, the term "antigen" refers to the binding site or epitope recognized by an antibody variable region.

As used herein, the term "treat," "treating," and "treatment" refer to therapeutic or preventative measures described herein. The methods of "treatment" employ administration to a subject, an antibody or antigen binding fragment thereof of the present invention, for example, a subject having an IL-6-associated disease or disorder (e.g. inflammation and cancer) or predisposed to having such a disease or disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disease or disorder or recurring disease or disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.

As used herein, the term "IL6-associated disease or disorder" includes disease states and/or symptoms associated with IL-6 activity. Exemplar} _' DL6- associated diseases or disorders include, but are not limited to, inflammatory diseases (e.g., inflammatory autoimmune diseases such as rheumatoid arthritis and systemic lupus eiythematosus), cancer (e.g., prostate cancer, diffuse large cell lymphoma, multiple myeloma, and renal cell cancer), and cancer-related disorders (e.g., anorexia and cachexia). As used herein, the term "effective amount" refers to that amount of an antibody or antigen binding fragment thereof that is sufficient to effect treatment, prognosis or diagnosis of an IL-6-associated disease or disorder, as described herein, when administered to a subject. A therapeutically effective amount will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readilv be determined bv one of ordinary skill in the art. The dosages for admin stration can range from, for example, about 1 ng to about 10,000 mg, about about 1 ug to about 5,000 mg, about 1 mg to about 1,000 mg, about 10 mg to about 100 mg, of an antibody or antigen binding fragment thereof according to the invention. Dosage regiments may be adjusted to provide the optimum therapeutic response. An effective amount is also one in which any toxic or detrimental effects (i.e., side effects) of a binding polypeptide are minimized and/or outweighed by the beneficial effects.

As used herein, the term "subject" includes any human or non-human animal.

As used herein, the term "surface plasmon resonance" refers to an optical

phenomenon that allows for the analysis of real-time interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore™ system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ).

As used herein, the term "KD" refers to the equilibrium dissociation constant of a particular binding polype tide/antigen interaction.

As used herein, the term "off-rate" is refers to the dissociation rate ( _off ) for a particular binding interaction.

II, IL-6 Binding Molecules

In one aspect, the invention provides binding molecules (antibodies or antigen binding fragments thereof) that specifically bind to and inhibit the activity of IL-6. Such binding molecules generally comprise at least one CDR region amino acid sequence set forth in Tables 13-18, herein.

Analysis of the crystal structure of human IL-6 in complex with the human IL-6 receptor has shown that 2 residues of the IL-6 receptor, F229 and I 279. are critical for the IL- 6/ IL-6 receptor interaction (see e.g., Boulanger et al., 2003, Science 27, 2101-2104, which is incorporated by reference herein in its entirety). In the IL-6/ IL-6 receptor complex, F229 and F279 are buried in separate cavities on the surface of IL-6. In certain embodiments, the binding molecules of the invention utilize these cavities on IL-6 to achieve high affinity binding. In one particular embodiment, binding molecules of the invention comprise an antibody CDR region, wherein the CDR region comprises an amino acid residue that is buried in the F229 cavity or the F279 cavity on IL-6 when the binding molecule to bound to IL-6.

In general, the binding molecules of the invention inhibit IL-6 activity (e.g., by antagonizing the binding of IL-6 to an IL-6 receptor). In certain embodiments, the binding molecules also inhibit binding of gpl30 to an IL-6 receptor. However, in other embodiments, the binding molecules can bind to IL-6 without inhibiting binding of gpl30 to an IL-6 receptor.

Binding molecules of the invention generally have a high affinity for IL-6 and are generally highly potent at inhibiting IL-6 activity in vivo and in vitro. In certain

embodiments, the binding molecules of the invention bind to human IL-6 with an off-rate (k _0ff measured by surface Plasmon resonance) of less than about 1 x 10 ^"4 s ^"1 (e.g., about 9 x 10 ^'5 , 8 x ΚΓ ^' , 7x 10 ^"5 , 6 x 10 ^'5 , 5 x 10 ^"5 , 4 x 10 ^"5 , 3 x 10 ^"5 , 2 x 10 ^"5 , and 1 x 10 ^"5 ). In other embodiments, the binding molecules of the invention inhibit IL-6-induced proliferation of B9 hybridoma cells with an IC50 of less than 0.1 pM. In certain other embodiments, the binding molecules of the invention compete with a predetermined antibody binding to IL-6 wherein such predetermined antibody containing a VH sequence and a VL sequence selected from VH and VL amino acid sequences set forth in Table3 13- 18. In certain other embodiments, the binding molecules of the invention compete away at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the binding of the predetermined antibody binding to IL-6. In certain other embodiments, the binding molecules of the invention compete with the binding of 20A4, 24D10, 68F2, 61117, 129D3 or 1 1 1 A7 to IL-6, e.g., compete away at least 50%, 60%, 70%, 80% or 90% of the binding of one of these antibodies to IL-6. In certain other embodiments, the binding molecules of the invention compete with the binding of 17F10, 24C9, 18C1 1 , 29B11, 28A6, or 126A3 to IL-6, e.g., compete away at least 50%, 60%, 70%, 80% or 90% of the binding of one of these antibodies to IL-6.

In general, the binding molecules of the invention also exhibit high thermal stability. In certain embodiments, the binding molecules exhibit a melting temperature (Tm) of greater than 55°C (e.g., at least 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75 C or higher). In certain exemplary embodiments, the IL-6 binding molecules of the invention are germlined variants which exhibit a thermal stability that is comparable to, or higher than, their parental, camelid-derived counterparts. In certain exemplary embodiments, thermal stability is measured following incubation in a suitable buffer (e.g., PBS) at a concentration of 100 ₍ ug/ml for 1 hour. In other exemplary, embodiments the thermal stability of the IL-6 binding molecule is that exhibited in a ful l-length IgG format (e.g., comprising an IgGl or IgG4 P ^' c region).

The binding molecules of the invention are also characterized by high expression levels of functional antibody, with low levels of non-functional contaminants such as high or low- molecular weight aggregates. For example, IL-6 binding molecules of the invention may be characterized by production levels of at least 20 mg/L (e.g., at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 mg/L or higher). In certain exemplary embodiments, the IL-6 binding molecules are germlined variants which exhibit an expression level that is comparable to, or higher than, their parental, camelid counterparts. In other exemplary embodiments, the expression level is determined using the full-length IgG format of an IL-6 binding molecule of the invention, e.g., by transient expression in a HEK293 cell.

Binding molecules of the invention are also generally characterized by low predicted immunogenicity. For example, IL-6 binding molecules of the invention exhibit EpiBase® scores (e.g., total DRB1 scores) of less than 15,0, least than about 12.0, or less than about 10.0. In certain exemplary embodiments, the binding molecules exhibit immunogenicity scores of about 9.0, about 8.0, about 7.0, or about 6.0. In yet other embodiments, the immunogenicity score is less than the immunogenicity score of Huniira®, e.g., about 6.0, about 5.0, or about 4.0.

Binding molecules of the invention can bind to any IL-6 including, without limitation, human and cvnomolgus monkey IL-6. Preferably, binding molecules can bind to both human and cynomolgus monkey IL-6.

0 IL-6 Antibodies or Antigen Binding Fragments Thereof

In certain embodiments, the invention provides antibodies or antigen binding fragments thereof that specifically bind to IL-6 (e.g., human IL-6) and antagonize the binding of IL-6 to an IL-6 receptor. The VH, VL and CDR sequences of exemplary Fab clones of the invention are set forth in Tables 13-18, Antibodies of the invention can comprise any of the framework and/or CDR amino acid sequences of these Fab clones.

Antibodies of the invention can comprise a CDR region sequence with an amino acid residue (e.g., an aromatic amino acid, such as tryptophan or tyrosine) that is buried in the F229 cavity on IL-6 when the antibody or fragment to bound to IL-6. Exemplary antibodies comprise a VH domain with a tryptophan at position 98 and/or VL domain with a tyrosine at position 30, according to Kabat. Such antibodies have particularly high affinity for IL-6,

Additionally or alternatively, antibodies of the invention can comprise a CDR region sequence with an amino acid residue that is buried in the F279 cavity on IL-6 when the antibody or fragment to bound to IL-6. Exemplary antibodies comprise a VH domain with a valine at position 99, according to Kabat.

In certain embodiments, the anti-IL-6 antibodies or fragments of the invention comprise a VH comprising 1 , 2, or 3 CDR amino acid sequences from a VH set forth in Tables 13-16.

In certain embodiments, the anti-IL-6 antibodies or fragments of the invention comprise a VL comprising 1, 2, or 3 CDR amino acid sequences from a VL set forth in Tables 13-16.

In certain embodiments, the anti-IL-6 antibodies or fragments of the invention comprise: a VH comprising 1, 2, or 3 CDR amino acid sequences from a VH set forth in Tables 13-18; and a VL comprising 1, 2, or 3 CDR amino acid sequences from a VL set forth in Tables 13-18. In a preferred embodiment all six CDRs are from the same Fab clone.

In certain embodiments, the anti-IL-6 antibodies or fragments of the invention comprise a VH set forth in Tables 13-16.

In certain embodiments, the anti-IL-6 antibodies or fragments of the invention comprise a VL set forth in Tables 13-16.

In certain embodiments, the anti-IL-6 antibodies or fragments of the invention comprise a VH and VL set forth in Tables 13-16.

In certain embodiments, the anti-IL-6 antibodies or fragments of the invention comprise a VH and VL from a single Fab clone set forth in Tables 13-16.

In certain embodiments, the invention provides antibodies or antigen binding fragments thereof that specifically bind to IL-6, the antibodies or fragments comprising a sequence variant of a CDR, VH, and VL amino acid sequences set forth in Tables 13-18.

In certain embodiments, the sequence variant comprises a VH and/or VL amino acid sequence with about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a VH or VL region amino acid sequences set forth in Tables 13-16.

In other embodiments, the sequence variant comprises a VH, VL, or CDR amino acid sequence selected from Tables 13-18 which has been altered by the introduction of one or more conservative amino acid substitutions. Conservative amino acid substitutions include the substitution of an amino acid in one class by an amino acid of the same class, where a class is defined by common physicochemical amino acid side chain properties and high substitution frequencies in homologous proteins found in nature, as determined, for example, by a standard Dayhoff frequency exchange matrix or BLOSUM matrix. Six general classes of amino acid side chains have been categorized and include: Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gin, Glu); Class IV (His, Arg, Lys); Class V (He, Leu, Val, Met); and Class VI (Phe, Tyr, Tip). For example, substitution of an Asp for another class III residue such as Asn, Gin, or Glu, is a conservative substitution. Thus, a predicted nonessential amino acid residue in an IL-6 antibody or antigen binding fragment thereof is preferably replaced with another amino acid residue from the same class. Methods of identifying amino acid conservative substitutions which do not eliminate antigen binding are well-known in the art (see, e.g., Brummell et ah, Biochem. 32: 1180-1187 (1993); Kobayashi el al. Protein Eng. 12(10):879-884 (1999): and Burks et a!, Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

In other embodiments, the sequence variant comprises a VH, VL or CDR amino acid sequence selected from Tables 13-18 which has been altered to improve antibody production and/or manufacturing, e.g., exchange of a methionine to alanine, serine or leucine. In certain other embodiments, the sequence variant comprises a VH, VL or CDR amino acid sequence selected from Tables 13-18 which has been altered to improve antibody production, e.g., exchange of glutamine to glutamic acid or asparagine to alanine or related amino acids. ii) IL-6 Binding Molecules with High Human Homology

In certain aspects, the IL-6 binding molecules of the invention are antibodies (or antigen binding fragments) with high human homology . An antibody will be considered as having "high human homology" if the VH domains and the VL domains, taken together, exhibit at least 90% amino acid sequence identity to the closest matching human germline VH and VL sequences. Antibodies having high human homology may include antibodies comprising VH and VL domains of native non-human antibodies which exhibit sufficiently high % sequence identity human germline sequences, including for example antibodies comprising VH and VL domains of camelid conventional antibodies, as well as engineered, especially humanised, variants of such antibodies and also "fully human" antibodies. In one embodiment the VH domain of the antibody with high human homology may exhibit an amino acid sequence identity or sequence homology of 80% or greater with one or more human VH domains across the framework regions FRl , FR2, FR3 and FR4. In other embodiments the amino acid sequence identity or sequence homology between the VH domain of the polypeptide of the invention and the closest matching human germline VH domain sequence may be 85% or greater, 90% or greater, 95 ' or greater, 97% or greater, or up to 99% or even 100%.

In one embodiment the VH domain of the antibody with high human homology may contain fewer than 10 (e.g. 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1) amino acid sequence substitutions across the framework regions FRl, FR2, FR3 and FR4, in comparison to the closest matched human VH sequence.

In another embodiment the VL domain of the antibody with high human homology may exhibit a sequence identity or sequence homology of 80% or greater with one or more human VI, domains across the framework regions FRl, FR2, FR3 and FR4. In other embodiments the amino acid sequence identity or sequence homology between the VL domain of the polypeptide of the invention and the closest matching human germline VL domain sequence may he 85%' or greater 90% or greater, 95% or greater, 97% or greater, or up to 99% or even 100%.

In one embodiment the VL domain of the antibody with high human homology may contain fewer than 10 (e.g. 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1) amino acid sequence substitutions across the framework regions FRl, FR2, FR3 and FR4, in comparison to the closest matched human VL sequence.

Antibodies with high human homology may also comprise hypervariable loops or CDRs having human or human-like canonical folds, as discussed in detail below. In one embodiment at least one hypervariable loop or CDR in either the VII domain or the VL domain of the antibody with high human homology may be obtained or derived from a VH or VL domain of a non-human antibody, for example a conventional antibody from a species of Cameiidae, yet exhibit a predicted or actual canonical fold structure which is substantially identical to a canonical fold structure which occurs in human antibodies.

It should be noted that antibodies with high human homology do not necessarily possess human or human-like canonical folds structures. For example, primate antibodies have high sequence homology to human antibodies yet often do not possess human or human-like canonical folds structures. It is well established in the art that although the primary amino acid sequences of hypervariable loops present in both VH domains and VL domains encoded by the human germline are, by definition, highly variable, all hypervariable loops, except CDR H3 of the VH domain, adopt only a few distinct structural conformations, termed canonical folds (Chothia et al, J. Mol. Biol. 196:901-917 ( 1987); Tramontane et al. Proteins 6:382-94 (1989)), which depend on both the length of the hypervariable loop and presence of the so- called canonical amino acid residues (Chothia et al., J. Mol, Biol. 196:901-917 (1987)).

Actual canonical structures of the hypervariable loops in intact VH or VL domains can be determined by structural analysis (e.g. X-ray crystallography), but it is also possible to predict canonical structure on the basis of key amino acid residues which are characteristic of a particular structure (discussed further below). In essence, the specific pattern of residues that determines each canonical structure forms a "signature" which enables the canonical structure to be recognised in hypervariable loops of a VH or VL domain of unknown structure;

canonical structures can therefore be predicted on the basis of primary amino acid sequence alone.

The predicted canonical fold structures for the hypervariable loops of any given VH or VL sequence in an antibody with high human homology can be analysed using algorithms which are publicly available from www.bioinf.org.ulc/abs/chothia.html,

www. biochem.ucl.ac.uk/~maitin/antibodies. html and

www.bioc.unkh.ch/antibody/Sequences These tools permit query VH or VL, sequences to be aligned against human VH or VL domain sequences of known canonical structure, and a prediction of canonical structure made for the hypervariable loops of the query sequence.

In the case of the VH domain, HI and H2 loops may be scored as having a canonical fold structure "substantially identical" to a canonical fold structure known to occur in human antibodies if at least the first, and preferable both, of the following criteria are fulfilled:

1. An identical length, determined by the number of residues, to the closest matching human canonical structural class.

2. At least 33% identity, preferably at least 50% identity with the key amino acid residues described for the corresponding human HI and H2 canonical structural classes. (note for the purposes of the foregoing analysis the HI and 112 loops are treated separately and each compared against its closest matching human canonical structural class)

The foregoing analysis relies on prediction of the canonical structure of the HI and H2 loops of the antibody of interest. If the actual structures of the H i and H2 loops in the antibody of interest are known, for example based on X-ray crystallography, then the HI and H2 loops in the antibody of interest may also be scored as having a canonical fold structure "substantially identical" to a canonical fold structure known to occur in human antibodies if the length of the loop differs from that of the closest matching human canonical structural class (typically by ±1 or ±2 amino acids) but the actual structure of the HI and H2 loops in the antibody of interest matches the structure of a human canonical fold.

Key amino acid residues found in the human canonical structural classes for the first and second hypervariable loops of human VH domains (HI and H2) are described by Chothia et al., J. ol. Biol. 227:799-817 (1992), the contents of which are incorporated herein in their entirety by reference. In particular, Table 3 on page 802 of Chothia et al., which is specifically incorporated herein by reference, lists preferred amino acid residues at key sites for HI canonical structures found in the human germline, whereas Table 4 on page 803, also specifically incorporated by reference, lists preferred amino acid residues at key sites for CDR H2 canonical structures found in the human germline.

In one embodiment, both HI and 112 in the VH domain of the antibody with high human homology exhibit a predicted or actual canonical fold structure which is substantiall identical to a canonical fold structure which occurs in human antibodies.

Antibodies with high human homology may comprise a VH domain in which the hypervariable loops HI and H2 form a combination of canonical fold structures which is identical to a combination of canonical structures known to occur in at least one human germline VH domain. It has been observed that only certain combinations of canonical fold structures at HI and H2 actually occur in VH domains encoded by the human germline. In an embodiment HI and H2 in the VH domain of the antibody with high human homology may be obtained from a VH domain of a non-human species, e.g. a Camelidae species, yet form a combination of predicted or actual canonical fold structures which is identical to a combination of canonical fold structures known to occur in a human germline or somatically mutated VH domain, in non-limiting embodiments HI and H2 in the VH domain of the antibody with high human homology may be obtained from a VH domain of a non-human species, e.g. a Camelidae species, and form one of the following canonical fold combinations: 1-1, 1-2, 1-3, 1-6, 1-4, 2-1, 3-1 and 3-5.

An antibody with high human homology may contain a VH domain which exhibits both high sequence identity/sequence homology with human VH, and which contains hypervariable loops exhibiting structural homology with human VH.

It may be advantageous for the canonical folds present at HI and H2 in the VH domain of the antibody with high human homology, and the combination thereof, to be "correct" for the human VH germline sequence which represents the closest match with the VH domain of the antibody with high human homology in terms of overall primary amino acid sequence identity. By way of example, if the closest sequence match is with a human germline VH3 domain, then it may be advantageous for HI and H2 to form a combination of canonical folds which also occurs naturally in a human VH3 domain. This may be particularly important in the case of antibodies with high human homology which are derived from non-human species, e.g. antibodies containing VH and VL domains which are derived from camelid conventional antibodies, especially antibodies containing humanised camelid VH and VL domains.

Thus, in one embodiment the VH domain of the IL-6 antibody with high human homology may exhibit a sequence identity or sequence homology of 80% or greater, 85% or greater, 90% or greater, 95% or greater, 97% or greater, or up to 99%' or even 100% with a human VH domain across the framework regio s FR1, FR2 , FR3 and FR4, and in addition HI and H2 in the same antibody are obtained from a non-human VH domain (e.g. derived from a Camelidae species), but form a combination of predicted or actual canonical fold structures which is the same as a canonical fold combination known to occur naturally in the same human VH domain.

For example, in one exemplary embodiment, the HI and H2 loops of an IL-6 antibody of the invention (e.g., 61H7) may comprise the 1-2 combination human canonical fold structures as found, for example, in the human antibody structure 1 DFB. In another exemplary embodiment, the HI and ί 12 of an IL-6 antibody of the invention (e.g., 68F2 or its germlined variant 129D3) loops may comprise the 3-1 combination of human canonical fold structures as found, for example, in the human antibody structure 1 ACY.

In other embodiments, LI and L2 in the VL domain of the antibody with high human homology are each obtained from a VL domain of a non-human species (e.g. a camelid- derived VL domain), and each exhibits a predicted or actual canonical fold structure which is substantially identical to a canonical fold structure which occurs in human antibodies,

As with the VH domains, the hypervariabie loops of VL domains of both VLarabda and VKappa types can adopt a limited number of conformations or canonical structures, determined in part by length and also by the presence of key amino acid residues at certain canonical positions.

Within an antibody of interest having high human homology, LI, L2 and L3 loops obtained from a VL domain of a non-human species, e.g. a Camelidae species, may be scored as having a canonical fold structure "substantially identical" to a canonical fold structure known to occur in human antibodies if at least the first, and preferable both, of the following criteria are fulfilled:

1. An identical length, determined by the number of amino acid residues, to the closest matching human structural class.

2. At least 33% identity, preferably at least 50% identity with the key amino acid residues described for the corresponding human LI or L2 canonical structural classes, from either the VLambda or the VKappa repertoire.

(note for the purposes of the foregoing analysis the Li and L2 loops are treated separately and each compared against its closest matching human canonical structural class).

The foregoing analysis relies on prediction of the canonical structure of the LI, L2 and L3 loops in the VL domain of the antibody of interest. If the actual structure of the Li, L2 and L3 loops is known, for example based on X-ray crystallography, then LI , L2 or L,3 loops derived from the antibody of interest may also be scored as having a canonical fold structure "substantially identical" to a canonical fold structure known to occur in human antibodies if the length of the loop differs from that of the closest matching human canonical structural class (typically by +1 or ±2 amino acids) but the actual structure of the Camelidae loops matches a human canonical fold.

Key amino acid residues found in the human canonical structural classes for the CDRs of human VLambda and VKappa domains are described by Morea et al. Methods, 20: 267-279 (2000) and Martin et al. , J. Mol. Biol, 263:800-815 (1996). The structural repertoire of the human VKappa domain is also described by Tomlinson et al. EM BO J. 14:4628-4638 (1995), and that of the VLambda domain by Williams et al. J. Mol. Biol, 264:220-232 (1996). The contents of all these documents are to be incorporated herein by reference.

LI and L2 in the VL domain of an antibody with high human homology may form a combination of predicted or actual canonical fold structures which is identical to a combination of canonical fold structures known to occur in a human germline VL domain. In non-limiting embodiments LI and L2 in the VLambda domain of an antibody with high human homology (e.g. an antibody containing a camelid-derived VL domain or a humanised variant thereof) may form one of the following canonical fold combinations: 11-7, 1.3- 7(A,B,C), 14-7(A,B), 12-11, 14-11 and 12-12 (as defined in Williams et al. J. Mol. Biol. 264:220 -32 (.1996) and as shown on

http://www.bioc.uzh.ch antibody/Sequences/Gen^ In non-limiting embodiments LI and L2 in the Vkappa domain may form one of the following canonical fold combinations: 2-1, 3-1, 4-1 and 6-1 (as defined in Tomlinson et al. EMBO .) . 14:4628-38 (1995) and as shown on

http://www.bioc.uzh.ch/antibod For example, in one exemplary embodiment, the LI. and L2 loops of an IL-6 antibody of the invention (e.g., 61H7) may comprise the 7λ-1 combination human canonical fold structures as found, for example, in the human antibody structure IMFA. In another exemplary embodiment, the LI and L2 of an IL-6 antibody of the invention (e.g., 68F2 or its germlined variant 129D3) loops may comprise the 6λ-1 combination of human canonical fold structures as found, for example, in the human antibody structure 3 MUG.

In a further embodiment, all three of LI, L2 and L3 in the VL domain of an antibody with high human homology may exhibit a substantially human structure. It is preferred that the VL domain of the antibody with high human homology exhibits both high sequence identity/sequence homology with human VL, and also that, the bypervariable loops in the VL domain exhibit structural homology with human VL. For example, in one exemplary embodiment, loops L1-L3 of an IL-6 antibody of the invention (e.g., 61H7) may comprise the 7λ- 1 -4 combination human canonical fold structures as found, for example, in the human antibody structure IMFA. In another exemplar}- ^' embodiment, the L1-L3 of an IL-6 antibody of the invention (e.g., 68F2 or its germlined variant 129D3) loops may comprise the 6λ-1 -5 combination of human canonical fold structures as found, for example, in the human antibody structure SMUG.

In one embodiment, the VL domain of a IL-6 antibody with high human homology may exhibit a sequence identity of 80% or greater, 85% or greater, 90% or greater, 95% or greater, 97% or greater, or up to 99% or even 100% with a human VL domain across the framework regions FR1 , FR2 , FR3 and FR4, and in addition hypervariabie loop LI and hypervariable loop L2 may form a combination of predicted or actual canonical fold structures which is the same as a canonical fold combination known to occur naturally in the same human VL domain.

It is, of course, envisaged that VH domains exhibiting high sequence

identity /sequence homology with human VH, and also structural homology with

hypervariable loops of human VII wil l be combined with VL domains exhibiting high sequence identity/sequence homology with human VL, and also structural homology with hypervariable loops of human VL to provide antibodies with high human homology containing VH/VL pairings (e.g came lid-derived VH/VL pairings) with maximal sequence and structural homology to human-encoded VH/VL pairings. iii). Non-immunoglobuiin Binding Molecules

In a further aspect, the invention provides non-immunoglobuiin binding molecules that specifically bind to IL-6. As used herein, the term "non-immunoglobuiin binding molecules" are binding molecules whose binding sites comprise a portion (e.g., a scaffold or framework) derived from a polypeptide other than an immunoglobulin, but which may be engineered (e.g., by the addition of CDR region sequences) to confer a desired binding specificity to the binding molecule. The non-immunoglobulin binding molecules of the invention generally comprise one or more of the CDR regions set forth in Tables 13-18 grafted into a non-immunoglobuiin polypeptide.

In certain embodiments, non-immunoglobulin binding molecules comprise binding site portions that are derived from a member of the immunoglobulin superfamily that is not an immunoglobulin (e.g. a T-cell receptor or a cell-adhesion protein (e.g., CTLA-4, N-CAM, telokin)). Such binding molecules comprise a binding site portion which retains the conformation of an immunoglobulin fold and is capable of specifically binding to IL-6 when modified to include one or more of the CDR region set forth in Tables 13-18. In other embodiments, non-immunoglobulin binding molecules of the invention comprise a binding site with a protein topology that is not based on the immunoglobulin fold (e.g. ankyrin repeat proteins, tetranectins, and fibronectins) but which nonetheless are capable of specifically binding to a target (e.g. IL-6) when modified to include one or more of the CDR region set forth in Tables 13-18.

In one embodiment, a binding molecule of the invention comprises a tetranectin molecule. Tetranectins are plasma proteins of trivalent structure. Each monomer of the tetranectin trimer comprises five distinct amino-acid loops that can be can be replaced by or engineered to contain antibody CDR sequences (e.g., CDR regions set forth in Tables 13-18). Methods for making tetranectin binding polypeptides are described, for example, in

US20110086770, which is incorporated by reference herein in its entirety.

In one embodiment, a binding molecule of the invention comprises a fibronectin molecule. Fibronectin binding molecules (e.g., molecules comprising the Fibronectin type I, II, or III domains) display CDR-like loops which can be replaced by or engineered to contain antibod CDR sequences (e.g., CDR regions set forth in Tables 13-18). Methods for making fibronectin binding polypeptides are described, for example, in WO 01/64942 and in U.S. Pat. Nos. 6,673,901 , 6,703,199, 7,078,490, and 7,119,171 , which are each incorporated herein by reference in their entirety.

In another embodiment, a binding molecule of the invention compri ses a binding site from an affibody. Affibodies are derived from the immunoglobulin binding domains of staphylococcal Protein A (SPA) (see e.g., Nord el al, Nat. Biotechnol., 15: 772-777 (1997)). Affibody binding sites employed in the invention may be synthesized by mutagenizing an SPA-related protein (e.g., Protein Z) derived from a domain of SPA (e.g., domain B) and selecting for mutant SPA-related polypeptides having binding affinity for IL-6. Other methods for making affibody binding sites are described in U.S. Pat. Nos. 6,740,734 and 6,602,977 and in WO 00/63243, each of which is incorporated herein by reference.

In another embodiment, a binding molecule of the invention comprises a binding site from an anticalin. Anticalins (also known as lipocalins) are members of a diverse beta-barrel protein family whose function is to bind target molecules in their barrel/loop region.

Lipocalin binding sites may be engineered to bind IL-6 by randomizing loop sequences connecting the strands of the barrel (see e.g., Schlehuber et al., Drag Discov. Today, 10: 23- 33 (2005); Beste et al, PNAS, 96: 1898-1903 (1999). Anticalin binding sites employed in the binding molecules of the invention may be obtainable starting from polypeptides of the lipocalin family which are mutated in four segments that correspond to the sequence positions of the linear polypeptide sequence comprising amino acid positions 28 to 45, 58 to 69, 86 to 99 and 114 to 129 of the Bilin-binding protein (BBP) of Pieris brassica. Other methods for making anticalin binding sites are described in W099/16873 and WO

05/019254, each of which is incorporated herein by reference.

In another embodiment, a binding molecule of the invention compri ses a binding site from a cysteine -rich polypeptide. Cysteine-rich domains employed in the practice of the present invention typically do not form an alpha-helix, a beta-sheet, or a beta-barrel structure. Typically, the disulfide bonds promote folding of the domain into a three-dimensional structure. Usually, cysteine-rich domains have at least two disulfide bonds, more typically at least three disulfide bonds. An exemplary cysteine-rich polypeptide is an A domain protein. A-domains (sometimes called "complement- ype repeats") contain about 30-50 or 30-65 amino acids. In some embodiments, the domains comprise about 35-45 amino acids and in some cases about 40 amino acids. Within the 30-50 amino acids, there are about 6 cysteine residues. Of the six cysteines, disulfide bonds typically are found between the following cysteines: CI and C3, C2 and C5, C4 and C6. The A domain constitutes a ligand binding moiety. The cysteine residues of the domain are disulfide linked to form a compact, stable, functionally independent moiety. Clusters of these repeats make up a ligand binding domain, and differential clustering can impart specificity with respect to the ligand binding.

Exemplary proteins containing A-domains include, e.g., complement components (e.g., C6, C7, C8, C9, and Factor I), serine proteases (e.g., enteropeptidase, matriptase, and corin), transmembrane proteins (e.g., ST7, LRP3, LR.P5 and LRP6) and endocytic receptors (e.g., Sortilin-related receptor, LDL-receptor, VLDLR, LRPL LRP2, and ApoER2). Methods for making A domain proteins of a desired binding specificity are disclosed, for example, in WO 02/088171 and W 04/044011, each of which is incorporated herein by reference.

In other embodiments, a binding molecule of the invention comprises a binding site from a repeat protein. Repeat proteins are proteins that contain consecutive copies of small (e.g., about 20 to about 40 amino acid residues) structural units or repeats that stack together to form contiguous domains. Repeat proteins can be modified to suit a particular target binding site by adjusting the number of repeats in the protein. Exemplary repeat proteins include designed ankyrin repeat proteins (i.e., a DARPins) (see e.g., Binz et al., Nat.

Biotechnol., 22: 575-582 (2004)) or leucine-ricli repeat proteins (i.e., LRRPs) (see e.g., Pancer et al., Nature, 430: 174-180 (2004)). All so far determined tertiary structures of ankyrin repeat units share a characteristic composed of a beta-hairpin followed by two antiparallel alpha-helices and ending with a loop connecting the repeat unit with the next one. Domains built of ankyrin repeat units are formed by stacking the repeat units to an extended and curved structure. LRRP binding sites from part of the adaptive immune system of sea lampreys and other jawiess fishes and resemble antibodies in that they are formed by recombination of a suite of leucine-rich repeat genes during lymphocyte maturation.

Methods for making DARpin or LRRP binding sites are described in WO 02/20565 and WO 06/083275, each of which is incorporated herein by reference.

Other non-immunoglobulin binding sites which may be employed in binding molecules of the invention include binding sites derived from Src homology domains (e.g. SH2 or SI B domains), PDZ domains, beta-lactamase, high affinity protease inhibitors, or small disulfide binding protein scaffolds such as scorpion toxins. Methods for making binding sites derived from these molecules have been disclosed in the art, see e.g., Panni et al, J. Biol. Chem., 277: 21666-21674 (2002), Schneider et al, Nat. Biotechnol., 17: 170-175 (1999): Legendre et al, Protein Sci„ 1 1: 1506-1518 (2002); Stoop et al, Nat. Biotechnol., 21: 1063-1068 (2003); and Vita et al., PNAS, 92: 6404-6408 (1995). Yet other binding sites may be derived from a binding domain selected from the group consisting of an EGF-like domain, a Kringle-domain, a PAN domain, a Gia domain, a SRCR domain, a Kunitz/B ovine pancreatic trypsin Inhibitor domain, a Kazal-type serine protease inhibitor domain, a Trefoil (P-type) domain, a von Willebrand factor type C domain, an Anaphylatoxin-iike domain, a CUB domain, a thyroglobulin type I repeat, LDL-receptor class A domain, a Sushi domain, a Link domain, a Thrombospondin type I domain, an Immunoglobulin-like domain, a C-type lectin domain, a MAM domain, a von Willebrand factor type A domain, a Somatomedin B domain, a WAP-type four disulfide core domain, a F5/8 type C domain, a Hemopexin domain, a Laminin-type EGF-like domain, a C2 domain, and other such domains known to those of ordinary skill in the art, as well as derivatives and/or variants thereof.

Non-immunoglobulin binding molecules may be identified by selection or isolation of a target-binding variant from a library of binding molecules having artificially diversified binding sites. Diversified libraries can be generated incorporation of a library of CDR sequences (e.g., selected from those CDR sequences set forth in Tables 13-18) and/or completely random approaches (e.g., error-prone PGR, exon shuffling, or directed evolution) and/or aided by art-recognized design strategies. For example, amino acid positions that are usually involved when the binding site interacts with its cognate target molecule can be randomized by insertion of degenerate codons, trinucleotides, random peptides, or entire loops at corresponding positions within the nucleic acid which encodes the binding site (see e.g., U.S. Pub. No. 20040132028). The location of the amino acid positions can be identified by investigation of the crystal structure of the binding site in complex with the target molecule. Candidate positions for incorporation of CDR sequences (e.g., selected from those CDR sequences set forth in Tables 13-18) and/or randomization include loops, flat surfaces, helices, and binding cavities of the binding site. In certain embodiments, amino acids within the binding site that are likely candidates for diversification can be identified by their homology with the immunoglobulin fold. For example, residues within the CDR-like loops of fibronectin may be randomized to generate a library of fibronectin binding molecules (see, e.g., Koide et al, J. Mol. Biol., 284: 1 141-1151 (1998)). Following incorporation of CDR sequences (e.g., selected from those CDR sequences set forth in Table 2-6) and/or randomization, the diversified library may then be subjected to a selection or screening procedure to obtain binding molecules with the desired binding characteristics, e.g. specific binding to IL-6. Selection can be achieved by art-recognized methods such as phage display, yeast display, or nucleic acid display. iv. Germlining of Canielid-Derived VH and VL Domains

Camelid conventional antibodies provide an advantageous starting point for the preparation of antibodies with utility as human therapeutic agents due to the following factors (discussed in US 12/497,239, which is incorporated herein by reference in its entirety):

1) High % sequence homology between camelid VH and VL domains and their human counterparts;

2) High degree of structural homology between CDRs of camelid VH and VL domains and their human counterparts (i.e. human-like canonical fold structures and human-like combinations of canonical folds).

The camelid (e.g. llama) platform also provides a significant advantage in terms of the functional diversity of the IL-6 antibodies which can be obtained.

The utility of IL-6 antibodies comprising camelid VH and/or camelid VL domains for human therapy can be improved still further by "germlining" of natural camelid VH and VL domains, for example to render them less immunogenic in a human host. The overall aim of germlining is to produce a molecule in which the VH and VL domains exhibit minimal immunogenic! ty when introduced into a human subject, while retaining the specificity and affinity of the antigen binding site formed by the parental VH and VL domains.

Determination of homology between a camelid VH (or VL) domain and human VH (or VL) domains is a critical step in the germlining process, both for selection of camelid amino acid residues to be changed (in a given VH or VL domain) and for selecting the appropriate replacement amino acid residue(s).

An approach to germlining of camelid conventional antibodies has been developed based on alignment of a large number of novel camelid VH (and VL) domain sequences, typically somatically mutated VH (or VL) domains which are known to bind a target antigen, with human germline VH (or VL) sequences, human VH (and VI.,) consensus sequences, as well as germline sequence information available for llama pacos.

The fol lowing passages outline the principles which can be applied to (i) select "camelid" amino acid residues for replacement in a camelid-derived VH or VL domain or a CDR thereof, and (ii) select replacement "human" amino acid residues to substitute in, when germlining any given camelid VH (or VL) domain. This approach can be used to prepare germlined variants of the VH and VL sequences set forth in Tables 13-16, herein.

Step 1. Select human (germline) family and member of this family that shows highest homology/identity to the mature camelid sequence to be germlined. A general procedure for identifying the closest matching human germline for any given camelid VH (or VL) domain is outlined below.

Step 2. Select specific human germline family member used to germline against.

Preferably this is the germline with the highest homology or another germline family member from the same family.

Step 3. Identify the preferred positions considered for germlining on the basis of the table of amino acid utilisation for the camelid germline that is closest to the selected human germline.

Step 4. Try to change amino acids in the camelid germline that deviate from the closest human germline; germlining of FR residues is preferred over CDR residues. a. Preferred are positions that are deviating from the selected human germline used to germlme against, for which the amino acid found in the camelid sequence does not match with the selected germline and is not found in other germlines of the same subclass (both for V as well as for J encoded FR amino acids). b. Positions that are deviating from the selected human germline family member but which are used in other germlines of the same family may also be addressed in the germlining process. c. Additional mismatches (e.g. due to additional somatic mutations) towards the selected human germline may also be addressed.

The following approach may be used to determine the closest matching human germline for a given camelid VH (or VL) domain:

Before analyzing the percentage sequence identity between Camelidae and human germline VH and VL, the canonical folds may first be determined, which allows the identification of the family of human germline segments with the identical combination of canonical folds for HI and H2 or LI and L2 (and L3). Subsequently the human germline family member that has the highest degree of sequence homology with the Camelidae variable region of interest may be chosen for scoring sequence homology. The determination of Chothia canonical classes of hypervariable loops LI , L2, L3, 111 and H2 can be performed with the bioinformatics tools publicly available on webpage

www.bioinf.org.uk/abs/chothia.html.page. The output of the program shows the key residue requirements in a datafile. In these datafiles, the key residue positions are shown with the allowed amino acids at each position. The sequence of the variable region of the antibody is given as input and is first aligned with a consensus antibody sequence to assign the abat numbering scheme, The analysis of the canonical folds uses a set of key residue templates derived by an automated method developed by Martin and Thornton (Martin et aL, J. Mol. Biol, 263:800-815 (1996)). The boundaries of the individual framework regions may be assigned using the IMGT numbering scheme, which is an adaptation of the numbering scheme of Chothia (Lefranc et al., NAR 27: 209-212 (1999); imgt.cines.fr).

With the particular human germline V segment known, which uses the same combination of canonical folds for HI and H2 or LI and L2 (and L3), the best matching family member in terms of sequence homology can be determined. The percentage sequence identity between Camelidae VH and VL domain framework amino acid sequences and corresponding sequences encoded by the human germline can be determined using bioinformatic tools, but manual alignment of the sequences could also be used. Human immunoglobulin sequences can be identified from several protein data bases, such as VBase (vbase.mrc--cpe.cam.ac.uk/) or the Pluckthun/Honegger database

(http://www.bioc.unizh.ch/antibody/Sequences/Germlines. To compare the human sequences to the V regions of Camelidae VH or VL domains a sequence alignment algorithm such as available via websites like www.expasy.ch/tools/#align can be used, but also manual alignment can also be performed with a limited set of sequences. Human germline light and heavy chain sequences of the families with the same combinations of canonical folds and with the highest degree of homology with the framework regions 1 , 2, and 3 of each chain may be selected and compared with the Camelidae variable region of interest; also the FR4 is checked against the human germline JH and JK or JL regions.

Note that in the calculation of overall percent sequence homology the residues of FR1, FR2 and FR3 are evaluated using the closest match sequence from the human germline family with the identical combination of canonical folds. Only residues different from the closest match or other members of the same family with the same combination of canonical folds are scored (NB ^■■ excluding any primer-encoded differences). However, for the purposes of germlining, residues in framework regions identical to members of other human germline families, which do not have the same combination of canonical folds, can be considered for germlining, despite the fact that these are scored "negative" according to the stringent conditions described above. This assumption is based on the "mix and match" approach for germlining, in which each of FR1, FR2, FR3 and FR4 is separately compared to its closest matching human germline sequence and the germlined molecule therefore contains a combination of different FRs as was done by Qu and colleagues (Qu et la., Clin. Cancer Res. 5:3095-3100 (1999,)) and Ono and colleagues (Ono et al., Mol. Immunol. 36:387-395 (1999)). IV. Modified Binding Molecules

In certain embodiments, binding polypeptides of the invention may comprise one or more modifications. Modified forms of binding polypeptides of the invention can be made using any techniques known in the art. i) Reducing Imniunogenicity Risk

In certain embodiments, binding molecules (e.g., antibodies or antigen binding fragments thereof) of the invention are modified to further reduce their imniunogenicity risk using art-recognized techniques. For example, antibodies, or fragments thereof, can be germlined according to the methods describe above. Alternatively, binding molecules of the invention can be chimericized, humanized, and/or deimmunized.

In one embodiment, an antibody, or antigen binding fragments thereof, of the invention may be chimeric. A chimeric antibody is an antibody in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a camelid (e.g., llama) monoclonal antibody and a human

immunoglobulin constant region. Methods for producing chimeric antibodies, or fragments thereof, are known in the art. See, e.g., Morrison, Science 229:1202 ( 1985); Oi et al, BioTechniques 4:214 ( 1986); Gillies et al, J. Immunol. Methods 125:191-202 (1989); U.S. Pat. Nos. 5,807,715; 4,816,567: and 4,816,397, which are incorporated herein by reference in their entireties. Techniques developed for the production of "chimeric antibodies" (Morrison et al, Proc. Natl. Acad. Sci. 81:851-855 ( 1984); Neuberger et al, Nature 312:604-608 (1984); Takeda et al, Nature 314:452-454 (1985)) may be employed for the synthesis of said molecules. For example, a genetic sequence encoding a binding specificity of a camelid anti- IL-6 antibody molecule may be fused together with a sequence from a human antibody molecule of appropriate biological activity. As used herein, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a camelid (e.g., llama) monoclonal antibody and a human immunoglobulin constant region, e.g., germlined or humanized antibodies.

In another embodiment, an antibody, or antigen binding portion thereof, of the invention is humanized. Humanized antibodies have a binding specificity comprising one or more complementarity determining regions (CDRs) from a non-human antibody and framework regions from a human antibody molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al, U.S. Pat. No, 5,585,089; Riechmann er /., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCX publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and

5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular

Immunology 28(4/5):489-498 ( 1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al, PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No.

5,565,332).

In some embodiments, de-immunization can be used to further decrease the immunogenicity risk of IL-6 binding molecules (e.g., antibody, or antigen binding portion thereof). As used herein, the term "de-immunization" includes alteration of polypeptide (e.g., an antibody, or antigen binding portion thereof) to modify T cell epitopes (see, e.g.,

W09852976A1, WO0034317A2). For example, VH and VL sequences from the starting II - 6-speeific antibody, or antigen binding portion thereof, of the invention may be analyzed and a human T cell epitope "map" may be generated from each V region showing the location of epitopes in relation to complementarity-determining regions (CDRs) and other key residues within the sequence. Individual T cell epitopes from the T cell epitope map are analyzed in order to identify alternative amino acid substitutions with a low risk of altering activity of the final antibody. A range of alternative VH and VL sequences are designed comprising combinations of amino acid substitutions and these sequences are subsequently incorporated into a range of IL-6-specific antibodies or fragments thereof for use in the diagnostic and treatment methods disclosed herein, which are then tested for function. Typically, between 12 and 24 variant antibodies are generated and tested. Complete heavy and light chain genes comprising modified V and human C regions are then cloned into expression vectors and the subsequent piasmids introduced into cell lines for the production of whole antibody. The antibodies are then compared in appropriate biochemical and biological assays, and the optimal variant is identified. ii) Effector Functions and Fc Modifications

In certain embodiments, binding molecules of the invention may comprise an antibody constant region (e.g. an IgG constant region e.g., a human IgG constant region, e.g., a human IgGl or IgG4 constant region) which mediates one or more effector functions. For example, binding of the CI component of complement to an antibody constant region may activate the complement system. Activation of complement is important in the opsonisation and lysis of cell pathogens. The activation of complement also stimulates the inflammatory response and may also be involved in autoimmune hypersensitivity. Further, antibodies bind to receptors on various cells via the Fc region, with a Fc receptor binding site on the antibody Fc region binding to a Fc receptor (FcR) on a cell. There are a number of Fc receptors which are specific for different classes of antibody, including IgG (gamma receptors), IgE (epsi!on receptors), IgA (alpha receptors) and IgM (mu receptors). Binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological responses including engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer and control of immunoglobulin production, In preferred embodiments, the binding molecules (e.g., antibodies or antigen binding fragments thereof) of the invention bind to an Fc-gamma receptor. In alternative embodiments, binding molecules of the invention may comprise a constant region which is devoid of one or more effector functions (e.g., ADCC activity) and/or is unable to bind Fey receptor.

Certain embodiments of the invention include anti-IL-6 antibodies in which at least one amino acid in one or more of the constant region domains has been deleted or otherwise altered so as to provide desired biochemical characteristics such as reduced or enhanced effector functions, the ability to non-covalently dimerize, increased ability to localize at the site of a tumor, reduced serum half- life, or increased serum half- life when compared with a whole, unaltered antibody of approximately the same immunogenicity. For example, certain antibodies, or fragments thereof, for use in the diagnostic and treatment methods described herein are domain deleted antibodies which comprise a polypeptide chain similar to an immunoglobulin heavy chain, but which lack at least a portion of one or more heavy chain domains. For instance, in certain antibodies, one entire domain of the constant region of the modified antibody will be deleted, for example, all or part of the CH2 domain will be deleted.

In certain other embodiments, binding molecules comprise constant regions derived from different antibody isotypes (e.g., constant regions from two or more of a human IgGl, IgG2, IgG3, or IgG4). In other embodiments, binding molecules comprises a chimeric hinge (i.e., a hinge comprising hinge portions derived from hinge domains of different antibody isotypes, e.g., an upper hinge domain from an IgG4 molecule and an IgGl middle hinge domain). In one embodiment, binding molecules comprise an Fc region or portion thereof from a human IgG4 molecule and a Ser228Pro mutation (EU numbering) in the core hinge region of the molecule.

In certain embodiments, the Fc portion may be mutated to increase or decrease effector function using techniques known in the art. For example, the deletion or inactivation (through point mutations or other means) of a constant region domain may reduce Fc receptor binding of the circulating modified antibody thereby increasing tumor localization. In other cases it may be that constant region modifications consistent with the instant invention moderate complement binding and thus reduce the serum half life and nonspecific association of a conjugated cytotoxin. Yet other modifications of the constant region may be used to modify disulfide linkages or oligosaccharide moieties thai allow for enhanced localization due to increased antigen specificity or flexibility. The resulting physiological profile, bioavailability and other biochemical effects of the modifications, such as tumor localization, biodistribution and serum half-life, may easily be measured and quantified using well know immunological techniques without undue experimentation.

In certain embodiments, an Fc domain employed in an antibody of the invention is an Fc variant. As used herein, the term "Fc variant" refers to an Fc domain having at least one amino acid substitution relative to the wild-type Fc domain from which said Fc domain is derived. For example, wherein the Fc domain is derived from a human IgGl antibody, the Fc variant of said human IgGl Fc domain comprises at least one amino acid substitution relative to said Fc domain.

The amino acid substiiution(s) of an Fc variant may be located at any position (i.e., any EU convention amino acid position) within the Fc domain. In one embodiment, the Fc variant comprises a substitution at an amino acid position located in a hinge domain or portion thereof. In another embodiment, the Fc variant comprises a substitution at an amino acid position located in a CH2 domain or portion thereof. In another embodiment, the Fc variant comprises a substitution at an amino acid position located in a CH3 domain or portion thereof, In another embodiment, the Fc variant comprises a substitution at an amino acid position located in a CH4 domain or portion thereof.

The binding molecules of the invention may employ any art-recognized Fc variant which is known to impart an improvement (e.g., reduction or enhancement) in effector function and/or FcR binding. Said Fc variants may include, for example, any one of the amino acid substitutions disclosed in International PCX Publications WO88/07089A1, W096/14339A1, WO98/05787A1, W098/23289A1 , W099/51642A1, W099/58572A1, WO00/09560A2, WO00/32767A1, WO00/42072A2, WO02/44215A2, WOG2/G60919A2, WO03/074569A2, WO04/016750A2, WO04/029207A2, WO04/035752A2,

WO04/063351A2, WO04/074455A2, WO04/099249A2, WO05/040217A2,

WO05/070963A1, WO05/077981 A2, WO05/092925 A2, WO05/123780A2,

WO06/019447A1, WO06/047350A2, and WO06/085967A2 or U.S. Pat. Nos. 5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871; 6,121,022; 6,194,551 ; 6,242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056: 6,821,505; 6,998,253; and 7,083,784, each of which is incorporated by reference herein. In one exemplary embodiment, a binding polypeptide of the invention may comprise an Fc variant comprising an amino acid substitution at EU position 268 (e.g., H268D or H268E). In another exemplary embodiment, a binding polypeptide of the invention may comprise an amino acid substitution at EU position 239 (e.g., S239D or S239E) and/or EU position 332 (e.g., I332D or I332Q).

In certain embodiments, a binding polypeptide of the invention may comprise an Fc variant comprising an amino acid substitution which alters the antigen-independent effector functions of the antibody, in particular the circulating half-life of the binding polypeptide. Such binding molecules exhibit either increased or decreased binding to FcRn when compared to binding molecules lacking these substitutions, therefore, have an increased or decreased half-life in serum, respectively. Fc variants with improved affinity for FcRn are anticipated to have longer serum half-lives, and such molecules have useful applications in methods of treating mammals where long half-life of the administered antibody is desired, e.g., to treat a chronic disease or disorder. In contrast, Fc variants with decreased FcRn binding affinity are expected to have shorter half-lives, and such molecules are also useful, for example, for administration to a mammal where a shortened circulation time may be advantageous, e.g. for in vivo diagnostic imaging or in situations where the starting antibody has toxic side effects when present in the circulation for prolonged periods. Fc variants with decreased Fc n binding affinity are also less likely to cross the placenta and, thus, are also useful in the treatment of diseases or disorders in pregnant women, In addition, other applications in which reduced FcRn binding affinity may be desired include those

applications in which localization the brain, kidney, and/or liver is desired, In one exemplary embodiment, the altered binding molecules (e.g., antibodies or antigen binding fragments thereof) of the invention exhibit reduced transport across the epithelium of kidney glomeruli from the vasculature. In another embodiment, the altered binding molecules (e.g., antibodies or antigen binding fragments thereof) of the invention exhibit reduced transport across the blood brain barrier (BBB) from the brain, into the vascular space, In one embodiment, an antibody with altered FcRn binding comprises an Fc domain having one or more amino acid substitutions within the "FcRn binding loop" of an Fc domain. The FcRn binding loop is comprised of amino acid residues 280-299 (according to EU numbering). Exemplary amino acid substitutions which altered FcRn binding activity are disclosed in International PCX Publication No. WO05/047327 which is incorporated by reference herein. In certain exemplary embodiments, the binding molecules (e.g., antibodies or antigen binding fragments thereof) of the invention comprise an Fc domain having one or more of the following substitutions: V284E, H285E, N286D, 290E and S304D (EU numbering). In yet other exemplary embodiments, the biding molecules of the invention comprise a human Fc domain with the double mutation H433 /N434F (see, e.g., US Patent No. 8,163,881).

In other embodiments, binding molecules, for use in the diagnostic and treatment methods described herein have a constant region, e.g., an IgGl or IgG4 heavy chain constant region, which is altered to reduce or eliminate glycosylation. For example, binding molecules (e.g., antibodies or antigen binding fragments thereof) of the invention may also comprise an Fc variant comprising an amino acid substitution which alters the glycosylation of the antibody Fc, For example, said Fc variant may have reduced glycosylation (e.g., N- or O- linked glycosylation). In exemplary embodiments, the Fc variant comprises reduced glycosylation of the N-linked glycan normally found at amino acid position 297 (EU numbering). In another embodiment, the antibody has an amino acid substitution near or within a glycosylation motif, for example, an N-linked glycosylation motif that contains the amino acid sequence NXT or NXS. In a particular embodiment, the antibody comprises an Fc variant with an amino acid substitution at amino acid position 228 or 299 (EU numbering). In more particular embodiments, the antibody comprises an IgGl or IgG4 constant region comprising an S228P and a T299A mutation (EU numbering). Exemplary amino acid substitutions which confer reduce or altered glycosylation are disclosed in International PCT Publication No. WO05/018572, which is incorporated by reference herein, in preferred embodiments, the antibodies, or fragments thereof, of the invention are modified to eliminate glycosylation. Such antibodies, or fragments thereof, may be referred to as "agly" antibodies, or fragments thereof, (e.g. "agly" antibodies). While not being bound by theory, it is believed that "agly" antibodies, or fragments thereof, may have an improved safety and stability profile in vivo. Agly antibodies can be of any isotype or subclass thereof, e.g., IgGl , IgG2, IgG3, or IgG4. In certain embodiments, agly antibodies, or fragments thereof, comprise an aglycosylated Fc region of an IgG4 antibody which is devoid of Fc-effector function thereby eliminating the potential for Fc mediated toxicity to the normal vital organs that express IL-6, In yet other embodiments, antibodies, or fragments thereof, of the invention comprise an altered glycan. For example, the antibody may have a reduced number of fucose residues on an N-glycan at Asn297 of the Fc region, i.e., is afucosylated. Afucosyiation increases FcRgll binding on the N cells and potently increase ADCC. It has been shown that a diabody comprising an anti-IL-6 scFv and an anti-CD3 scFv induces killing of IL-6 expressing cells by ADCC. Accordingly, in one embodiment, the afucosylated an anti-IL-6 antibody is be used to target and kill IL-6-expressing cells. In another embodiment, the antibody may have an altered number of sialic acid residues on the N-glycan at Asn297 of the Fc region. Numerous art-recognized methods are available for making "agly" antibodies or antibodies with altered glycan s. For examples, genetically engineered host cells (e.g., modified yeast, e.g., Picchia, or CHO cells) with modified glycosylation pathways (e.g., glycosyltransferase deletions) can be used to produce such antibodies.

Hi) Covaknt Attachment

Binding molecules of the invention may be modified, e.g., by the covalent attachment of a molecule to the binding polypeptide such that covalent attachment does not prevent the binding polypeptide from specifically binding to its cognate epitope. For example, but not by way of limitation, the antibodies, or fragments thereof, of the invention may be modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, aeetylation, formylation, etc.

Additionally, the derivative may contain one or more non-classical amino acids.

Binding polypeptide (e.g., antibodies, or fragments thereof) of the invention may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions. For example, anfi-IL-6 antibodies may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drags, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995: and EP 396,387.

Binding molecules may be fused to heterologous polypeptides to increase the in vivo half life or for use in immunoassays using methods known in the art. For example, in one embodiment, PEG can be conjugated to the binding molecules of the invention to increase their half-life in vivo. Leong, S. R., et al, Cytokine 16:106 (2001); Adv. in Drug Deliv. Rev. 54:531 (2002): or Weir et ., Biochem. Soc. Transactions 30:512 (2002).

Moreover, binding molecules of the invention can be fused to marker sequences, such as a peptide to facilitate their purification or detection. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif, 91311), among others, many of which are commercially available. As described in Gentz et ah, Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et ., Cell 37:767 (1984)) and the "flag" tag.

Binding molecules of the invention may be used in non-conjugated form or may be conjugated to at least one of a variety of molecules, e.g., to improve the therapeutic properties of the molecule, to facilitate target detection, or for imaging or therapy of the patient. Binding molecules of the invention can be labeled or conjugated either before or after purification, when purification is performed. In particular, binding molecules of the invention may be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG.

The present invention further encompasses binding molecules of the invention conjugated to a diagnostic or therapeutic agent. The binding molecules can be used diagnostically to, for example, monitor the development or progression of a immune cell disorder (e.g., CLL) as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment and/or prevention regimen. Detection can be facilitated by coupling the binding molecules to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials,

bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. See, for example, U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and

avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rbodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 1251, 1311, 11 lln or 99Tc.

Binding molecules for use in the diagnostic and treatment methods disclosed herein may be conjugated to cytotoxins (such as radioisotopes, cytotoxic drags, or toxins) therapeutic agents, cytostatic agents, biological toxins, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, immunologically active ligands (e.g., lymphokin.es or other antibodies wherein the resulting molecule binds to both the neoplastic cell and an effector cel l such as a T cell), or PEG.

In another embodiment, an anti-IL-6 antibody for use in the diagnostic and treatment methods disclosed herein can be conjugated to a molecule that decreases tumor cell growth. In other embodiments, the disclosed compositions may comprise antibodies, or fragments thereof, coupled to drugs or prodrugs. Still other embodiments of the present invention comprise the use of antibodies, or fragments thereof, conjugated to specific biotoxins or their cytotoxic fragments such as ricin, gelonin, Pseudomonas exotoxin or diphtheria toxin. The selection of which conjugated or unconjugated antibodyto use will depend on the type and stage of cancer, use of adjunct treatment (e.g., chemotherapy or external radiation) and patient condition. It will be appreciated that one skilled in the art could readily make such a selection in view of the teachings herein.

It will be appreciated that, in previous studies, anti-tumor antibodies labeled with isotopes have been used successfully to destroy tumor cells in animal models, and in some cases in burnans. Exemplary radio sotopes include: 90Y, 1251, 1311, 1231, l l lln, ! 05Rh, 153Sm, 67Cu, 67Ga, 166Ho, 177Lu, 186Re and 188Re. The radionuclides act by producing ionizing radiation which causes multiple strand breaks in nuclear DNA, leading to cell death. The isotopes used to produce therapeutic conjugates typically produce high energy alpha- or beta-particles which have a short path length. Such radionuclides kill cells to which they are in close proximity, for example neoplastic cells to which the conjugate has attached or has entered. They have little or no effect on non-localized cells. Radionuclides are essentially non -immunogenic.

V. Expression of Binding molecules

Following manipulation of the isolated genetic material to provide binding molecules of the invention as set forth above, the genes are typically inserted in an expression vector for introduction into host cells that may be used to produce the desired quantity of the claimed antibodies, or fragments thereof.

The term "vector" or "expression vector" is used herein for the purposes of the specification and claims, to mean vectors used in accordance with the present invention as a vehicle for introducing into and expressing a desired gene in a ceil. As known to those skilled in the art, such vectors may easily be selected from the group consisting of plasmids, phages, viruses and retroviruses. In general, vectors compatible with the instant invention will comprise a selection marker, appropriate restriction sites to facilitate cloning of the desired gene and the ability to enter and/or replicate in eukaryotic or prokar otic cells.

Numerous expression vector systems may be employed for the purposes of this invention. For example, one class of vector utilizes DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia vims, baculovirus, retroviruses (RSV, MMTV or MOMLV) or SV40 vims. Others involve the use of poivcistronic systems with internal ribosome binding sites. Additionally, cells which have integrated the DNA into their chromosomes may be selected by introducing one or more markers which allow selection of transfected host cells. The marker may provide for prototrophy to an auxotrophic host, biocide resistance (e.g., antibiotics) or resistance to heavy- metals such as copper. The selectable marker gene can either be directly linked to the DNA sequences to be expressed, or introduced into the same cell by cotransformation. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include signal sequences, splice signals, as well as transcriptional promoters, enhancers, and termination signals. In particularly preferred embodiments the cloned variable region genes are inserted into an expression vector along with the heavy and light chain constant region genes (preferably human) synthetic as discussed above.

In other preferred embodiments the binding molecules, or fragments thereof, of the invention may be expressed using polycistronic constructs. In such expression systems, multiple gene products of interest such as heavy and light chains of antibodies may be produced from a single polycistronic construct. These systems advantageously use an internal ribosome entry site (IRES) to provide relatively high levels of polypeptides of the invention in eukaryotic host cells. Compatible IRES sequences are disclosed in U.S. Pat. No. 6,193,980 which is incorporated by reference herein. Those skilled in the art will appreciate that such expression systems may be used to effectively produce the full range of polypeptides disclosed in the instant application.

More generally, once a vector or DNA sequence encoding an antibody, or fragment thereof, has been prepared, the expression vector may be introduced into an appropriate host cell. That is, the host cells may be transformed. Introduction of the plasmid into the host cell can be accomplished by various techniques well known to those of skill in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus. See, Ridgway, A. A. G. "Mammalian

Expression Vectors" Chapter 24.2, pp. 470-472 Vectors, Rodriguez and Denhardt, Eds.

(Butterworths, Boston, Mass. 1988). Most preferably, plasmid introduction into the host is via electroporation. The transformed cells are grown under conditions appropriate to the production of the light chains and heavy chains, and assayed for heavy and/or light chain protein synthesis. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or iioureseence-activated cell sorter analysis (FACS), immunohistochemistry and the like.

As used herein, the term "transformation" shall be used in a broad sense to refer to the introduction of DNA into a recipient host cell that changes the genotype and consequently results in a change in the recipient cell,

Along those same lines, "host cells" refers to cells that have been transformed with vectors constructed using recombinant DNA techniques and encoding at least one

heterologous gene. In descriptions of processes for isolation of polypeptides from

recombinant hosts, the terms "cell" and "cell culture" are used interchangeably to denote the source of antibody unless it is clearly specified otherwise. In other words, recovery of polypeptide from the "cells" may mean either from spun down whole cells, or from the cell culture containing both the medium and the suspended cells.

In one embodiment, the host cell line used for antibody expression is of mammalian origin; those skilled in the art can determine particular host cell lines which are best suited for the desired gene product to be expressed therein. Exemplary host cell lines include, but are not limited to, DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR minus), HELA (human cervical carcinoma), CVI (monkey kidney line), COS (a derivative of CVI with SV40 T antigen), R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line), SP2/0 (mouse myeloma), BFA-lclRPT (bovine endothelial cells), RAJI (human lymphocyte), 293 (human kidney). In one embodiment, the cell line provides for altered glycosyiation, e.g., afucosylation, of the antibody expressed therefrom (e.g., PER.C6.RTM. (Crucell) or FUT8 -knock- out CHO cell lines (Potelligent.RTM. Cells) (Biowa, Princeton, N.J.)). In one embodiment NSO cells may be used. CHO cells are particularly preferred. Host cell lines are typically available from commercial services, the American Tissue Culture Collection or from published literature,

In vitro production allows scale-up to give large amounts of the desired polypeptides. Techniques for mammalian cell cultivation under tissue culture conditions are known in the art and include homogeneous suspension culture, e.g. in an airlift reactor or in a continuous stirrer reactor, or immobilized or entrapped cell culture, e.g. in hollow fibers, microcapsules, on agarose microbeads or ceramic cartridges. If necessary and/or desired, the solutions of polypeptides can be purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, chromatography over DEAE-celiuIose and/or (immuno-)affinity chromatography.

Genes encoding the binding molecules, or fragments thereof, of the invention can also be expressed non-mammalian cells such as bacteria or yeast or plant cells. In this regard it will be appreciated that various unicellular non-mammalian microorganisms such as bacteria can also be transformed: i.e. those capable of being grown in cultures or fermentation.

Bacteria, which are susceptible to transformation, include members of the enterobacteriaceae, such as strains of Escherichia co!i or Salmonella; Bacillaceae, such as Bacillus subtilis;

Pneumococcus; Streptococcus, and Haemophilus influenzae. It will further be appreciated that, when expressed in bacteria, the polypeptides can become part of inclusion bodies. The polypeptides must be isolated, purified and then assembled into functional molecules. In addition to prokaryotes, eukaryotic microbes may also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among eukaryotic microorganisms although a number of other strains are commonly available. For expression in Saccharomyces, the plasmid YRp7, for example, (Stinchcomb et ah, Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)) is commonly used. This plasmid already contains the TRP1 gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4--1 (Jones, Genetics, 85:12 (1977)). The presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

VI, Pharmaceutical Formulations and Methods of Administration of Binding Molecules.

In another aspect, the invention provides pharmaceutical compositions comprising a binding molecule (e.g., an antibody, or antigen binding fragment thereof).

Methods for preparing and administering binding molecules of the invention to a subject are well known to or are readily determined by those skilled in the art. The route of administration of the antibodies, or fragments thereof, of the invention may he oral, parenteral, by inhalation or topical. The term parenteral as used herein includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. The intravenous, intraarterial, subcutaneous and intramuscular forms of parenteral administration are generally preferred. While all these forms of admini stration are clearly contemplated as being within the scope of the invention, an exemplary form for

admi istration would be a solution for injection, in particular for intravenous or intraarterial injection or drip. Usually, a suitable pharmaceutical composition for injection may comprise a buffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), optionally a stabilizer agent (e.g. human albumin), etc. In other methods compatible with the teachings herein, the polypeptides can be delivered directly to the site of the adverse cellular population thereby increasing the exposure of the diseased tissue to the therapeutic agent. For example, the high thermal stability and solubility properties of the binding molecules of invention make them ideal agents for local administration, e.g., via subcutaneous (Sub-Q) injections. In addition, the extremely high affinity and potency of the antibodies of the invention allow the use of a lower effective dose, thereby simplifying subcutaneous injection. Accordingly , the binding molecules are particularly well-suited for the treatment or prevention of inflammatory-related disorders (e.g., rheumatoid arthritis), cancers or associated symptoms (e.g., cachexia or anorexia) for which localized delivery of the binding molecule may be desirable.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. In the subject invention, pharmaceutically acceptable carriers include, but are not limited to, 0.01-0.1M and preferably 0.05M phosphate buffer or 0.8% saline. Other common parenteral vehicles include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishes, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present such as for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. More particularly, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the

extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and will preferably be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens,

chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

In any case, sterile injectable solutions can be prepared by incorporating an active compound (e.g., an antibody by itself or in combination with other active agents) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization, Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze- drying, which yields a powder of an active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparations for injections are processed, filled into containers such as ampoules, bags, bottles, syringes or vials, and sealed under aseptic conditions according to methods known in the art. Further, the preparations may be packaged and sold in the form of a kit such as those described in co-pending U.S. Ser. No. 09/259,337 and U.S. Ser. No, 09/259,338 each of which is incorporated herein by reference. Such articles of manufacture will preferably have labels or package inserts indicating that the associated compositions are useful for treating a subject suffering from, or predisposed to autoimmune or neoplastic disorders.

The binding molecules of the invention can be formulated to a wide range of concentrations for pharmaceutical use. For example, the binding molecule may be formulated to a concentration of between 5 mg/ml and 50 mg/ml (e.g., 5, 10, 20, 50mg/ml), Alternatively, the binding molecules of the invention may be adapted to higher concentration formulations for local (e.g., subcutaneous) administration, For example, the binding molecule may be formulated to a concentration of between 50 mg/ml and 200 mg/ml, e.g, about 50, about 75, about 100, about 150, about 175 or about 200 mg/ml),

Effective doses of the binding molecules of the present invention, for the treatment of the above described conditions, vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human, but non-human mammals including transgenic mammals can also be treated. Treatment dosages may be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.

For passive immunization with an antibody of the invention, the dosage may range, e.g., from about 0.0001 to 100 mg kg, and more usually 0,01 to 5 mg/kg (e.g., 0.02 mg kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, etc.), of the host body weight. For example dosages can be 1 mg kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg, preferably at least 1 mg/kg. Doses intermediate in the above ranges are also intended to be within the scope of the invention.

Subjects can be administered such doses daily, on alternative days, weekly or according to any other schedule determined by empirical analysis. An exemplary treatment entails admin stration in multiple dosages over a prolonged period, for example, of at least six months. Additional exemplary treatment regimes entail administration once per every two weeks or once a month or once every 3 to 6 months, Exemplary dosage schedules include 1- 10 mg kg or 15 mg/kg on consecutive days, 30 mg/kg on alternate days or 60 mg/kg weekly. In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered may fall within the ranges indicated.

Binding molecules of the invention can be administered on multiple occasions.

Intervals between single dosages can be, e.g., daily, weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of polypeptide or target molecule in the patient. In some methods, dosage is adjusted to achieve a certain plasma antibody or toxin concentration, e.g., 1-1000 ig/ml or 25-300 _, ug/ml. Alternatively, antibodies, or fragments thereof, can he administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient. In general, germlined or humanized antibodies show the longest half- life, followed by chimeric antibodies and nonbuman antibodies. In one embodiment, the antibodies, or fragments thereof, of the invention can be administered in unconjugated form. In another embodiment, the antibodies of the invention can be administered multiple times in conjugated form. In still another embodiment, the antibodies, or fragments thereof, of the invention can be administered in unconjugated form, then in conjugated form, or vise versa.

The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, compositions containing the present antibodies or a cocktail thereof are administered to a patient not already in the disease state to enhance the patient's resistance. Such an amount is defined to be a "prophylactic effective dose." In this use, the precise amounts again depend upon the patient's state of health and general immunity, but generally range from 0.1 to 25 mg per dose, especially 0,5 to 2.5 mg per dose, A relatively low dosage is administered at relatively infrequent intervals over a Song period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage (e.g., from about 1 to 400 mg kg of antibody per dose, with dosages of from 5 to 25 mg being more commonly used for radioimmunoconjugates and higher doses for cytotoxin-drug conjugated molecules) at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patent can he administered a prophylactic regime.

In one embodiment, a subject can be treated with a nucleic acid molecule encoding a polypeptide of the invention (e.g., in a vector). Doses for nucleic acids encoding polypeptides range from about 10 ng to 1 g, 100 ng to 100 mg, 1 g to 10 mg, or 30-300 _t ug DNA per patient. Doses for infectious viral vectors vary from 10-100, or more, virions per dose.

Therapeutic agents can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal or intramuscular means for prophylactic and/or therapeutic treatment. Intramuscular injection or intravenous infusion are preferred for administration of an antibody of the invention. In some methods, therapeutic antibodies, or fragments thereof, are injected directly into the cranium. In some methods, antibodies, or fragments thereof, are administered as a sustained release composition or

TV ¹

device, such as a Medipad ^{1 Λ} device.

Agents of the invention can optionally be administered in combination with other agents that are effective in treating the disorder or condition in need of treatment (e.g., prophylactic or therapeutic). Preferred additional agents are those which are art recognized and are standardly administered for a particular disorder.

Effective single treatment dosages (i.e., therapeutically effective amounts) of ⁹⁰ Y- labeled antibodies of the invention range from between about 5 and about 75 mCi, more preferably between about 10 and about 40 mCi. Effective single treatment non-marrow ablative dosages of ^L,l I-iabeled antibodies range from between about 5 and about 70 mCi, more preferably between about 5 and about 40 mCi. Effective single treatment ablative dosages (i.e., may require autologous bone marrow transplantation) of ^ljl I-labeled antibodies range from between about 30 and about 600 mCi, more preferably between about 50 and less than about 500 mCi.

While a great deal of clinical experience has been gained with ^m I and ⁹⁰ Y, other radiolabels are known in the art and have been used for similar purposes. Still other radioisotopes are used for imaging. For example, additional radioisotopes which are compatible with the scope of the instant invention include, but are not limited to, ¹ I, "" ^" Ί, ³² P, ⁵⁷ Co, ¾, ⁶⁷ Cu, ⁷⁷ Br, ^8l Rb, ^8f Kr, ⁸⁷ Sr, ^{1 ϊ 3} ίη, ¹²⁷ Cs, ¹²⁹ Cs, ⁱ³² I, ¹⁹⁷ Hg, ²⁰³ Pb, ²⁰⁶ Bi, ^!7? Lu, ¹⁸⁶ Re, ^2¾2 Pb, ^2¾2 Bi, ⁴⁷ Sc, ¹⁰⁵ Rh, ' "'hi. ¹⁵³ Sm, ^¾88 Re, ¹⁹⁹ Au, ²²⁵ Ac, ^{21 1} A ²ⁱ³ Bi, In this respect alpha, gamma and beta emitters are all compatible with the instant invention. Further, in view of the instant disclosure it is submitted that one skilled in the art could readily determine which radionuclides are compatible with a selected course of treatment without undue experimentation. To this end, additional radionuclides which have already been used in clinical diagnosis include ¹²⁵ I, ^12j I, 99Tc, ^",3 K, ⁵² Fe, ⁶⁷ Ga, ⁶⁸ Ga, as well as ^{Xl l} ln. Antibodies have also been labeled with a variety of radionuclides for potential use in targeted

immunotherapy (Peirersz et al. Immunol. Cell Biol. 65: 111-125 (1987)). These radionuclides include ¹⁸ Re and ⁱ⁸⁶ Re as well as ¹⁹⁹ Au and ^6/ Cu to a lesser extent. U.S. Pat. No. 5,460,785 provides additional data regarding such radioisotopes and is incorporated herein by reference.

As previously discussed, the binding molecules of the invention can be administered in a pharmaceutically effective amount for the in vivo treatment of mammalian disorders. In this regard, it will be appreciated that the disclosed antibodies, or fragments thereof, will be formulated so as to facilitate administration and promote stability of the active agent.

Preferably, pharmaceutical compositions in accordance with the present invention comprise a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic butlers, preservatives and the like. For the purposes of the instant application, a

pharmaceutically effective amount of a antibody of the invention, conjugated or unconjugated to a therapeutic agent, shall be held to mean an amount sufficient to achieve effective binding to a target and to achieve a benefit, e.g., to ameliorate symptoms of a disease or disorder or to detect a substance or a cell. In the case of tumor cells, the polypeptide will be preferably be capable of interacting with selected immunoreactive antigens on neoplastic or

immunoreactive cells and provide for an increase in the death of those cells. Of course, the pharmaceutical compositions of the present invention may be administered in single or multiple doses to provide for a pharmaceutically effective amount of the polypeptide.

In keeping with the scope of the present disclosure, the binding molecules of the invention may be administered to a human or other animal in accordance with the

aforementioned methods of treatment in an amount sufficient to produce a therapeutic or prophylactic effect. The polypeptides of the invention can be administered to such human or other animal in a conventional dosage form prepared by combining the antibody of the invention with a conventional pharmaceutically acceptable carrier or diluent according to known techniques. It will be recognized by one of skill in the art that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. Those skilled in the art will further appreciate that a cocktai l comprising one or more species of polypeptides according to the present invention may prove to be particularly effective.

VII. Methods of Treating IL-6- Associated Disease or Disorders

The binding molecules of the invention are useful for antagonizing IL-6 activity. Accordingly, in another aspect, the invention provides methods for treating IL-6-associated. diseases or disorders by administering to a subject in need of thereof a pharmaceutical composition comprising one or more binding molecules of the invention.

IL-6-associated diseases or disorders amenable to treatment include, without limitation, inflammatory diseases and cancer.

One skilled in the art would be able, by routine experimentation, to determine what an effective, non-toxic amount of antibody (or additional therapeutic agent) would be for the purpose of treating an IL-6-associated disease or disorder. For example, a therapeutically active amount of a polypeptide may vary according to factors such as the disease stage (e.g., stage I versus stage IV), age, sex, medical complications (e.g., immunosuppressed conditions or diseases) and weight of the subject, and the ability of the antibody to elicit a desired response in the subject. The dosage regimen may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. Generally, however, an effective dosage is expected to be in the range of about 0.05 to 100 milligrams per kilogram body weight per day and more preferably from about 0.5 to 10, milligrams per kilogram body weight per day.

VO L Exemplification

Example 1 Generation and Selection of IL~6-Specific Antagonistic Fabs

Llamas were immunized with human IL-6 (either produced in E.coli purchased from MACS Miltenyi Biotec (Cat. No.130-093-934) or produced in Human Embryonic kidney cells purchased from Humanzyme (Cat. No.HZ-1044)). Immunization of llamas and harvesting of peripheral blood lymphocytes (PBLs), as well as the subsequent extraction of RNA and amplificat on of antibody fragments, were performed as described by De ilaard and colleagues (De Haard H, et al., JBC. 274:18218-30, 1999). After the last immunization, blood was collected and total RNA extracted from PBLs prepared using a Fieoll-Paque gradient and the method described by Chomczynski P et al. (Anal. Biochem. 162: 156- 159, 1987). The extracted RNA was then used for random cDNA synthesis and PGR.

amplification of the V-regions of the heavy and the light chains (Υλ and VK) in order to construct Fab-containing phagemid libraries as described by De Haard H, et al. (Biol. Chem. 274, 1999).

Phage expressing Fabs were produced according to standard protocols and further selected on immobilized human IL-6 either biotinylated and captured by neutravidine or directly coated on maxisorp plates. Total or competitive elution of the IL-6 binding phage with trypsin was performed according to standard phage display protocols.

IL-6-specific Fabs were next screened for cross-competition with the IL-6

neutralizing antibody, B-E8 and IL-6 receptor using an ELISA-based competition assay. The VH and VL amino acid sequences of exemplary antagonistic IL-6-specific Fabs identified using this assay are set forth in Table 13 below.

The binding kinetics of IL-6 -specific Fabs that were able to cross-compete with the B- E8 antibody was assessed using surface plasraon resonance (Biacore). Specifically, biotinylated (prokaryotie) IL-6 was captured on a streptavidin biacore sensor chip (CM5-SA) and different concentrations of purified Fabs were injected during 3 minutes following by a 5 minute wash with buffer. From the washing phase the οίϊ-rate (kd) was determined, while from the injection phase the on-rate (ka) was calculated using the concentration and off-rate as parameters. The measured off-rates and on-rates and calculated affinities of antagonistic Fabs are shown in Table 2. Fabs 24C9 and 24D10 have off-rates in the 10 ^"5 s ^"1 range and have affinities of 660 and 270 M, respectively . The binding kinetics of antagonistic purified Fabs (Table 3) and periplasmic fractions of Fab-expressing bacteria (Table 4) were also evaluated by surface plasraon resonance (Biacore) using both bacterially and

eukaryotically produced human IL-6 directly coated on a CMS chip. The Fabs tested had off- rates between 6x10 and 2x1ο ⁵ s ^"1 . Table 2 Binding Kinetics of Selected Purified Antagonistic Fab Clones

Fab ka kd Rmax KA KD

(1/s) (RU) (1 M) Chi2

17B3 3.02E+04 2.92E-03 35.7 1.04E+07 9.65E-08 2.67

18C7 L05E+05 1.85E-03 43.9 5.70E+07 1.75F.-08 6.98

18F8 4.84E+04 7.84E-04 51.5 6.18E+07 1.62E-08 8.43

18C9 5.56E+04 1.19E-03 49.9 4.67E+07 2.14E-08 9.51

18C11 5.52E+04 7.84E-04 22.1 7.05E+07 1.42E-08 0.2

28C6 1.01E+05 2.50E-03 22.6 4.04E+07 2.48E-08 1.2

20A4 2.88E+05 1.68E-03 48.2 1.72E+08 5.82F.-09 2.56

29B11 1.30E+05 2.98E-03 23.3 4.36E+07 2.29E-08 1.02

24C9 1.33E+05 8.77E-05 38.1 1.51E+09 6.61E-10 2.21

24D10 6.27E+04 1.69E-05 40.6 3.70E+09 2.70E-10 3.48

24E9 1.Γ7Ε+05 1.39E-04 41 8.38E+08 1.19E-09 3.87

Table 3 Binding Kinetics of Selected Purified Antagonistic Fab Clones

Table 4 Binding Kinetics of Fab-Containing Periplasmic Fractions

ratio average neg

Example 2. VH /VL Shuffling for Improved Affinity,

VL chain shuffling was used to improve the affinity of the Fabs 17F10, 18C7, 18C9, 18C11, 20A4, 29B11, 16D2 and 28A6. In this method, the heavy chain of these clones (as a VHCH.1 fragment) was reintroduced into the primary phagemid-Iight chain library (see Example 1). Affinity selections were perfomied to select for chain shuffled Fabs with an improved affinity for IL-6. The binding kinetics of chain shuffled Fabs were evaluated by surface plasmon resonance (Biacore) using both bacterially and eukaryotically produced human IL-6, and cynomolgus monkey IL-6 (Tables 5-7). The VH and VL amino acid sequences of exemplary IL-6-specific Fabs selected by the VL chain shuffling method are set forth in Table 14 below.

VH chain shuffling was also used to improve the affinity of the Fab 24D10. In this method, the light chain of 24D10 was reintroduced into the primary phagemid-heavy chain library (see Example 1 ) and selected using an off-rate assay. In this type of selection, the phage were allowed to bind to antigen on a substrate for 1.5 to 2 hours. At round 2, after 15 washes with PBS-Tween, an additional wash was performed with the presence of excess soluble IL-6. The principle is that phage antibodies with inferior off-rates and therefore dissociating more rapidly are captured by the excess of soluble target and are removed during washing. This procedure avoids re- binding of such phage to the coated target. The time of the additional wash step was increased with the number of rounds performed and the temperature was also increased to 37 °C, to select for more stable Fab variants. The binding kinetics of chain shuffled Fabs and the benchmark antibodies, BE8 and GL18, were evaluated by surface plasmon resonance (Biacore) using both bacterially and eukaryotically produced human IL-6 (Tables 8 and 9). The VH and VL amino acid sequences of exemplary IL-6- specific Fabs selected by the VH chain shuffling method are set forth in Table 14 below.

Table 5 Binding Kinetics of Purifed 17F10, 18C11, 18C7 and 20A4 Chain- Shu filed Fabs

Table 6 Binding Kinetics of Periplasmic Fractions Containing 29 Bl 1 Chain Shuffled Fabs

29B11 ka ( i ./Ms) kd (1/s) KA i l/M) KD (M) Chi2

bact X5IE+05 1.31.E-04 1. 1 E+09 S.24E-1.0 35

euk L 87E+05 1. 2E- 4 ) .83E+09 S.46E-10 48.4

c 1. I3E+05 7.46E-04 1.51E+08 6.61E-09 59.5 FOLD IMPROVEMENT

60 55Cla ka (1/Ms) kti (Us) KA ( 1 /M) KD (M) Chi2 ka (1 /Ms) kd (Us) KD (M) baci 5.6Ϊ Ε+05 7.38E-05 7.60E+09 1..32E-10 21 ? 2" 1.78 3.97 euJ 4.40E+05 7.93E-05 5.55E+09 1.80E-1.0 37.7 2.35 1 .29 3.03 c no .75E+05 3.81E-04 1.25E+09 8.01E-10 32.4 4.20 1.96 8.25

55Ε21» ka (1/Ms) kd (Us) KA ( 1/M) KD (M) Chi2 ka (1/Ms) kd (Us) KD (Mi baci 4.89E+05 6.72E-0S 7.28E+09 1.37E-10 ^■ 5 ,6 1.95 1.95 3,82 euk 4. Ϊ4Ε+05 6.53E-05 6.34E+09 1.58E-10 29.2 2.21 1 .56 3.46 cy 4.6SE+05 3.36E-04 1 .39E+09 7.17E-10 24.1 4.14 2.22 9.22

55Hlc ka ( I Ms) kd (Us) KA ( 1/M) KD (M) Chi2 ka (1/Ms) kd 1 !/··,! KD (M) baci 1.28E+06 1.27E-04 l .OOE+10 9.97E-U 17.2 5.10 1 .03 5.26 euk 1.13E+06 1.11E-04 1.02E+10 9.77E-11 1 1 .7 6.04 0.92 5.59 cy I.16E+06 6.15E-04 1.89E+09 S.29E-10 29,9 10.27 1.21 12.50

47C2

ka (1/Ms) kd (Us) KA ( 1/M) KD (M) Chi2 ka (1/Ms) kd (Us) KD (Mi (R4)

baci 1.12E+06 3.1SE-04 3.56E+09 2.8IE-10 18.3 4.46 0,42 1.86 euk 8.49E+05 2.40E-04 3.54E+09 2.82E-10 ^■ 6,6 4.54 0.43 1 ,94 cy 8.5 IE+05 1.31E-03 6.48E+08 1.54E-09 18.7 -T5 0.57 4.29

48C10 ka (1/Ms) kd (Us) KA ( i /M) KD (M) Chi2 ka i l /Ms ) kd (Us) KD (M) baci 2.62E+05 1.S4E- 4 i .43E+09 7.01E-10 29 1.04 0.71 0.75 euk bad fit...

cy 1.80E+05 4.4 Ε-Θ4 4.10E+08 2.44E-09 5.1 1 1.59 1.70 2.71

le 7 Binding Kinetics of Periplasmic Fractions Containing 28A6 Chain-Shuffled Fabs

28A6

ka (1/Ms) kd (Us) KA (1/M) KB (M) C i2

(parental)

bad 2.38E+05 1.78E-04 1.34E+09 7.49E-10 40,7

euk 1.82E+05 1.05E-04 1.73E+09 5.80E-10 63.1

cyno 1.28E+05 6.73E-04 1.91E+08 5.25E-09 74.5 FOLD IMPROVEMENT

SSAild ka (1 Ms) kd (Us) KA (1/M) KD (M) Chi2 ka (1/Ms) kd (Us) KB <M)

BACT 1.S8E+05 6.76E-0S 2.78E+09 3.59E-10 14,5 0.79 2,63 2.09 euk 1 .0J.E+05 6.36E-05 1.59E+09 6.31E-10 9.18 0.55 1.65 0 92 cyno 1.44E+05 8.17E- 5 I .7 E+09 5.66E-10 5,69 1. 13 8.24 9.28

55C10e ka (l /Ms ) kd a/s) KA (1/M) KD (M) C i2 ka i l /Ms ) kd (Us) KD (M) bact 1 .7 IE 05 6.99E-05 2.44E+09 4.09E-10 7.83 0.72 2 55 1.83 euk 1.39E+05 6.14E-0S 2.26EH-09 4.43E-10 15.2 0.76 1.71 1.31 cy 1.36E÷05 8.67E-0S 1.56E+09 6.39E-10 6.97 1.06 7,76 8.22

55Cllf ka (1/Ms) kd (l/s) KA (1/M) KD (M) C i2 ka (1/Ms) kd (Us) KD (M) baci 2.28E4-05 6.94E-05 3.29E+09 3.04E-10 6.56 0.96 2.56 2.46 euk 2.01E+05 6.47E-05 3. i0E 09 3.23E-10 13.4 1.10 1 .62 1.80 cy 2.85E4-05 8.90Ε-Θ5 3.20E4-09 3.13E-3.0 26.7 2.23 7.56 16.77

SSEl Ogl ka (1 Ms) kd (1/s) KA (1 M) Kl (M) Chi2 ka (1/Ms) kd (Us) KD (M) bact 1.93E+05 6.09Ε-Θ5 3.18E+09 3.ISE-10 10.4 0.81 2.92 2.38 euk 1.46E+05 5.59E-0S 2.62E409 3.82E-10 8.45 0.80 1 ,88 1.52 cyno 1 45E4-05 6.88E-05 2. 10E+09 4.76E-10 9.6 1.13 9.78 1 1 .03

55Ellg3 ka ( i/Ms) kd (l/s) KA (1 /M) KD (M) Chi2 ka (1/Ms) kd (Us) KD (M) bact 1.68E+05 7.05E-05 2.38E 09 4.21E-10 4.98 0.71 2.52 1.78 euk 1.38E 05 S.1.4E-05 2.68E4-09 3.73E-10 8.62 0.76 2.04 1.55 cy 1.39E+05 7.46E-0S 1.86E+09 5.38E-10 7.34 1.09 9.02 9.76

48Hlg2,R4 ka (1/Ms) kd (Us) KA (1/M) KD (M) Chi2 ka (1/Ms) kd (Us) KB <M) ί bad 2.06B+O5 7. 4Ε-Θ5 2.93E+09 3.41E-1 8.93 0.87 2.53 2.20

I euk 1.06E+05 6.S E- 5 1.56E+09 6.40E-1 19.9 0.58 J .54 0.91

! cy 1 .J 2E+05 9.46E-05 1.18E+09 8.45E-10 15.7 0.88 7.11 6.21

Binding Kinetics of Pexiplasmic Fractions Containing 24D10 Chain- Shuffled

Fabs

RANKED Average

euk binding

62B6 9.07E-05 5.58E-05 7.49E-05 214

62c6 1.20E-04 6.1 1E-05 1.05E-04 150

62f6 1.13E-04 7.19E-05 l .OlE-04 154

CAT- 9.41E-05 7.30E-05 1.20E-04 547

62g6 1.78E-04 7.67E-05 8.58E-05 132

62f5 1.63E-04 7.96E-05 1.15E-04 139

62h5 1.57Ε- 4 8.28E-05 1.28Ε- 4 139

BE8-Fab 9.70Ε- 5 8.39E-05 1.18E-G4 338

62h6 1.53E-04 8.46E-05 9.76E--G5 196

BE8-Fab 1.03E-04 8.72E-05 1.28Ε- 4 340

BB8-Fah 9.29E--G5 8.96E-05 Ι .04Ε- 4 347

61h7 7.54Ε- 5 9.32E-05 9.45Ε- 5 635

CAT- 9.98E-05 9.33E-05 1.40E-04 486

GLlSFab

62d6 1.30E-04 9.53E-05 1.02E-04 134

62g5 1.68E-04 9.78E-05 1.31E-04 150

61B7 9.07E-05 9.79E-05 1.16E-04 597

55Hlc 1.35E-04 l .OlE-04 4.17E-04 773

62A6 1.62E-04 l .OlE-04 1.39E-04 138

62A5 1.48E-04 1.05E-04 1.29E-04 138

62X3 1.75E-04 1.05E-04 1.16E-04 109

55Hlc 1.40E-04 1.06E-04 4.30E-04 770

62c5 9.95E-05 1.09E-04 1.17E-04 131

61g7 1.19E-04 l . lOE-04 3.18E-04 139

62B5 1.30E-04 l . l lE-04 1.46E-04 170

6tf6 1.12F,04 1.13E-04 1.25E-04 606

55H l c 1.26E-04 1.13E-04 3.90E-04 873

61g3 1.39E-04 1.15E-04 5.28E-04 687

6IA3 1.34E-04 1.18E-04 5.19E-04 694

62d5 1.63E-04 1.18E-04 1.47E-04 129

62c2 1.37E-04 1.20E-04 5.44E-04 619

62g l 1.38E-04 1.21E-04 5.38E-04 629

61el 1.39E-04 1.21E-04 5.36E-04 690

61 B4 1.55E-04 1.21E-04 5.68E-04 561

62h ! 1.41E-04 1.24E-04 5.40E-04 647

61g ! 2 1.42E-04 1.27E-04 4.30E-04 138

62g4 1.51E-04 1.27E-04 5.51E-04 605

61g ! 0 1.45E-04 1.30E-04 3.99E-04 159

61h9 1. 1E-04 1.31E-04 4.12E-04 152

62e5 1.48E-04 1.32E-04 1.88E-04 148

62 A 1 1.73E-04 1.32E-04 6.41E-04 597

61g5 1.30E-04 1.39E-04 3.69E-04 229

61cl2 1.43E-04 1.39E-04 4.44E-04 267 Table 9 Binding Kinetics of Purified 24D10 Chain-Shuffled Fabs

Example 3 Crystal Structure of Fab 61H7 in complex with IL-6

i) IL-6/Fah 61H7 Complex Characterization

Size exclusion chromatography was performed on an Alliance 2695 HPLC system (Waters) using a Silica Gel KW803 column (Shodex) eluted with 50 mM Tris-HCl pH 7.5, 150 mM NaCI at a flow rate of 0.5 ml/rnin. Detection was performed using a triple-angle light scattering detector (Mini- DAWN™ TREOS, Wyatt technology, Sanata Barbara, USA). Molecular weight determination was performed by ASTRA V software (Wyatt technology). ii) Crystallization

Initial crystallizat on screening of the IL-6/Fab 61117 complex was performed with commercial kits Structure screen 1 and 2, Proplex screen and Stura Footprint screen

(Molecular Dimensions Ltd). Drops were set-up with a l :l(v:v) ratio of protein (8.45 rag/ml) to mother liquor in a total volume of 200 nl on Greiner 96- well plates using a Cartesian MicroSys SQ robot. Diffraction-quality crystals of complex were obtained by sitting-drop vapor diffusion at 277 K after optimization in 27.14% PEG MME 2K, 0.1M Na Flepes pEI 7.14. Crystals belong to the C2 space group with unit cell dimensions: a- 108.2 A, b- 47.5

A, and c=148.3 A and β=97°. They contain on ILF/Fab complex per asymmetric unit with a

3

Vm value of 2.36 A /Da which correspond to a solvent content of 48%. Hi) Analysis of the Structure of the IL-6:61H7 Complex The canonical structures predicted for the CDR loops of 611:17 mAb are 1 and 3 for HI and H2, respectively, and 7, 1, and 4 for LI, L2, and L3, respectively

(www.bioinf.org.uk/abs/chothia.html). The overlay of the 61.H7 VH with Protein Data Bank (PDB) entiy ldfb (derived from a patient derived antibody against the HIV- l protein gp41) shows that HI and 1 12 adopt the predicted canonical folds (see Figure 3A). The overlay of the 61H7 VL with Protein Data Bank (PDB) entiy Imfa demonstrates that all three light chain CDRs adopt, the predicted conformations (see Figure 3B). Accordingly, structural analysis confirms that the VH (a VH3 family member) of 61H7 belongs to the human 1-3 combination of canonical H1-H2 structures, while the VL (a VL8 family member) of 61H7 belongs to the human 7λ1-4 combination of human canonical structures.

IL-6 was previously crystallized and classified as a four helix bundle linked by loops and an additional mini-helix (Somers el ah, 1997, EMBO journal 16, 989-997, which is incorporated herein by reference in its entirety). Superposition of the apo IL-6 (pdb 1ALU) with the IL-6 from the IL-6:61 H7 complex shows good agreement between both models (rms

0.54 A). This confirms that the 61H7 Fab recognizes and binds the native conformation of IL-6.

The crystal structure of the IL-6 in complex with its receptor and the signalling receptor gpl30 showed a hexameric complex (Boulanger et al., 2003, Science 27, 2101-2104, which is incorporated herein by reference in its entirety). IL-6 forms a non- signalling complex with IL-6R through site L Site II is a composite epitope formed by the binary complex of IL-6 and IL-6R. Interaction of site III with gpl30 forms the signalling complex.

Superposition of the IL-6:IL-6R structure (pdb 1P9M) with the IL-6:61H7 structure shows good agreement (rms 1.2 A) between both I L-6 structures. Two loops differ in conformation. The first loop covering residues Asn48-Asn61, is a long loop that is unstructured in the apo IL-6 and IL-6:61 H7 complex. This loop is stabilized in the IL-6:IL-6R structure by the binding of the IL-6R. The second loop that differs in conformation is the so called BC loop.

The crystallographic asymmetric unit contains a single 1 : 1 complex. The global architecture of the IL-6:61 H7 complex shows that both the VH (60%) and the VL (40%) contribute to the large interaction surface (940 A ² ). From the crystal structure the residues important, for the interaction with IL-6 were determined, The hydrogen bonds and salt bridges formed between the 61117 Fab and the cytokine are listed in Table 10. The interactions are limited to the CDRl and CD 3 of the light chain and the CDR1, CDR2 and CDR3 of the heavy chain.

Table 10. Hydrogen bonds and salt bridges I the 61H7:lL-6 complex

Overlay of the IL-6:61H7 complex and the IL-6:IL-6R complex shows that there is sterical hindrance between the 61 H7 Fab and the IL-6R. It is mainly the VL that gives a sterical clash with the IL-6R. Epitopes of IL-6R and 61 H7 will be very close to each other. To verify if there is overlap between both epitopes, residues within 4.0 A of the IL-6R and residues within 4.0 A of the 61 H7 Fab were mapped and searched for overlap between both epitopes. The overlap between both epitopes is rather small and is mainly formed by the VH paratope. The overlap concentrates around a cavity occupied by both the HCDR3 loop of the 61H7 and the IL-6R molecule. This binding site of IL-6R on IL-6 has been referred to as site I (Boulanger et ah, 2003, Science 27, 2101-2104, which is incorporated herein by reference in its entirety), The cavity forming site I is occupied by the hydrophobic side chain of Phe 229 of IL-6 . This amino acid is called the hotspot residue by Boulanger et ah because mutagenesis studies have shown its critical role in the interaction between the receptor and the cytokine. Mutation of this residue to valine or serine completely abolished the IL-6R binding to IL-6 (Kalai et aL, 1997, Blood, 1319-1333, which are both incorporated herein by reference in their entirety), Trp98 in the center of the heavy chain CDR3 loop of Fab 61H7 occupies the same cavity, suggesting that it hits thus the critical epitope in IL-6 to block its interaction with IL-6R (shown in Figure 5). Trp98 is likely a key residue for the ultra high affinity of Fab61H7 for IL-6.

Example 4 Crystal Structures of Fabs 68F2 and 1291)3 in complex with IL-6 i) Generation, Data Collection and Structure determination of the IL-6:68F2 Crystal

8 mg of 68F2 mAb (4 mg/ml) in Dulbecco's Phosphate buffered saline (d-PBS) pH 7.2 were buffer-exchanged to digestion buffer containing 20mM evstein-HCi on a Zeba TM Desalt Spin Column (Pierce Fab Preparation Kit Thermo Scientific), Sample was incubated with Immobilized Papain (Pierce Thermo Scientific) and digested for 6 hours at 37°C. The Fc fragments were separated from the Fab fragments using a CaptureSelect human Fc affinity- matrix (BAC BV Unilever) equilibrated in d-PBS. Fab fragments were recovered in the flow- through and Fc fragments were eluted using 0.1M glycine pH 2,0. Protein concentration was determined by UV spectrometry from the absorbance at 280 nm, 4.6 mg (>50%) of purified Fab 68F2 was recovered and concentrated to 1.53 mg/ml on an Amicon-Ultra (cut-off 10 kDa).

2.5 mg of rh IL-6 (Immunotools) was incubated with 2,6 mg of Fab 68F2 in

Dulbecco's Phosphate buffered saline (d-PBS) pH 7.2 for 1 hour at 4°C before being concentrate to 1 ml on a Amicon-Ultra (cut-off lOkDa). The IL-6:68F2 complex was then separated from excess free IL-6 by gel filtration chromatography on a Superdex75 column in d-PBS and finally concentrated to 8.1 mg/ml on an Amicon-Ultra concentrator (cut-off 10 kDa). Purification of the complex was evaluated on SDS-PAGE.

Size exclusion chromatography was performed on an Alliance 2695 HPLC system (Waters) using a Silica Gel KW803 column (Shodex) eluted with 50 mM Tris-HCl pH 7.5, 150 mM NaCi at a flow rate of 0.5 ml/min. Detection was performed using a triple-angle light scattering detector (Mini-DAWN ⁱ TREOS, Wyatt technology, Santa Barbara, USA). Molecular weight determination was performed by ASTRA V software (Wyatt technology). Initial crystallization screening of the IL-6:68F2 complex was performed with commercial kits Structure screen 1 and 2, Proplex screen and Stura Footprint screen (Molecular

Dimensions Ltd). Drops were set-up with a 1 : 1 (v/v) ratio of protein (8.1 mg/ml) to mother liquor in a total volume of 200 nl on Greiner 96- well plates using a Cartesian MicroSys SQ robot. A diffraction -quality crystal of complex was obtained by sitting-drop vapor diffusion at 277 K after 9 months in 25% PEG 4K, 0.15M (NH ₄ ) ₂ S0 ₄ , 0.1M MES pH 5.5.

Crystals for data collection were transferred to liquor mother with 7.5% ethylene glycol and flash-frozen in liquid nitrogen. Diffraction data were collected under standard cryogenic conditions on beamline ID 14-4, using an ADSC Quantum 4 detector at the ESRF synchrotron (Grenoble, France), processed using XDS and scaled with XSCALE. The crystal structure of IL-6 in complex with Fab 68F2 was determined from single-wavelength native diffraction experiments by molecular replacement with Fab 129133 and the IL-6 structure using MOLREP (table 4). Refinement was performed with BUSTER. The IL-6:129D3 Crystal was similarly produced.

Hi) Analysis of the Structure of the IL~6 68F2 and IL-6 129D3 Complex

The crystal structure of the IL-6:68F2 complex has a resolution of 2.9 A. The model was refined to an R-factor of 26.7% and an R _frse -factor of 29.6% with reasonable

stereochemistry (meaning that more than 95% of the residues adopt allowed conformations). The crystal structure of the IL-6:129D3 complex has a resolution of 2.8 A. The model was refined to an R-factor of 28.5% and an R _free -factor of 31.3% with reasonable stereochemistry .

An overlay of the crystallized 68F2 and 129D3 Fabs (r.m.s.d. 1.4 A), VH domain (r.m.s.d. 0.5 A) and of the VL domain was made (r.m.s.d. 0.4). These superpositions show that there is no significant difference between the parental 68F2 and germlined 129D3 Fab structures,

The canonical structures predicted for the CDR loops of the 68F2 mAb and its germlined variant 129D3 are 3 and 1 for HI and H2, respectively, and 6λ, 1 and 5 for LI, L2 and L3, respectively. The canonical folds of the heavy chain were predicted by the server at www.bioinf.org.uk/abs/chothia.html, the canonical folds of the light chain were manually determined. The reference Fab (PG16) for comparison of the light chain canonical folds, was found manually by searching the Antibody Structure Summary Page (www.bioinf.org.uk abs/sacs/antibody_stracture_summary_page.htmlj. The overlay of the 68F2 and 129D3 VL with the VL of PG16 (Protein Data Bank (PDB) entry SMUG) demonstrates that all three CD 's adopt the predicted conformations (see Figure 4 A). The overlay of the 68F2/129D3 VH with the reference PDB entry 1ACY shows that HI and H2 adopt the predicted canonical folds (see Figure 4B). Accordingly, structural analysis confirms that the VL (a VL2 family member) of 68F2/129D3 belongs to the human 6λ-1-5 combination of canonical L1-L3 structures, while the VH (a VH4 family member) of 68F2/129D3 belongs to the 3-1 combination of human canonical H1-H2 structures.

The crystallographic asymmetric unit contains a single 1:1 complex. The global architecture of the IL-6:68F2 complex shows that the VH (50%) and the VL (50%) contribute equally to the large interaction surface (1156 A ² ). The interaction surface is slightly bigger than the interaction surface of the 61H7 with IL-6 (940 A ² ). Analogous to the 61H7:IL-6 complex, only the L2 loop is not directly involved in the interaction with IL-6.

The interface between the heavy and light chain corresponds to 1764.7 A ² for the

68F2 and 1768.5 A ² for the 129D3 structure. This interface area was comparable to the areas measured for the 61H7 (1580.6 A ² ) and the 27B3 (1700.3 A ² ) antibodies. All interface areas were calculated with i -.ΜΒί . web sendee program PISA.

From the crystal structure the residues important for the interaction with IL-6 were determined. The hydrogen bonds and salt bridges formed between the 68F2 Fab and the cytokine are listed in Table 11. The interactions are limited to the CDR1 and CDR3 of the light chain and the CDR1, CDR2 and CDR3 of the heavy chain.

The VL shuffling of the 20A4 clone resulted in the 68F2 clone which has a lOx better affinity than the parental 20A4. Both antibodies differ mainly in their LI and L2 CDRs. The light chain CDR2 loop does not contribute to the binding of IL-6, thus, the improvement in affinit is to be attributed to mutations in the LI loop. Two out of the three residues that make important interactions with IL-6 are not conserved in the 20A4 clone: Asn26 that makes a hydrogen bond with Ser76 of IL-6 is a Ser in 20A4; and Thr31 that makes two hydrogen bonds with IL-6 is a Gly in the 20A4 parental LI . The addition of three new hydrogen bonds when changing these two residues likely explains some gain in affinity observed after the VL shuffling. Most importantly, the changes in LI probably stabilize and/or position Y30 (Kabat numbering) properly in the F229 cavity allowing for extra high potency. Table 11. £ ydrogen bonds anc i salt bridges in the ί »8F2:IL-6 cc implex.

68F2 residue Ka at Distance IL-6 residue structure mimbering (A)

Hydrogen Light chain-CDRl ASN 26[0] ASN 27 3.60 SER 76[OG] bonds TYR 32[OH] TYR 30 3.43 SER 176[0]

THR 1 N] THR 29 3.55 SER 76[OG]

THR 1 0] THR 29 3.42 GLN 751 X 1

Light chain-CDR3 ASN 95[ND2] ASN 93 3.77 MET 67 [O]

ASN 97[0D1] ASN 95 3.73 ARG 179[NH2]

Heavy chain-CDRl TYR 33 [OH] TYR 33 3.47 ARG 30[O]

TYR 33! ί >i i | TYR 33 2.16 ASP 34[0D1]

ARG 32[NE] ARG 32 3.20 ASP 34[OD2]

ARG 32[NH2] ARG 32 3.28 ASP 34[OD2]

Heavy chaiii-CDR2 TYR 60[OH] TYR 58 2.59 GLU 172[OE2]

ASP 54[OD2] ASP 52 3.37 LYS 171 [NZ]

ASP 56[0D1] ASP 54 2.90 SER 37 [OG]

ASP 58[0D1] ASP 56 3.86 HIS !64[NE2]

ASP 58[OD2] ASP 56 3.42 LYS 171 [NZ ]

THR 59 [0] THR 57 2.52 ARG 168[NH2]

TYR 60[OH] TYR 58 3.69 ARG 168[NE ]

TYR 60[OH] TYR 58 3.15 LYS 17I [NZ ]

Heavy chain-CDR3 ASP 102[OD2] ASP 97 2.77 ARG 30[NH2]

VAL 104[O] VAL 99 3.69 ARG 179[NH1]

Salt bridges Heavy cbain-CDR ! ARG 32[NE] ARG 30 3.20 ASP 34[OD2]

ARG 32[NH2] ARG 30 3.28 ASP 34[OD2]

Heavy chain-CDR2 ASP 54[OD2] ASP 52 3.37 LYS 171 [NZ]

ASP 58[ODI] ASP 56 3.86 HIS 164ENE2]

ASP 58[OD2] ASP 56 3.42 LYS 171 [NZ]

Heavy chain-CDR3 ASP 102[OD1] ASP 97 3.40 ARG 30[NH2]

ASP 102[OD2] ASP 97 2.77 ARG 30[NH2]

Overlay of the IL-6:68F2 complex and the IL-6:IL-6R complex shows that there is steric hindrance between the 68F2 Fab and the IL-6R, as was observed for the 61H7:IL-6 complex. However, in contrast to the 61H7:IL-6 complex, it is mainly the VFI of 68F2 that gives a sterical clash with the IL-6R in the 68F2:IL-6 complex. 68F2 Fab interacts with IL-6 exactly at the same site as IL-6R. Furthermore, 68F2 does not overlap with the gpl30 binding sites and therefore competes specifically and only with the IL-6R.

Overlay of the IL-6:IL-6R and the IL-6:68F2 complexes suggests that the epitopes of IL-6R and 68F2 will be very close to each other. The residues belonging to both epitopes were mapped on IL-6 and the overlap determined. The overlap of the 68F2 epitope with the IL-6R is almost complete. The binding site of IL-6R on IL-6 has been called site I

(Boulanger et /., 2003, Science 27, 2101 -2104). The cavity forming site I is occupied by the hydrophobic side chain of Phe 229 of IL-6R. This amino acid is called the hotspot residue by Boulanger et al, since mutagenesis studies have shown its critical role in the interaction between the receptor and the cytokine. Mutation of this residue to valine or serine completely abolished the IL-6R binding to IL-6 (Kalai et ah, 1997, Blood, 1319-1333). Inspection of the cavity described as site I in the IL-6:68F2 structure reveals that is occupied by the CDRl loop of the light chain of Fab 68F2. In particular, Tyr32 (position 30 in Kabat numbering) in the CDRl of the light chain plays a crucial role in binding this site (Figure 5C).

Another key residue in the interaction between IL-6 and IL-6R is Phe279 of IL-6R. This residue represents 20% (129 A ² ) of the total binding interface (compared to 28% (174

A ² ) for Phe 229) making it the second most important interaction. Like Phe229, Phe279 also binds in a cavity formed on the surface of IL-6. This cavity is also occupied by a 68F2 residue, more particularly by Vall04 (position 99 in Kabat numbering) of the CDR3 loop of the heavy chain (Figure 6).

IL-6 was previously crystallized and classified as a four helix bundle linked by loops and an additional mini-helix (Somers et ah, 1997, EMBO Journal 16, 989-997). The crystal structure of IL-6 in complex with, its receptor and the signalling receptor gp.130 has also been solved (Boulanger et ah, 2003, Science 27, 2101-2104). IL-6 forms a non- signalling complex with IL-6R. through site L Site II is a composite epitope formed by the binary complex of IL- 6 and IL-6R. Interaction of site III with gpl30 forms the signalling complex.

Comparison of the structure of the IL-6:61H7 complex with the IL-6:68F2 structure shows that although both mAbs compete with IL-6R for binding to site I of IL-6, the two antibodies bind IL-6 at two different epitopes. 68F2 binds on the side of the barrel shaped IL- 6 and exclusively competes for IL-6R binding, while 61 H7 interacts more at the base of the barrel shaped IL-6, competing with both IL-6R and gpl30. Interestingly, the unique overlapping epitope on IL-6 is the cavity that is filled by the hot spot residue Phe229 of IL- 6R (Figure 5). This suggests that binding to this cavity is key to the high potency observed for 68 F2 and 61 H7.

Example 5 Structure function Analysis of W98 in HCD 3 of 61H7

A striking feature of the extremely highly potent antibodies disclosed herein is their capacity to occupy the cavit on IL-6 where F229 of the IL-6R binds (herein referred to as the F229 cavity). For 61H7 (and its germlined variants e.g., 111A7), the F229 cavity is occupied by the tryptophan 98 residue (W98) of the HCDR3. To assess the functional importance of W98 to 61H7 binding to IL-6, mutants of 111A7 were generated in which VII position 98 was mutated towards all the possible amino acids in the background of 100A or M100L, The binding kinetics of periplasmic fractions of bacteria containing the mutant Fabs were tested using surface Plasmon resonance (Biacore) and the off-rate for each mutant was determined. The results of the mutational analysis are set forth in Table 12. The data clearly show that the tryptophan (W) at position 98 is the best possible amino acid to achieve the best off-rate. Mutation of W98 was always detrimental to the binding of 1 11A7 to IL-6.

Table 12. Off-rate of 61H7 Fabs with VH mutations at Positions 98 and 100

Example 6 Germlining of the VH and VL of Fab Clones 61 H7 and 68F2

The VH and VI, sequences of clones 68F2 and 61H7 were aligned against human germline VH and VL sequences to identify the closest related germline sequences. The germlining process was performed as described in WO 2010/001251 and by Baca el al. (J. Biol, Chem. (1997) 272: 10678-10684) and Tsurushita et al. (J. Immunol. Methods (2004) 295: 9- 19). A library/phage display approach was used, in which the deviating FR residues for both the human and the llama residues were incorporated.

The camelid derived IL-6 antibodies of the invention were remarkably human-like in sequence and structure. As a result, only a minimal number of sequence alterations (via germlining) were incorporated into final germlined variants, For example, of the 87 (VH) and 79 (VL) amino acids in VH and VL framework regions of the parental 68F2 antibody, only 6 (VH) and 7 (VL) amino acid changes were introduced (a total of 13 amino acid changes), resulting in a final germlined lead ( 129D3) with 93.1 % and 91.1% identities in their respective VH and VL frameworks (see alignment of Figure 10A). Similarly, of the 87 (VH) and 79 (VL) amino acids in the VH and VL framework regions of the parental 61117 antibody, only 8 (VH) and 5 (VL) amino acid changes were introduced (a total of 13 amino acid changes), resulting in a final germlined lead ( 11 1 A7) with 90.8 and 93.7 % identities in their respective VH and V L frameworks (see alignment of Figure 1GB).

By contrast, art- ecogn zed IL-6 antibodies require an extensive amount of engineering (CDR-grafting) and sequence alterations (backmutations) to generate variant suitable for therapeutic use. For example, the reference mouse IL-6 antibody CNTO-328 required 14 (VH) and 22 (VL) amino acid alterations (a total of 36) to generate the humanized variant CNTQ136 with only 84 and 72.5% homology in its VH and VL frameworks (for alignment, see Figure 11 A). Another reference rabbit IL-6 antibody, ALD518, required a total of 46 framework changes (26 in VH and 20 in VL) to generate a final humanized variants with only 70.5% and 74% sequence homology to the parental antibody. Therefore, the IL-6 antibodies clearly require only minimal engineering and result in molecules which are much more human in sequence.

Furthermore, the small number of FR residues to be changed makes it possible to incorporate changes in CDR residues into the germlining process. Such CDR mutations can be used to remove amino acid introducing production variability (glycosylation site, oxidation, isomerisation, etc) or to change CD residues toward amino acids found in different variant of the antibody to gerrnline.

Phage display, applying stringent selection conditions, was used to select for additional functional Fabs. Individual clones were screened for off-rate and the best hits were sequenced to determine the human sequence identity. The VH and VL amino acid sequences of exemplary germlined IL-6- specific Fabs are set forth in Tables 15 and 16 below.

CDR region sequences from all identified VH and VL domains that are variant Fabs of 129D3 and 111A7 were compared and CDR amino acid consensus sequences determined. CDR variants of 1291)3 and 1 11A7 and derived CDR consensus sequences are set forth in Tables 17 and 18 below.

Example 7 In vitro Potency Assay

The in vitro efficacy of clones 129D3, 68F2, 61H7, 133A9, 133H2, 133E5 and 132E7 were determined using a cell-based neutralizing bioassay using the B9 cell line, essentially as described in Helle et a!, 1988, Eur. J. Immunol 18;1535-1540. B9 cells are derived from the murine B cell hybridoma cell line B 13.29, which require IL-6 for survival and proliferation and respond to very low concentrations of human IL-6. The assay was performed using 10 pg/ml (or 0.5 pM) human IL-6 and a concentration series of purified 129D3, B-E8,

CAT6001, CNT0136, 61H7, UCB124,gl, B9 cells were seeded in IL-6-free medium at 5000 cells/200 micro! in flat-bottom wells in the presence of IL-6, with and without antibodies. Proliferation was measured by a [3H]thymidine pulse at 64-72 h. The results (shown in Figure 1 and Table 23) demonstrate that all clones have high potency, with 129D3 having an IC50 in this assay of less than O.lpM. Interestingly, as shown in Figure 1, the IC50 of clone 129D3 was superior to the benchmark CNT0136, CAT6001, UCB124, and B-E8 antibodies.

The m vitro efficacy of clones 133E5, 133 A9, 133H2, i l l Bl, 104C1 , 129D3, 68F2, 61F17, were also determined using a cell-based neutralizing bioassay using the 7TD1 cell line, essentially as described in Van Snick et al. PNAS; 83, :9679 (1986), which is hereby incorporated by reference in its entirety. 7TD1 cells are a murine hybridoma cell line formed by fusion of the mouse myeloma cell line Sp2/0-Agl4 with spleen cells from a C57BL/6 mouse immunized with Escherichia coli lipopolysaccharides three days before fusion. The 7TD1 cell line is dependent on IL-6 for its growth and IL-6 withdrawal leads to cell death by apoptosis. The assay was performed using 75 pg/ml human IL-6 and a concentration series of purified 133E5, 133A9, 133H2, 111B1, 104C1, 129D3, 68F2, 61H7, B-E8, GL18LB, CNT0136 and hul U.

Briefly, 7TD1 cells (7.10E3) were incubated for 2-4h in RPMI1640 medium +10%FCS before addition of IL-6 (75 pg/ml final concentration ) in microtiter plates (200ul final volume), The cells were incubated 3 clays at 37C, before washing with PBS and addition of 60ul of substrate solution (p-nitrophenyl-N-acetyl- -D-glucosaminide (Sigma N-9376); 7,5 mM substrate in 0.05 M NaCitrate pH 5; 0.25 vol% triton X- 100) for 4h. The enzymatic reaction was stopped with 90ul of stop solution (100 mM glycine + 10 mM EDTA pH 10,4) and OD at 405nm measured. The results (shown in Table 24) demonstrate that all clones have high potency in this assay, with IC50s of less than IpM.

Example 8 Epithelial Ovarian Cancer Mouse Xenograft Assay

The in vivo potency of clone 129D3 was determined using a mouse xenogaft model. IGROV- 1 epithelial ceils (5 x 10°) were injected subcutaneously into nude mice. After three days, mice were administered 129D3 or CNT0328 biweekly, at a dosage of 4 or 20 mg kg. There were 5 mice per group and 10 mice in the control group. Percentage survival of mice in each study group was determined each week and the results plotted as a survival curve. The results (shown in Figure 2) demonstrate that 1 291 )3 exhibits an in vivo potency that is superior to the benchmark CNT0328 IL-6 antibody.

Example 9, Immimogenicity Analysis

VH and VL regions of the IL-6 antibodies of the invention were scored for the presence of potential immunogenic sequences (e.g., putative HLA class II restricted epitopes, also known as TIL epitopes) and compared with immunogenicity scores for a variety of commercially- available reference antibodies using the Epibase® profiling method.

Profiling was done at the allotype level for 18 DR.B .1 , 6 DRB3/4/5, 13 DQ and 5 DP, i.e. 42 HLA class II receptors in total. Strong and medium binders of DRB1, DRB 3/4/5 were identified, as well as the strong binders of DQ and DP epitopes. Epitope counting was done separately for strong and medium affinity DRB1 binders. Peptides binding to multiple allotypes of the same group were counted as one. An approximate score expressing a worst- case immunogenic risk was calculated as follows: Score =∑ (epitope count x allotype frequency). In other words, the number of epitopes affecting a particular HLA allotype is multiplied by the allele frequency of the affected allotype, For a given sequence, the products were summed for all DRB1 allotypes used in the study that are present in 2% or more of the Caucasian population.

DRB1 scores for the IL-6 antibodies of the invention and representative reference antibodies are provided in Figure 9. Total DRB1 scores were a composite of the VH and VL scores for each antibody and low scores indicate low iramunogenicity for the antibody, Accordingly, Figure 9 demonstrates that IL-6 antibodies of the invention are equal to or less immunogenic than benchmark IL-6 antibodies as well as other commercially-available antibodies (e.g., Humira and Remicade).

Example 10. Manufacturability

The VH and VL of the germiined versions of 68F2 were recloned in pUPE Heavy Chain and Light Chain expression vectors, respectively, for transient expression of full-length IgGl antibodies. After transient expression in HEK293E cells, IgGl antibodies were purified with protein A and quantified by measurement of OD280. Table 19 below summarizes the production levels of the germiined derivatives together with the levels of the 68F2 parental antibody. Potencies (in pM) were also measured in a 7TD1 based proliferation assay for each antibody.

The variants 126A3, 127F1, 129D3 and 129F1, all selected from the germiined libraries under stringent conditions, were found to have similar potencies as the wild type 68F2 (i.e. between 0.5 and 0.7 pM). Moreover, all germiined variants expressed very well, i.e. between 24 and 28 jig/ml. The exception was germiined variant 129F2 that gave a production yield of 9 ug/rnl.

Example 11. Stability Analysis

To examine the thermal stability of the germiined and the parental versions of 68F2 in full length human IgGl format, antibody samples were incubated at a concentration of 100 ,Lig/mI (in PBS) at 4, 50, 55, 60, 65, 70 and 75°C for 1 hour. Following this, the samples were cooled down slowly during a period of 15 minutes to 25 °C and kept at this temperature for 2 hours, after which they were stored overnight at 4°C. Following centrifugation to remove precipitates (of denatured antibody), the concentration of functional antibody remaining in solution was measured using Biacore (1 /10 dilution in PBS and 1/10 in HBSEP). The slope of the association curve obtained after injection on the IL-6 immobilized chip is a measure of the concentration of functional antibody.

As shown in Figure 7A, the melting curves and the melting temperatures of wild type 68F2 and its germlined derivatives were clearly unaffected by germlining. Quite

unexpectedly, the melting temperatures even seemed to be improved. For example, 129D3 has a I ^' m of around 70°C, which is 3°C higher than the parental 68F2 antibody. The melting curves of the germline variant 129D3 and the parental 68F2 together were also compared with the reference antibodies GL18 and CNT0136 (see Figure 7B). The favorable thermal stability of the SIMPLE antibody 68F2 and especially of its germlined variant 129D3 (Tm of 70 ^' C s was striking when compared to Tm of 65°C for CNT0136 and the Tm of 61"C for the GL18 antibody. Surprisingly, the extensive antibody engineering (e.g., humanization) and in vitro affinity maturation applied to both reference antibodies strongly affected their stability, whereas the in vivo generated SIMPLE antibody and the minimal engineering by germlining resulted in extremely good thermal stabilities.

The serum stability of the full length human IgG.1 versions of 68F2 and its germline variant (129D3) (and the germlined variant 103A1 derived from SIMPLE™ antibody lead 61H7) were compared to those of the reference antibodies. Following incubation at 37°C in human serum, functional concentration of antibody was measured at weeks 1, 2, 4, 8, 12, 16, 24, 32 and 56 and compared to a pre-aliquoted standard. As depicted in Figure 8, the serum stability for the antibodies of the invention compared favourably to that reference antibodies.

Example 12. CMC Optimization

Several residues or motifs are not recommended for CMC-quality manufacturing of antibodies. Among them is the presence of Methionine in the CD loops of the antibody. Methionine can be oxidized leading to chemically-altered variant of the antibody with altered properties such as affinity, potency, and stability. Accordingly, the methionine present in CDR3 of 111 A7 (and its germlined variant of 61H7) was mutated to Alanine (1 1 1A7MA), leucine (1 1 1A7ML) or Serine (11 IA7MS). The resultant CMC-optimized sequences are provided in Table 20 below. As shown in Table 21, the mutation of the methionine residue has a negligible effect on the binding to IL-6. Example 13, Pharmacokinetic (PK) study in Cynomoigus Monkeys of Clone 1291 ⁾ 3 and Fc Mutants Thereof

Pharmacokinetic analysis of antibody clone 129D3 formatted as various IgG! molecules was performed. The following antibodies were analysed: Wild-type IgGl 1291 )3 (12 D3-WT), IgGl 129D3 with the mutations M252Y/S254T/T256E in the Fc region (129D3-YTE), and IgGl 1291 )3 with the mutations H433K and N434F in the Fc region (129D3-HN).

Cynomoigus monkeys (3 per antibody tested) were injected intravenously with a single 5 mg/kg dose of 129D3-WT, 129D3-YTE, or 129D3-HN. Samples were taken at different time points and tested for plasma concentration of mAb by the ELISA. Specifically, a micro titerplate (Maxisorb Nunc) was coated with 1 ug/ml IL-6 (Imrmmotools) in PBS overnight at 4C. The plate was washed 2 times with PBS-Tween and blocked for 2 hours with 300 μΐ PBS-l%casein. After 2 washes with PBS-Tween, the samples were applied. All dilutions were made in ! % pooled blank plasma (this is a pool from 3 naive cynomoigus monkeys, see chapter 4.2). The samples were allowed to bind for 2 hours at RT. Plates were then washed 5 times with PBS-Tween and goat biotinilated anti-human IgG heavy and light chain monkey adsorbed polyclonal antibodies were applied at a 1000-fold dilution (Bethyl, catno: A80-319B) and allowed to bind for 1 hour at RT. After washing the plates 5 times with PBS-Tween, streptavidin conjugated with HRP (Jackson Immunoresearch 016-030-084) was applied at a 300,000-fold dilution and allowed to bind for 1 hour at RT. Plates were then washed 5 times with PBS-Tween and a 1: 1 mixture of TMB (calhiochem CLQ7)-s(HS)TMB weakener (SDT, #sTMB-W) was added. The staining was allowed to proceed for 10 minutes and then stopped with 0.5 M H2SO, after which the Optical Density was measured at 450nm. The samples were analysed four times and 129D3-WT (from the same batch that was injected into the animals) was used for a standard curve.

The relevant PK parameters for the non-compartmental analysis are shown in Table 22 below. The pharmacokinetic profiles for the different 129D3 IgGl antibodies are shown graphically in Figure 12 (the results are shown are the average result of the group of monkeys). This data clearly shows that 129D3-YTE and 129D3- N have a longer mean residency time (MRT) than the parental 129D3-WT antibody. Moreover, 129D3-YTE and 129D3-HN have a slower elimination rate and thus a substantially prolonged half-life as compared to 129D3- WT. Interestingly, although both antibodies contain a wild-type IgGl Fc region, the half- life of 129D3-WT is significantly longer than the half-life of the Medlmmune anti-IL-6 IgGl antibody (GL18) described in US201200344212. Specifically, 129D3-WT has a half-life of about 15.6 days as compared to about 8.5 days for antibody GL18. Thus, the extended half- life of the antibodies of the invention appears to be due to the properties of their respective Fab regions.

Example 14. Serum Amyloid A (SAA) Mouse Model

The in vivo efficacy of clones 68F2 and 61H7 was further investigated by measuring the ability of these antibodies to block serum amyloid A (SAA) induction in response to injected IL-6, General methods for performing this assay are set forth in

WO2006/1191 15A2, which is hereby incorporated by reference in its entirety. Specifically, Balb/c mice were injected intravenously with 68F2, 61H7, the benchmark antibodies GL18, or CNTC3136, or salt solution (control). Four hours after administration of the antibody, the mice were injected with 0.1 ug of IL-6. After a further 16 hours, blood was taken from the mice and the concentration of Serum Amyloid A was determined by ELISA. The experimental groups, dosages and results are set forth in Table 26, herein. The dose responses are also graphically depicted in Figure 13. The results show that antibody clones 68F2 and 61H7 have in vivo efficacy at least equal to the high potency benchmark controls GL18 and CNT0136.

Example 15, Humanized Moose Psoriasis Xenograft Model

A mouse xenograft model was employed to evaluate the prophylactic efficacy of clone 68F2 on the development of induced psoriatic lesions. Specifically, BNX mice were transplanted with 5mm diameter full-thickness skin biopsies from non-involved skin from psoriasis patients (1 per mouse). After 3 weeks, the transplants were injected with 0.5x106 activated PBMCs. Treatment of mice with clone 68F2, anti-TNF antibody (Remicade), or betamethasone dipropionate (positive control) was begun 1 clay before the activated cells were injected into the transplants. Details of the treatment groups and regimes are shown in Table 25.

Treatment efficacy was determined by epidermal ridge thickness, as measured by light microscopy. Significance between groups was analyzed statistically using analysis of variance (ANOVA) followed by post-hoc Least Square Difference (LSD) tests to establish statistical significance differences between treatment groups. A value of p<0.05 denoted a significant difference between groups.

The results of these experiments are set forth in Figure 14, herein. The epidermal ridge thickness of the control group (group 2) was 156μηι ± 4 (mean ± s.e.m.). Treatment with betamethasone (group 1, n=3) significantly reduced epidermal ridge thickness to 83μηι ± 13 (p< 0.05) (Figures 12 and 13). The average epidermal ridge thicknesses of the

Remicade treatment group (group 3) was 125μηι ± 12 and the 68F2 treatment group (group 4) was 125 πι ± 12. These data show that clone 68F2 is as efficacious as the anfi-TNF antibody Remicade in a humanized mouse psoriasis model.

Example 16, Renal Cell Cancer Mouse Xenograft Model

The in vivo efficacy of clones 68F2 was investigated in a renal cell cancer mouse xenograft model, General methods for performing this assay are set forth in

WO2008/144763, which is hereby incorporated by reference in its entirety. Briefly, RXF393 cells (2 x 10°) were injected subcutaneously at both lateral sides of a nude mouse, lumors were allowed to grow to a volume between of 50 en 300 mm ³ , prior to antibody administration. 90% of the injected mice developed a tumor. 40 mice were split into 5 groups of 8 mice. Each group was received intraperatoneal injection of either PBS (control) or a specific dose of clone 68F2 (1, 3, 10, or 30 mg/kg). Tumor size and survival was monitored twice per week.

The survival data, set forth in Figure 15 herein, show that 68F2 is effective at delaying death of mice relative to control. Specifically, the observed median survival times were 15.5 days for the PBS group, 21 days for the 1 mg/kg, 3 mg/kg and 30 mg/kg 68F2 group, and 27.5 days for the 10 rag/kg 68F2 group. The survival data, set forth in Figure 18 herein, show that 129D3 and 1 11A7 dosed at 3 mg/kg are effective at delaying death of mice relative to control. The tumor growth rate data, set forth in Figure 16 herein, shows that clone 68F2 is effective at inhibiting tumor outgrowth in a dose-dependent manner with saturating effects at lOmg/kg dose. The tumor growth rate data, set forth in Figure 17 herein, shows that clone 129D3 and 11 1 A7SDMA>A are effective at inhibiting tumor outgrowth when dosed at 3 mg/kg. Additional Tables.

le 13. VH and VL Amino Acid Sequences of Exemplary Anti-IL-6 Neutralizing Fabs.

FAB VH SEQUENCE SEQ VL SEQUENCE SEQ CLONE ID !D

HQ MO

17F10 EVQLQESGPGLVKPSQTLSLTC!VSGGSI 1. QAGLTQPPSVSGSPGKTVTI SCAGTTSD G 233.

ATSYYAWS IRQPPGKGLEWMG IDYDGD TGNF SWYQQLPGMAPKLLIYDVNKRASGI TYYKPSLKSRTSISRDTSKNQFSLQLSSV ADRFSGSKSGNTASLTISGLQSEDEADYYC TPΞDTA.VYYCARAGLGDSYYLGTYYAMDY ASYRSL NWFGGGTHLTVLG

WGKGTLVTVSS

18C11 EVQLVESGGGLVQPGGSLRLSCAASGFTF 2. QSWTQPSALSVTLGQTAKI CQGGGLRSS 234.

SSYAMSWVRQAPGKGLEWVSAIMSGGGST YAHWYQQKPGQAPVLV YDDDSRPSGIPER SYADSVKGRF I SRDNAKNTLYLQMNSLK FSGSSSGGRATLTI SGAQAEDEGDYYCQSA PEDTAYYYCAKEGDTGWKDPMYDYWGQGT DSSGNAAVFGGGTHLT LG QVTVSS

18C7 EVQLVESGPGLVKPSQTLSLTCTVSGGSI 3, QAGLTQPSALSVTLGQTAKITCOGGSLGSS 235.

TASFDAWSWIRQPPGKGLEWMGVTAYDGS YAHWYQQKPGQAPVLVIYDDDSRPSGIPER TYYSPSLKSP.TS I SRDTSK QFSLQLSSV FSGSSSGGRATLTI SGAQAEDEGDYYCQSA TPEDTAVYYCARKSSWLIGYGMDYWGKGT DSSGNAAVFGGGTHLTVLG LVTVSS

18C9 EVQLVESGGGLVQPGGSLRLSCA SGFTF 4. LNFMLTQPSALSVTLGQTAKITCQGGSLGS 236.

SRNAMSWVRQAPGKGLEWVSAINSGGGST RYAHWYQQKPGQAPVLVIYDDDSRPSGIPE SYADSVKGRFT SRDNAK TLYLQMNSLK RFSGSSSGGRATLTI SGAOAEDDGDYYCQS PEDTAVYYCAKEGYTGWKDPMYDYWGQGT ADSSGNASVFGGGTKLTVLG QVTVSS

18F8 EVQLVESGGGLVQPGGSLRLSCAASGFTF 5. QSALTQPSALSVTLGQTAKITCQGGSLGSR 237.

SRNAMSWVRQAPGKGLEWVS INSGGGST YAHWYQQKPGQA.PVLV YDDDSRPSGIPER SYADSVKGRFTI SRD AKNTLYLQM SLK FSGSSSGGRATLT SGAQAEDEGDYYCQSA PEDTAVYYCAKEGYTGWKDPMYDYWGQGT DTSΞH VFGGGTHLTVLG QVTVSS

20G2 EVQLVESGGGLVQPGGSLRLSCAASGFTF 6. ALNFMLTQPSALSVTLGQTAK TCQGGSLG 238.

SRNAMSWVRQAPGKGLEWVSAIMSGGGST SSYAHWYQQKPGQAPVLVIYDDDSRPSGIP SYADSVKGRFIISRDNAKNTLYLQM SLK ERFSGSSSGGRATLTI SGAQAEDEGDYYCQ PEDTAVYYCAKEGYTGWKDPMYDYWGQGT SADSSGNA.VFGGGTHLTVLGQ

QVTVSS

18E12 EVQLQESGPGLVKPSQTLSLTCTVSGGSI 7 , QSALTQPPSMSGTLGKTLTI SCAGTSSD G 239.

TTRYYAWSWIR.QPPGKGLΞWMGVIDYDGD YGDYVSWYQQLPGTAPK.I,LIYKVSTRASGI TYYSPSLKSRTS SWDTSKNQFSLQLSSV PDRFSGSKSGNTASLTISGLQSEDEADYYC TPEDTAVYYCARDPDWTGFHYDYWGQGT ASYRHYNNAVFGGGTHLTVLG QVTVSS

20A4 EVQLQESGPGLVKPSQTLSLTCTVSGGSI 8. ALNFMLTQPPSVSGTLGKTVTI SCAGTSSD 240.

TTRYYAWSWIRQPPGKGLEWMGV DYDGD IGGYNYVS YQQLPGTAPKLLIHRVSTRAS TYYSPSLKSRTSISWDTSKNQFSLQLSSV GIPDRFSGSKSG TASLTI SGLRSEDEA Y TPEDTAVYYCARDPDWTGFHYDYWGQGT YCA.SYRNFNNAVFGGGTQLTVI,G

QVTVSS

22C10 QVQLQESGPGLVKPSQTLSLTCTVSGGS 9. ALPVLTQPPSVSGSPGQKFTISCTGSSSNI 241.

TTSYYA. S IRQPPGKGLΞWMGVIGYDGS GENYVNWYQQLPGMAPKLLIYSNTNRA.SGV TYYSPSLKSRTS I SRDTSKNQFSLQLSSV PDRFSGSKSGSSASLTITGLQVEDEADYYC TPEDTAVYYCARDAGWYVGYEYDYWGQGT SSWDDSLSGLVFGGGTKLTVLG QVTVSS

22D11 QLQLVESGGGLVQPGGSLRLSCAASGFTF 10. ALNFMLTQPPSLSASPGSSVRLTCTLSSGN 242.

DDYA SWVRQAPGKGLEWVSDI S NGGNT SVGSYDI SWYQQKAGSPPRYLLYYYSDSYK YYAESMKGRFΪI SRDNAKNTLYLQMNSLK HQGSGVPSRFSGSKDASANAGLLLI SGLQP SEDTAVYYCAKEGGAWAGTVGYYGMDYW EDEAAYYCSAYKSGSYVFGGGTKLTVLG GKGTLVTVSS A3 QVQVQESGGGLVQPGGSLRLSCAASGFTF 11. ALNFMLTQPPSVSGSPGQKFTIRCTGSFRS 243. SNYA SWVRQAPGKGLEWVSGI SFRGGMI DSYVNWYQQLPGTAPKLLINYDDRRVSGVP S Y VD SVKGRF T I S RDNAKN T LYLQMM S LK SRFSGSKSGNSASLTIDGLQAEDEAEYYCS P Ei D T A V Y Y C AK S G S S R S N A L D A. G Q G T L FWD.HTFGGHVFGGGTKLTVLG

VTVSS

B9 QVQLQESGGGLVQPGESLRLSCVASGFTF 12. Q T V V T Q E P S L S V S P G G T V T L T C G L S S G S V T 244.

S SHRMYWVRQPPGKGLEWVS AI S S SGVST ASNYPGWFQQTPGQAPRALIYSTNDRHSGV YYTDSVKGRFTI S D AKNTVYLQMNSLK P SRFSGS I SGNKAALT I TGAQPEDEADYYC FEDTALYYCKRRTWYAGEYDYWGQGTQVT ALD I GD I TEFGGGTKLTVLG VSS

C9 QVQLVE S GGGLVQP GG S LRLS CAASGFTF 13. QTWTQEPSLSVSPGGTVTLTCGLSSGSVT 245.

S S YRMYWVRQP P GKGLE WVS AI SAGGG S T ASM YP GWFQQTP GQAP RAL I Y S TNDRH S GV Y YGD SVKGRF T I S RD AK T V Y LQM S LK P SRFSGS I SGNKAALT I TGAQPEDEADYYC PEDTALYYCKKSTWADGESDYWGQGTQVT ALD I GD I TEFGGGTKLTVLG VSS

D10 QLQVVE S GGGLVQP GG S LR.L S C AASGF TF 14. QTWTQEP SLSVSPGGTVTLTCGLS SGSVT 246.

S S YAMSWVRQAPGKGPEWVSRI S SGGGST ASNYPGWFQQTPGQAPRALIYSTNDRHSGV S YAD SVKGRF T I S RDNAKNT LYLQM S LK P SRFSGS I SGNKAALT I TGAQPEDEADYYC PEDTAVYYCANRAGWGMGDYWGQGTQVTV ALD I GD I TEFGGGTKLTVLG

s s

D9 EVQVQE S GGGLVQP GE SLRLS C AASGF TF 15. QTWTQEPSLSVSPGGTVTLTCGLSSGSVT 247.

S S HRMY VRQP P GKGLE WVS AI S S SGVST ASN YP GWFQQTP GQAP RAL I Y S TMDRK S GV Y Y AD S VKGRF T I S RDNAKM TVYLQMM S LK P SRFSGS I SGNKAALT I TGAQPEDEADYYC P E D T LYYCKRRT W Y G G E Y D Y W G Q G T Q VT ALD I GD I TEFGGGTKLTVLG VSS

E9 EVQLVESGGGLVQPGGSLRLSCAASGFTF 16. QTWTQEPSLSVSPGGTVTLTCGLSSGSVT 248.

S T YAMS WVRQAP GKGP E WVS R I S SGGG S T ASNYPGWFQQTPGQAPRALIYSTNDRHSGV NYADSVKGRFTI SRD AKKTLYLQMKSLK P SRFSGS I SGNKAALT I TGAQPEDEADYYC PEDTAVYYCANRAGWGMGDYWGQGTQVTV ALD I GD I TEFGGGTKLTVLG SS

F4 EVQLVE S GGGLVQP GE S LRLS C AASGF TF 17. QTWTQEPSLSVSPGGTVTLTCGLSSGSVT 249.

S S H RM Y VRO P P G KG L E V S A I S S SGVST ASNYPGWFQQTPGQAPRALIYSTNDRHSGV YYA.DSVKGRFT I SRDNAKNTVYLQMNSLK P SRFSGS I SGNKAALT I TGAQPEDEADYYC P E D T A L Y Y C K R R T W Y G G E Y D Y W G Q G T Q L T ALD I GD I TEFGGGTKLTVLG VAS

G3 QLQVVESGGGLVQPGSSLRLSCGASGFTF 18. QTWTQEP SLSVSPGGTVTLTCGLS SGSVT 250.

S S H RM Y W VP. Q P P G KG L E WV S A I S S SGVST ASN YP GWFQQTP GQAP RAL I Y S TNDRH S GV Y YAD SVKGRF T I S RDNAKNT VYLQMN S LK P SRFSGS I SGNKAALT I TGAQPEDEADYYC PEDTALYYCKRRTWYGGEYDYWGQGTQVT ALD I GD I TEFGGGTKLTVLG VSS

B3 EVQLVE S GGGLP XP GE S LRL S C AASGF TF 19. QTWTQEPSLSVSPGGTVTLTCGLSSGSVT 251.

S S HRMY VRQP P GKG LEW VS AI S S SGVST ASNYPGWFQQTPGQAPRALIYSTNDRHSGV Y Y AD S VKGRF T I S RDM AK T VYLQMN S LK P SRFSGS I SGNKAALT I TGAQPEDEADYYC PEDTALYYCKRRTWYGGEYDYWGQGTQLT ALD I GD I TEFGGGTKLTVLG VAS

B11 EVQLVESGGGLVQPGGSLRLSCAASGFTF 20. QSVLTQPPSVSGSPGQTVTISCAGTSEDVG 252.

S S YAMS WVRQAP GKGLE WVS R I S S GG I S T Y GN Y V S W Y Q Q L P G MAP K L L I Y D VN K P. A.3 G I Y YAD SVKGRF T I S RDNAKNT LYLQMN S LK ADRFSGSKSGNTASLTISGLQSEDEADYYC P E D T AVY YC VR Y AW G VQ WA.F DFWGQGTQV AS YRRT I D I FGGGTHLTVLG

TVSS

C6 QVQLVE S GGGLVQP GG S LRLS C AASGF TF 21. DIVMTQTPSSLSASLGDRVTITCQASQSIS 253.

SNYYMTWVROAPGKGLEWVSSIYSFSGDT T ELS W Y 0 Q KP G Q T P K L L I Y G A S R L Q T G VP A AYA.DSVKGRFTISRDNAKNTLYLQMNKLK RF S G S G S G T S F T L T I S G L E AE D LATYYCLQ SEDTAVYYCTRDLGGVWTANGYDYWGQG D Y S W PYSFGSGTRL TQVTVSS

B6 QVQLVE S GGGLVQP GG S LR.L S C AASGF TF 99 D I Q L T Q S P S S L S S L G D R VT I T C Q A S Q S I S 254.

SNYYMTWVRQAPGKGLEWVSSIYSFSGDT TEL S W Y Q Q K P G Q T P K L L I Y G A S R L Q T G VP A AY AD SVKGRF T I S RDNAKNT LYLQMN S LK R F S G S G S G T S F T L T I S G L Ξ AE D L A T Y Y C L Q SEDTAVYYCTRNLGGVVVTTNGYDYWGQG D Y S W P Y S F G S G T R L TQVTVSS

E6 QVQLVESGGGLVQPGGSLRLSCAASGFTF 23. DIQMTQSPSSLSTSLGDRVTITCQASQAIT 255.

SNY ^" YMTWVRQAPGKGLEWVSS IYSFSGDT TΞLS YQQKPGQPPKLLIYGTSRLQTGVPS AYADSVKGRFTI SRDNAKNTLYLQMMSLK RFSGTGSGTSFTLTISDLEAEDLATYYCLQ SED AVYYCTRNLGGVVVT NGYDYWGQG DYGWPFTFGQGTKV TQVTVSS

F6 QVQLVESGGGLVQPGGSLRLSCAASGFTF 24. DIVMTQSPSSLSASLGDRV ITCQTSO IS 256.

SNYYMTWVRQAPGKGLEWVSS IYSFSGDT TELS YQQKPGQAPKLLIYGASRLQTGVPS AYADSVKGRFTT SRDNAK TLYLQMNSLK RFSGSGSGTSFTLTI SGLEAEDLATYYCLQ SΞDT-VY ^" YCTRNLGGWVTTNGY ^" DYWGQG DYSWPFTFGQGTKV

TQVTVSS

A2 ELQLVESGGGLVQPGGSLRLSCAASGYTF 25. DIVMTQSPFSLSASLGDRVTITCQASESIL 257.

DDYAMGWVRQAPGKGLEWVSS IYSYSSDT TEVSWYQQKPGQTPKLLIYGASGLQTGVP YYADSVKGRF I SRD AQ T YLQ TSLK RFSGSGSGTSFTLTISGLEAEDLATYYCLQ PEDTALYYCARCARDIGSAWCGGVDY ^' GK DYRWPLTFGQGTKVELKR

GTLVTVSS

B1 ELQLVESGGGLVQPGGSLRLSCAASGYTF 26. DIVM QSPSSLTASLGDRVTITCQASQS IR 258.

DDYAMGWVRQAPGKGLEWVSS IYSYSSDT TDI SWYQQKPGQTPKLLIYAASP,LQTGVPS YYADSVKGRFTI SRDNAQNTVYLQMTSLK RFSGSGSG SFTLTI SGLEAEDLGTYYCLQ PEDTALYYCARCARDIGSA CGGVDYWGK DYSWPLTFGQGTKVELKR GTLVTVSS

D2 ELQLVESGGGLVQPGGSLRLSCAASGYTF 27. DIVMTQSPSSLSASLGDRVTITCQASQS I S 259.

DDYAMGWVRQAPGKGLEWVSSIYSYSSDT TELSWYQQKPGQTPKLLIYGASRLQTGVPS

YYADSVKGRFTI SRDNAQN VYLQMTSLK SFSGSGSGTSFTLTISGLEAEDLATYYCLQ PEDTALYYCARCARDIGSAWCGGVDYWGK DYNWPFTFGQGTKVELKR GTLVTVSS

G3 ELQLVESGGGLVQPGGSLRLSCAASGFTF 28. DIQMTQSPSSVTASVGEKVTLNCKSSQSW 260.

SSYAMSWVRQAPGKGLE VSRI SSGGI ST VP,SDQKSYLNWYQQRPGQSPRLLIYY STQ YYADSVKGRFTI SRDNAKNTLYLQM SLK ESGIPDRFSGSGSTTDFTLTINSVQPEDAA PEDTAVYYCARYAWGVQWAFDFWGQGTQV VYYCQQASSAPYNFGSGTRL TVSS

Table 14. VH and VL Amino Acid Sequences of Exemplary Anii-IL-6 Neutralizing Fabs Generated by VH or VL Shuffling

VH SEQUENCE SEQ ID VH SEQUENCE SEQ !D

FAB

(clone name given in piace of sequence if sequence ideniicai NO (cione name given in piace of sequence if sequence NO CLONE

to VII in another done) identical to VL in another done)

QSALTQPPSMSGTLGKTLTISCNGTSSDIGSGDYVSWYQ 261.

35C1 VH_1 ^' 7F10 QLPGITPKLLIEGVTTRASGIPDRFSASKSDNTASLIIS

GLOSEDEATY CAFlYRFINiNVVFGGGTHLTVLS

QAVLIQPPSMSGTLGKTLTISCNGTSSDIGSGNYVSWYO 262.

35B1 VH_17F10 QLPGTTPKLL1EGVITRVSGIPDRFSGSK3D TASLTIS

GLQSEDFATTYCASYRETF^VVFGGGTHLTVLG

QAGLTOPPSVSGSPGKTVTISCAGTSSDVGYGNYVSWYQ 263.

35F5 VH_1 F10 QLPGMAPKLLIYDVNKRASGIADRFSGSKSGNTASLIIS

RLOSEDEADY CAFlYKTYNNVVFGGGTHiTCVLG

2. QSWTQPPSVSGTLGKTVTISCAGTTSDIGGYNYVSWYQ 264.

35 1 VH_ ^' I7F10 QLPGTAPKFLI YEVSKRAAGIPDRFLTSSKSGSTASLTIS

GLQSEDFADYYCA3YRL)TA^/VFGGGTHLTVLG

ALNFMLTQPSALSVTLGQTAKITCQGGSLG SYAHWYOQ 265.

37A1 VEi__13Cll KPGQAPVLVIYDDDSRPSGIP RFSGSSSGGRAILTISG

AQAED GD YYCOSADSSGNAVFGGGTHFTVLG

2. AQSALTQPSALSVTLGQTAKITCQGGSLGTRYAHWYQQK 266.

36A; VM__1SC1 ; PGOAPVFVIYDDDSRPSGIPERF3GS3SGGRATI,T ISGA

QAEDEGDYYCQSADSSGNASVFGGGTHLTVLG

ALNFMLTQPSALSVTLGQTAKITCQGGSLGSRYAHWYOQ 267.

36F3 __13Cll KPGQAPVLVIYDDDSRPSGIP RFSGSSSGGRAILTISG

AQAED GDYYCOSADSSGNAAVFGGGTHLTVFG

2. AQAGLTQPSALSVTLGQTAKITCQGGSLGSSYAFl*YQQK 263.

3 D1 vM__ i sci ; PGOAPVLVIYDDDSRPSGIPERF3GS3SGGRATI,Ϊ ISGA

QAEDEGDYYT!QSADSSGNAIVFGGGTHLTVLG

2 , AQAV:FIQPSAFSV ;3¾TA:KITCQGG3IR3SYAHWYQOK 269.

37G1 __13Cll PGQAPVLVI YDDD3RP3GIPE RFSGSSSGGRATL ISGA

QAEDEGDYYCQSADSSG ASVFGGGTHLTVLG

2. ALNFMLTQPSALSVT LGQTAKITCOGGSLGSSYABWYQO 270.

20G2 VM__1SC1 ; KPGQAPVLVIYDDDSRPSGI PERESGSSSGGRAT LTISG

AQAFDEGDYYCQSADS3GNAVFGGGTHLTVLG

AQSALTQF'SA.LSVTLGQI TCQGG3LG3S YAHWYQO'K 27 ; .

44C6 __13C7 PGQAPVLVI YDDD3RP3GIPE RFSGSSSGGRATLT ISGA

QAEDEGDYYCQSADSSG ASVFGGGTHLTVLG

3. AL E LTQPSALSVTLGQTAKITCQGGSLGSSYAHWYQQ 272.

44E7 VH_18C7 KPGOAPVLVIYDDDSRPSGIPERESGSSSGGRA.TLSI SG

AQAEDEGDYYCOSGDSSGNAAVFGGGTKLTVLG

SVQLQESGPGI 'KPSQTLSLTCTVSGGSITTRYYASS IK 8. OSALTQPPLVSGTPGQTVTISCAGANNDIGTYAYVSWYQ 273.

QLPGTAPKLLiYKVTTRASGIPSRFSGSKSG TASLTIS

68F2 QPPGKGLBW GVIDYDGDTYYSPSLKSRTSISW TSK QP

SLQLSSVTPEDTAVYYCARDPDWTGFHYDYWGQGTQVTV GLQSEDEADYYCASYRNF NAVFGRG'IHL'iyLG

8S (Identical to VB__2CA4)

8. QSALTQPPSVSGTLGKTLTISCAGTSSDVGYGNYVSWYQ 274.

71C8 VH_20A4 QLPGTAPKLL1YRVSTRASGIPDRFSGSK5G TASLTIS

GLQSEDEADYYCA8YR8SNNAVPGGGTHLTVLG

8. QSVLTQPPSVSGTLGKTVTISCAGTSSDVGYGNYVSWYQ 275.

7 OH2 VH_20A4 QLPGTAPKLLIYAVSYRVSGiPDRFSGSKSGNTASLTIS

GLOSEDEADYYCASYRNRNNaVFGGGTHLTVEG

8. QAVLTQPPSVSG1LGKTVTI SCAGTSSDVGYG YVSWY0 276.

71D12 VH_20A4 QLPGTAPKLyiYAVNYRASGIPDRFSGSKSG TASLFIS

GLQSEDEAD YCASYRDFNNAVPGGGTHLTVLG

8. QAVLΪQPPSVSGSPGKTVTI SCAGTSSDVGFGNYVSWYQ 277.

7CE2 VH_20A4 QLPGMAPKLLIYEVNKRTSGiPDRFSGSKSGNTASLTIS

GLOSEDEADYilCASYRNENNaVEGGGTHLTVEG

8. QSALTQPPSVSGSPGKTVTISCAGTSSDVGYGNYVSWYQ 273.

71C3 V1-L20A4 QLPGMAPKPL; YDVNKRASGIADRFSGSKSGNTASLT Is

RLQSEDEaD YCASYKTYNNVVPGGGTHLTVLG

8. QSALTQPPSVSGTLGKTVTISCAGTS5DVGYGNYVSWYQ 279.

69H4 VEi__20A4 QLPGTAPKLLIYAVS RASG : PDRESGSK SGNTAS L 1 S

GLOSEDEADYYCASYRYENNAVEGGGTHLTVEG

27' . LDIVMTQTPS5LSASLGDRVTITC0ATQN1NTELSKYQ0 280.

66 8 VEL16D2 KPGQTPKLLIYDTSRLQTGVPSRFSGSGSRTTFTLTiSG

ITIAEDIATY CMQDY WPLTFGQGTKVELKR

27 , LDIVMTQTPSSLSASLGDPVTITCQASQSISTELSKYOO 231.

63E10 VEi__16D2 KPGQSPKLLlYGASRLQiGVPSRFSGSGSGTSFTLTISG

LEADDLATYYCLQDYNWPLSFGSGTRLEIK

27' . LDIQMTQSPSSLSASLGDRVTITCOASQSISTELASYQO 282.

69H7 VEL16D2 KF'GQ P LLI YGA.S LQTGVPSRFSGSGSGTSF7 ¹ LTiSG

ITIAEDIATY CLQDY WPETFGQGTKVELKR

27 , LDIVMTQTPSSLSASLGDPVAITCQASQSI VDVSKYOQ 233.

7 CBS VEi__16D2 KPGQTPKLLIYAA8RLQTGVPSRFSGSGSGTSFALTISG

LEAEDLASYYCLQDYSWPLTFGQGTKVEL R

27' . LDIQMTQSPSSLSVFLGDRVTITCOASQRISTELSSYQO 284.

7 GO5 VEL16D2 KPGQ' pKLLIWGASRLQTlWPSRFSGSGSGTSFTLTiSG

LEAEDLATY CLQDYSWPLTl'GQGTKVELKR

27. LDIVMTQ3PS3LSASLGDRVTITCQASQ IITELSWYQQ 285. CC6 VH_16D2 KPGQTPKLLIYGASRLQTGOTSRFSG3GSGTSFTLTISG

LEAEDLATYYCLQDYNWPLTFGOGTKVELKR

27. LDIVMTQTPSSLSA3LGDRVTITCQA3QNINTDLSWYQQ 286. 0H4 VH_16B2 KPGQTPKLLFYGASGLOTGIPSRESGSGSGTSETLAISG

LEAEDLATY YCLODYNWPLTFGQGTKVELKR

27. LEIV TQSPSSLSAS-/GDRVTITCQASQSISTELSWYQQ 287. 2A4 VH_16D2 KPGOTPKLLIYDASRLQTGVPSRFSGSRSGTTFTLTISG

LEAEDLATYYCLQDYNWPLTFGOGTKVELK

27. LDIVMTQSPSSLSASLGDRVTITCQATQSISTELSWYQQ 288. 2B VH_1 D2 KPGQAPKLLIYDASKLQTGVPSRFSG3GSGRSFTLTISG

LEAEDSATY YCLODYNWPLSFGSGTRLEIK

27. LDIQLTQ3P33L3A3LGDRVTITCQA3031 IDLSWYQQ 289. 2D2 VH_16D2 KPGQTPKLLFYGA3GLQAGVPSRFSGSGSGTSFTLTING

LEASDLAT YCLQDYNWPLTFGQGTKVELKR

27. LETTLTQSPSSLSVSLGDRVTITCQASQRISTELSWYQQ 290.2G1 VH_1 D2 KPGQAPKLLIYDASTLQTGVPFRFGGSGSGTSFTLTISG

LEAEDLALY YCLODYSWPLTFGQGTKVELNR

20. ALSYDLTQPP3VSGSPGKTVTISCAGT3SDVGYG YVSW 291. 7C2 VH_29B11 YQQLPGMAPKILIYDVNKRASGIADRFSGSKSGNTASLT

ISGLQSEDEADYYCAS RRGETIVPGGGTHI/TVLG

20. A.L,SYELTQPPSVSGSPGKTVTISCAGTSSDVGYGSYVSS 292. 7C3 VH_29Bli YQ0LPG APKLLIYDVNKPASGIADRFSG3KSGNTASLT

I SGLQSEDEADY CASYRDGNKYVFGGGTKLTVLG

20 , AQSVLTQPPSVSGSPGQTVTISCAGT3EDVGYGNYVSKY 293. 3C1C VH_29B11 QQLPG APKLLIYDVNKRASGIADRFSGSKSGNTASLTI

8GLQSEDEAD YCASYRRT; DNIFGGGTHL'iVLG

20. AQSALTQPPSVSGSPGKTVTISCAGTSS IGYGNYVSWY 294. 7B2 VH_29B11 QQFPG APKFLIYDVHRRASGIADRFSGSKSGNTASLTI

SGLQPEDEAVYYCASYRRGSNAVFGGGTBLTVLG

20 , ALNFMLTQPPSVSGSPGKTVTISCAGTSSDVGYGSYVSW 295.5C1 VH_29B11 YQQLPGTAPKLLIYMVNKRASGITDRFSGSKSGNTASLT

ISGLQSEDEADYYCA3YRTGDNAAFGGGT LTVLG

20. AQSVLTQPPS SGSPGKTVTISCAGTSSDVGYGNYVS*Y 296. 5E2 VH_29B11 QQLPG APKLLIYDVNKRASGIADRFSGSKFAfTIASLTI

SGLQSEDEADYYCASYKRGDNAVFGGGT LTVLG

20 , AQSVVTQPPSVSGSPGKTVTISCAGT3SDVGYGNYVSKY 297.5H1 VH_29B11 QQLPG APKLLIYDVSKRASGIADRFSGSKSGNTASLTI

8GLQSEDEAD yCASYRRGGTAVFGGGTHLTVLG

QVQVOE S G GGLVEP GE SLRL S CAAS GE FS S ERMYWVRQP 29. 12.

65E7 VL_24B3

LQ NSLKPEDTALYYCKRR'IWYGGEYDYSGQGTQVTVSP

Ξ VQ Ε Έ S GGALVEPGGSLRL 3 CAJVS GFTFSS ERMYWVRQP 30. 12.

65B12 PGKGLEWVSAISSSGVSTYYADSVKGRFTISr<DNAi;NTVY VL_24B9

LQM SLKPEDTALY YCKERTWYGGEYD YWGQGTQVT VSS

QVQLVE S G GGLVEP GE SLRL S CAAS GF TF S S ERMYWVRQP 31. 12

65H8 PGKGLESVSAISSSGVSTYYADSVKGRF ISRDKA AITVY VL_24B3

LQMNSLKPEDTALYYCKRR'IWYGGEYDYSGQGTQVTVSS

EVQLV^SGGGLVQPGGSLRLSCAASGFTFSS YAMS WVRQA 12

77 1 PGKGLEWVSR1SSGGISTYYADSVKGRFTISRDNAKNTLY VL_24B9

LQMJ SLKPED AVY YGVRYAJWGVQWAJE'DF'WGQG QV VSS

E VQLVE S G GGLVQP GG SLRL S CAAS GF TF S S YAMS WVRQA 33. 12

77 6 PGKGLESVSRISSGGISTYYADSVKGRFTISRDNAKNTLY VL_24B9

LQMNSLKPEDTAVYYCVRYAWGVQWAFDEKGQGTQVT SS

0 VQ L VE S GG G LVQP G G S LRL S C VAS GFTFSS YAMS WVE QA 34. 12

61A7 PGKGPEKVSR.i SSGGGSTS YADS VKGRE'T ISRDNAKNTLY VL_24B9

LQM SLKPEDTAVYYCMIRAGWGMGDYWGQGTQVTVSS

ELQLVESGGGLVQPGGSLRLSCAA3GFTFS3 YAMS WVRQA 35. 12

61E7 PGKGPESVSRISSGGGSAYYADSVKGRFTISRDNAKNTLY VL_24B9

LQMNSE PEDTaVYYCANRAGWGMGDYWGQGTQVTVSS

0 VQ L VE S GG G L VHP G G S LRL S CAAS GF FSS YAMS SVE QA 36. 12

65F9 P GKGPE VSR 1 S SG GG SAY YAD S VKGRE' T I SRDNAKNT L Y VL_24B9

LQM SLKPEDTAVYYCANRAGWGMGDYWGQGTQVTVSS

37 , QTVVTQEPSLSVSPGGT VTLTCGLS SGSVTASNi YPGWEQ 12

FVQLVBSGGGLVQPGGSLELSCAASGFTFSSYEMYWVRQP QTPGQAPRALIYST DRHS GVP S RE S G S I S GNKAALT I T

61H7 PGKGLEWVSATSAGGGSTYTGDSVKGEFTISEDN¾KNTVY

GAQPEDEADYYCALDIGD TTEFGGGTHLTVLG LQMSSLKPEDTAVYYCSNKAGWG GDY GQGTQVTVSS

(IDENTICAL TO VL__24B9)

EVQLVESGGGLVQPGESLRLSCVASGFTESSERMYKVRQP 36 , 12 b.¾j / PGKGLEWVSAISSSGVSTYYTDSVKGRFTT!SRDNAK^iVY VL__24B9

LQMNSE PEDTaVYYCANRAGWGMGDYWGQGTQVTVSS

22. DVVMTQSPSSLPTSLGDSVTITCQASQSISDELSWYQQK 293.

46H ; VH__28E56 P GO Ϊ P KL L I Y GA S K LQ T GVP S R F G S G S GT S E Ϊ L T I S Gl

AED LAT Y CLQGY SWPFMFGQ'GT KVE L

22 , DIOMTOSPSSLPTSLGDSVTITCQASQSISDELSWYQOK 299.

55E10 VEi__23B6 PGQTPKLLl YGASKLQTGVPSRPSGSGSGTSFTLTISGL

EAE D LAT Y YCLQG Y SWP FMF GQGTKVE LK

22. DIQMTQSPSSLPT8LGDSVTITCQASQSISDELSWYQQK 300.

55 ; 1 VH__28E56 PGQTPKLLl YGASRLQTGVPSRF8GRGSGTSETLT ISGL

AED LAT Y CLQGY SWPFMFGQGT KVE LK

22. DIQLTQSPSSLSASLGDSVTITCQASQSISDELSSYQQi; 301.

55CI1 VH_28B6 PGQTPKLLIY GA S K LQTG VP SRFSGSGSGTSFTLTISGL

EAEDLATYiCLQGYRWPFMFGQGTKVEL

2„.. DIQMTQSPSSLSTSLGDRVTITCQASOSISTELSSYQQK 302.

55C10 VH_2SB6 P G Q T P KL L I Y G A S R L Q T G VP S RF S G X G S G T S F T L T I S GM

E AYE D LAY Y Y C L Q D Y S W P Y XF G X G T R VE I K

Table 15. Vii and VL Amino Acid Sequences of Exemplary Germlined Variants of Fab Clone 61H7.

EVQLLESGGGLVQPGGSLRLSCAA3GFTFS3YAMSWVRQ 46. 310.

QTVVrQEPSLTVSPGGTVTLTCGLSSGSVTASNYPGWFQQK AP'GKGPEWVSKlSSGGGSTYYGDSyKGRFTISRD SK T

; GIG;8 P'GQAPRALIYSTNDRRSGiTPSRFSGSTiSGG AALT TLGAQP

VYLQMN3LKTENT7AVY YCANRAiGWG GDYKGQGTQVTVS

EDEAF;YYCALDIGDITEFGGGTKLTVLG

S

EVQLVESGGGLVQPGGSLRLSCSASGFTFSSYRMSWVRQ 47. 311.

QTVVTQEPSLSV3FGGTVTI7ICGLSSGSVTASNYPGSYQQT PPGKGLEWVSPISAGGGSTYYGDSVKGRFTISRDNAKNT

; G4A5 PGQAPRALIYSTNDRHSGVPDRFSGS I LGNKAALT TTGAQP

LYLQKNSLKPEDTAVYYCA RAGWGMGDYWGQGTQVTVS DDEADYYCALDIGDITEFGGGTQLTVLG S

EVOLLESGGGLyQPGGSLRLSCAASGFTFSSYAMYiiVRQ 48 , 312. APGKGLEWVSR : SACGGSTY YGDSVK.GRFTISRDNAKNT QIVVTQEPSICWSPGGTVTLTCGLSSGSVIASNYPGWYQQT

1 C4C1

VYLQKNSLKPEDTAVYYCA RAGWGMGDYWGQGTQVTVS P'GQAvPRAvLI YSTNDRHSGVPDRFSGS I SGNKAAvLT I TGAvQA. S DDESDYYCALDIGDITEFGGGTKLTVLG

EVOLVESGGGLVQPGGSLRLSCAASGF' FSS RMSWVRQ 49 , 313.

OTVVTQEPSFSVSF'GGTVTLTCCTJSSGSVTASNYPGWYOCT PPGKGLEWVSA : SACGGSTY YGDSVK.GRFTISRDNAKNT

104C5 PGQAPRTLIYSTNDRHSGVPSRFSGSISGNKAALTITGAQP

VYLQKNSLKPEDTAVYYCANRAGWGMGDYBGQGTQVTVS EDEADYYCALDIGDITEFGGGTHLTVLG S

EVOLVESGGGLVQPGGSLRLSCAASGF' FSS AMYWVRQ 50 , 314.

QTVVTQEPSFSVSPGGIVTLTCGLS8GSVTA8NYPGKYQQT APGKGLEWVSR I SAG1GGSTYYGDSVKGRF ISRDNS NT

104C7 PGQAPRALIYSTNDRHSGVPDRFSGSISGNKAALTITGAQA

VYLQKSSLKPEDTAVYYCANRAGSGMGDYSGQGTQVTVS DDESDYYCALDIGDITEF'GGGT LTVLG

S

EVQLVESGGGIA/QPGGSLRLSCAASGFTFSSYAMYBVRQ 51. 315.

QTVVTQEPSFSVSPGGIVTLTCGLS8GSVTA8NYPGKYQQT APGFvGPESySRISAiGGGSTYYGDSVKGRFTISRD AK T

104D1 PGQAPRTLlYSTNlARHSGVPSRFSGSISGNKAALTITGAQP

LrvdjQKSSLRAEDTAVYYCANRAGSGMGDYSGQGTQVTVS

s EDEADYYCALDIGDITEFGGGTHLTVLG

EVQLWSGGGLVQFGGSLRLSCAJiSGFTFSSYA SWVRQ 52. 316.

QTVVTQEPSi'SVSPGGTVILT'CGLSSGSVT'ASNYPGWYQQT APGKGLEWVSRISAGGGSTYYGDSVKGRFTISRDNSKNT

10 D5 PGQAPRALlYSTNlA HSGVPDRFSGSILGNKAALTITGAQA

LYLOMNSLKTEDTAVYYCANRAGViG GDYKGQGTQVTVS

s EDESDYYCALDIGDITEFGGGTHLTVLG

EVQLLESGGGLVQFGGSLRLSCAJiSGFTFSSYA SWVRQ 53. 317.

QTVVTQEPSLSVSPGGTV'rLT'CGLSSGSVT'ASNYPGWYQQT APGKGPEWVSRISAGGGSTYYGDSVKGRFTISRDNAKNT

1C4 11 PGQAPRALIYSTNDRHSGVFin¾FSGSISGNKAALT:iTGAQP

VYLQFJNSLRAEDTAVYYCANRAGWGMGDYWGQGTQVTVS DDESDYYCALDIGDITEFGGGTOLTVLG S

EyQLVESGGGLVOPGGSLRLSCAASGFIFSSYAKYKVRO 54. 318,

QTVVTQEPSFSVSPGGTVTLTCGI,S3GSVTA3NYPG;WYQQT APGKGPEWVSRISAGGGS YGDSVKGRFT ISRDNSKNT

104F7 PGQAPRALIYSTNDRHSGVPSRFSGSISGNKAALTITGAQA

VYLOKNSLKPEDTAVYYCANRAGWGMGDYWGQGTQVTVS EDESDYYCALDIGDITEFGGGTHLTVLG S

EVQ /ESGGGLVQPGGSLRLSCAASGFIFSSYAKSWVRQ 55. 3 : 9 ,

QT TQEPSFSVSPGGTVTLTCGLSSGSVTASNYPGl'iYQQT APGKGPEWVSRISAGGGSTYYGDSVKGRFT ISRDNSKNT

1G4G7 PGQAPRTLIYSTNDRHSGVPSRFSGSILGNKAALTITGAQA

LYLQM SLRAEDTAVYYCANRAGWGMGDYKGQGTQVTVS EDEADYYCALDIGDITEFGGGTQLTVLG

S

3VQLVESGGGLVQPGGSLRLSCAA _L SGFTFS8YA_M£viVRQ 56. 320.

QTVVTQEPSLSVSPGGTVTLTCGLSSGSVIASNYPGWYQQT AR'GKGLEWVSRISAGGGSTYYGDSyKGRFTISRDMSKMT

; G5A1 PGQAPRALIYSTNDRHSGVPSRFSG3 I SGNKAALT !TGAQA.

LYLQM[\ ^T 8LRAEDTAVY YCA RAGWG GDYKGOGTQVTVS

EDEADYYCALDIGDITEFGGGTQLTVLG S

FVQLVESGGGLVQPGGSLRLSCAA _L SGFTFS8YA_M£viVRQ 57. 321.

QTVVTQEPSLSV3FGGTVTLTCGLSSGSVTASNYPGSFQQT APGKGLEWVSRISAGGGSTYYGDSVKGRFTISRDNSKNT

! 05A5 PGQAPRTLIYSTNDRHSGVPSRFSG3 I SGNKAALT TGAQA

VYLQKNSLKTEDTAVYYCA RAGWGMGDYWGQGTQV VS DDEADYYCALDIGDITEFGGGTHLTVLG S

EVOLLESGGGLVQPGGSLRLSCAASGFTFSSYAtiSiiVRQ 58 , 322.

QAVVTQEPSLSV3FGGTVTLTCGLSSGSVTASNYPGSFQQK APGKGLEWVSR : SAGGGS Y YGDSV .GRFT1SRDNS N

1 C 5A7 PGOAPRALIYST DRHSGTPSRFSGSLSGNKAALTILGA _L QP

VYLQKNSLKPEDTAVYYCA RAGWGMGDYWGQGTQV VS EDEADYYCALDIGDITEFGGGTHLTVLG S

EVQLVESGGGIA/QPGGSLRLSCAASGPTFSSYAKSWVRQ 5 ^c ; 323.

QAWTQEPSLTVSPGGTVTLTCGLSSGSVTASNYPGi'iFQQ?; APG GPEWVSR : SSGGGSTY YGDSVK.GRFT SRDNSKNT

105311 PGQAPRALIYSTNDRHSGVPDRFSGSISGNKAALTITGAQP

LYLQKNSLRAEDTAVYYCANRAGWGMGDYBGQGTQVTVS EDEAEYYCALDIGDITEFGGGTHLTVLG S

EVQLVESGGGIA/QPGGSLRLSCAASGPTFSSYAKYWVRQ 60 , 324.

QTVVTQEPSLTVSPGGIVTLTCGLS8GSVTA8NYPGKFQQT PPGKGPEWVS I SAGGGSTYYGDSVKGRFTISRDNSKNT

10535 PGQAPRALIYSTNDRHSGVPARFSGSISGGKAALTLLGAQP

LYLQKSSLKPEDTAVYYCANRAGSGMGDYSGQGTQVTVS EDEAEYYCALDIGDITEFGGGTHLTVLG S

EVQLVESGGGLVQPGGSLRL3CAASGFTFSSYAMSBVRQ 61. 325.

QTVVTQEPSLTVSPGGIVTLTCGLS8GSVTA8NYPGKFQQK APGrGPESVSR SSGGGSTYYGDSVKGRFTISRD AK T

10537 PGQAPRALlYSTNllRHSGVPARFSGSLLGGKAALT LGAQA

VYLQKSSLRAEDTAVYYCANRAGSGMGDYSGQGTLVTVS

s DDEAEYYCAL IGDITEFGGGTQLTVLG

EVQ1,^SGGGLVQPGGSLRL8 A_A.£GFTFSSYAJ^YSVRQ 62. 326.

QTVVTQEPSI ;VSPGGIVTLTCGLS8GSVTA8NYPGWPQQT AJGKGLEWSRISAGGGSTYYGDSWGRFTISRDNSKNT

105C1 PGQAPRALlYSTNllRHSGVPARFSGSLSGGKAALT TGAQA

VYLOM SLKPEDTAVYYCANRAGWG GDYSGQGTLVTVS

s EDEAEYYCALDIGDITEFGGGTQLTVLG

EVQ1,LESGGGLVQPGGSLRL8 A_A.£GFTFSSYAJ^SSVRQ 63. 327.

QTVVTQEPSFSVSPGGIVTLTCGLS8GSVTA8NYPGWPQQ APGKGPEWVSRISSGGGSTYYGDSVKGRFTISRDNAKNT

105C7 PGQAPRALIYSTNDRHSWV ARFSGSLSGGKAALTLLGAQP

VYLOMNSLKTEDTAVYYCANRAGViG GDYKGQGTLVTVS

EDEAEYYCALDIGDITEFGGGTHLTVLG S

ELQLVESGGGLV0PGGSLRLSCAASGFIFS8YR SKVR0 64. 328 ,

QTVVTQEPSLTVSPGGTVTLTCGLS3GSVTA3NYPGWFQQX APG GPEWVSAISAGGGS YYGDSVKGRFTISRDNSKNT

105D1 PGQAPRALIYSTNDRHSviVPARFSGSLSGNKAALTLTGAQP

LYLOKNSLRAED AVYYCANRAGWGMGDYWGQG QV VS EDEAEYYCALDIGDITEFGGGTHLTVLG S

EVQL^/ESGGGLVOPGGSLRLSCAASGETFSSYAKYWVRO 65. 329 ,

QA_ TQEPSLSVSPGGTVTLTCGLSSGSVTASNYPG\'iFQQK APG GPEWVSRISAGGGS YYGDSVKGRFTISRDNSKNT

105Ξ5 PGQAPRALIYSTNDRHSGVPARFSGSISGGKAALTLTGAQP

VYLQM SLJ^TEDTAVYYCANRAGWGMGDYKGQGTQVTVS

DDEAEYYCALDIGDITEFGGGTKLTVLG

S

ELGLLESGGGLVQPGGSLRLiSCAASGFTFSSYAMSWVRQ 66. 330.

QIVyTQEPSLTVSPGGTVTI/ICGiLSSGSVIASNYPGWFQQK AR'GKGPEWVSRISAGGGSTYYGDSyKGRFTISRDMAKMT

; C5G1 F'GQAPRALIYST1\TRHSVvVPSRFSGSLSGGlKAALTLLGAQP

LYLQMNSLKPEDTAyYYCANRAGWG GDYiiGOGTQVTyS

EDEAEYYCALDIGDITEFGGGTQLTVLG S

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAliSWVRQ 67. 331.

QTVVTQEPSLSV3FGGTVTLTCGLSSGSVTASNYPGSFQQT APGKGPEWVSRISSGGGSTYYGDSVKGRFTISRDNSKNT

; G5H11 P'GQAPRALIYSTNDRRS iVPARFSGS I SGGKAALTLLGAQP

LYLQKNSIdiAIDTAVY CA RAGWGMGD WGQGTQVTVS

EDEAEYYCALDIGDITEFGGGTQLTVLG S

EVOLVESGGGLVQPGGSLRLSCAASGFTFSSYR SiiVRQ 63 , 332.

QAVVTQEPSLSV3FGGTVTLTCGLSSGSVTASNYPGSFQQT APGKGPEWVSA : SSGGGSTY YGDSVKGRFTISRDNSKNT

1 C5H5 PGQAFRALIYSTNDRH3GOTARFSG3ILGGKAALTILGAQP

VYDQKNSLKPEDTAVY CA RAGWGMGD WGQGTLVTVS NDEAEYYCALDIGDITEFGGGTHLTVLG S

EVQLVELSGGGIA/QPGGSLRLSCAASGPTFSSYAKYWVRQ 69 , 333.

QTWTQEPSLTVSPGGTVTLTCCTjSSGSVTASNYTGSFOQK PPGKGLEWVSR : SAGGGS Y YGDSV .GRFTISRDNS N

93C1C PGQAPRALIYSTNDRHSGVPARFSGSLLGGKAALTILGAQA

VYLQMNS ^' [,KPEDTAVYYCAKRAGWGMGDYSGQGTQVTVS

DDEAEYYCALDIGDITEFGGGTQLTVLG S

ELOLVESGGGLVQPGGSLRLSCAASGPTFSSYAKYWVRQ 70 , 334.

QTVVTQEPSLSVSPGGIVTLTCGLS3GSVTA3NYPGKFQQK PPGKGLFWVSR I SAG1GGSTYYGDSVKGRF ISRDNS NT

93E1C PGQAPRALIYSTNDRHSGVPARFSGSLSGNKAALTITGAQA

VYLQKSSLKTEDTAVYYCANRAGSGMGDYSGQGTQVTVS EDEADYYCALDIGDITEFGGGTQLTVLG S

EVQLVESGGGIA/QPGGSLRLSCAASGFTFSSYA^WBVRQ 71. 335.

QAVVTQEPSLTVSPGGIVTLTCGLS3GSVTA3NYPGKFQQT FPGKGPEWSRISAGGGSTYYGDSWGRFTISRDNAJNT

93F2 PGQAPRALlYSTNllRHSGTPARFSGSLSGNKAALT TGAQP

LrYLQKSSLKTEDTAVYYCANRAGSGMGDYSGQGTQVTVS

s EDEADYYCALDIGDITEFGGGTKLTVLG

ELQLLESGGGLVQFGGSLRLSCAASGFTFSSYA YWVRQ 72. 336.

QTVVTQEPSLSVSPGGTV'rL 'CGLSSGSV'iASNYPGWFQQK APGKGLEWVSRISAGGGSTYYGDSVKGRFTISRDNSKNT

99C10 PGQAPRALlYSTNllRHSWVPARFSGSISGGKAALTLTGAQP

VYLOMNSLKPEDTAVYYCANRAGViG GDYKGQGTLVTVS

EDEAEYYCALDIGDITEFGGGTQLTVLG T

EVQLWSGGGLVQPGGSLRLSCAASGFTFSSYR SSVRQ 73. 337.

QTVVTQEPSFSVSPGGIVTLTCGLSSGSVTASNYPGWYQQT APGKGPEWVSAISSGGGSTYYGDSVKGRFT1SRDNAKNT

1C4G5 PGQAPRALIYSTNDRHSGVFDRFSGSILGNKAALTITGAQA

VYLOMNSLRAEDTAVYYCANRAGViG GDYKGQGTQVTVS

DDESDYYCALDIGDITEFGGGTHLTVLG S

EyQLVESGGGLVOPGGSLRLSCAASGFIFSSYR SKVRO 74. 333 ,

QTVVTQEPSFSVSPGGTVTLTCGI,S3GSVTA3NYPG;WYQQT APGKGiTEWySAISAGGGST YGDSVKGRFT I SRDNAKNT

108A1 PGQAPRALIYSTNDRHSGVPSRFSGSISGNKAALTITGAQA

LYLQKNSLRAEDTAVYYeANRAGWGMGDYWGQGTQVTVS

EDEADYYCALDIGDITEFGGGTKLTVL S

EVQLVESGGGLVOPGGSLRLSCAASGFTFSSYRKSWVRO 339 ,

QT TQEPSFSVSPGGTVTLTCGLSSGSVTASNYPGl'iYQQT APGKGPEWySAISAGGGST YGDSVKGRFT ISRDNSKNT

103A3 PGQAPRALIYSTNDRHSGVPSRFSGSILGNKAALTITGAQA

LYLQMNSLRAEDTAVYYCANRAGWGMGDYKGQGTQVTVS EDEADYYCALDIGDITEFGGGTKLTVL

S

EVQLVESGGGLVQPGGSLRLSCAA3GFTFS3YRMSWVRQ 76. 340.

QTVVTQEPSFSVSPGGTVTLTCGDSSGSVIASNYPGWFQQT PPG E V AI G8 YY 1

; G 3A5 PGQAPRTLIYSTNDRHSGVPSRFSGS LGNKAALT ITGAQP

LYLQMNSLKPEDTAVY YKGQGTQVTVS EDESDYYCALDI ITEFGGGTHLTVL

EVQLLESGGGLVQPGGSLRLSCAA3GFTFS3YRMYWVRQ 77. 341.

QAVVTQEPSLSV3FGGTVTLTCGLSSGSVTASNYPGSFQQT APGKGLEWVSAISAGGGS'TYYGDSVKGRFTISRDNSKNT

; G 3A9 P'GQAPRALIYSTNDRRS i GSliSGNKAALT ILGAQP

VYDQKNSLKPEDT'AVY CAKRAGWGMGD WGQGTQVTVS

EDE YYCALDIGDITEFGGGTQLTVL S

EVOLLESGGGLVQPGGSLRLSCAASGFTFSSYRMSiiVRQ 78 , 342.

QTVVTQEPSFSV3FGGTVTLTCGLSSGSVTASNYPGSYQQT

1 C831 P IYST S S SGS LG KAALTITGAQR

VYLQKNSLRAiDlAVY CA RAGWGMGD WGOG'rQV'rVS

EDESDYYCALDIGDITEFGGGTHLTVL

EVQLLESGGGIA/QPGGSLRLSCAASGPTFSS AKSWVRQ 79 - 343.

QTWTQEPRLSVSPGGTVTLTCGLSSGSVTAS YPGi'iYOQT

10833 PGQAPRALIYSTNDRHSGVPDRFSGSISGNKAALTIT'GAQA

1YLQMNSI

DDEADYYCALDIGDITEFGGGTQLTVL

EVQLVESGGGIA/QPGGSLRLSCAASGPTFSS RMSWVRQ 80 , 344.

QTVVTQEPSLTVSPGGIVTLTCGLS3GSVTA3NYPGKFQQT

1083 ^' / PGQAPRALIYSTNDRHSSVPARFSGSLSGGKAALTILGAQP

LYLQKiiSL PEDTAVYYCAiRAGWGMGDYWGQGTLVTVS

EDEAEYYCALD 'THL'TVL

EVQLLESGGGLVQPGGSLRL3CAASGFTFSSYAMSBVRQ 81. 345.

QAVVTQEPSLSVSPGGIVTLTCGLS3GSVTA3NYPGKFQQK AJGKGPEWSRISAGGGSTYYGDSWGRFTISRDNSKNT

10839 PGQAPRALIYS 'TILGAQP

LrYLQKSSLKPEDTAVYYCANRAGSGMGDYSGQGTQVTVS

s EDEADYYCALD

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYR SSVRQ 82. 346.

Q'TVVTQEPSFSVSPGGIVTLT'CGLSSGSVTASNYPGWPQQT GLiE V3A.IS G YYG VK F

108C5 PGQAPRTLlYSTNDRHSGVPDRl'SGSISGNKAALTIT'GAQA

LYLOMNSLKPED'TAVYYCANRAGViG GDYKGQGTQVTVS

s EDESDYYCALDIGDITEFGGGTHLTVL

ELQLLESGGGLVQFGGSLRLSCAJiSGFTFSSYA YWVRQ 83. 347.

Q'TVV'TQEPSI iVSPGGTVT'LT'CGLSSGSV' ASNYPGWFQQK APGKGLEWVSRI

108C9 PGQAPRALIYS'TNDRHSGVFSRFSGSLSGNKAAL'TLTGAQP

LYLOMNSLRAED'TAVYYCANRAGViG GDYKGQGTQVTVS

EDEAEYYCALDIGDITEFGGGTQLTVL

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYRMSWVRQ 84. 348 ,

3 3

.GGGSTYYCIOSVKGRFT S DN3KN

111A11 PGQ LI S SGSIL LTITGAQA

EDEADYYCALDIGDITEFGGGTHLTVL

0 8 0 65. 349 ,

QT TQEPSLSVSPGGTVTLTCGLSSGSVTAS YPGl'iYQQT PP S DN3KN

111A5 PGQ LIYSTNDRHSGVPD SGSIS LTITGAQA

YYCALDIGDITEFGGGTKLTVL

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAJMSWVRQ 86. 350.

QIVVTQEPSFSVSPGGTVIXTCGDSSGSVIASNYPGWYQQT APGKGFEWVSX1SAGGG8TYYGDSVKGRFT1SRDMSKMT

111 A7 FGQAPRALIYSTMDRHSGVPDRFSG3 I SGMKAALT !TGAQ .

LYLQMMSLRAEDTAVY YCAMPAGWG GDYKGQGTLVTVS EDEADYYCALDIGDITEFGGGTQLTVL S

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAliYWVRQ 87. 351.

QTVVTQEPSFSV3FGGTVTLTCGLSSGSVTASNYPGSFQQT PPGKGLEWVSRISAGGGSTYYGDSVKGRFTISRDNSKNT

111 Bl PGQAPRALIYSTMDRHSGVPDRFSG3 I LGNKAALT 1TGAQA

LYDQKNSITiAiDTAVY CANRAGWGMGD WGQGTLVTVS

EDEADYYCALDIGDITEFGGGTQLTVL S

EVOLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSiiVRQ 88 , 352.

QTVVTQEPSLSV3FGGTVTLTCGLSSGSVTASNYPGSYQQT APGKGPEWVSR : SAGGGSTY GDSVKGRFT1SRDNSKNT

11 IB11 PGOAPRALIYST DRHSGVPDRFSGS ISG KAALTITGAQP

VYDQKNSLKPEDTAVY CANRAGWGMGD WGQGTQVTVS DDESDYYCALDIGDITEFGGGTKLTVL S

EVQLIT:SGGGIA;QPGGSLRL8CAASGFTFSSYRMSWVRQ 89 , 353.

QTWTQEPSFSV3PGGTVTLTCGLSSGSVTASNYPGSYOQT APGKGLEWVSA : SSGGGSTY GDSVKGRFT1SRDNSKNT

11 IB5 PGQAPRALIYSTNDRHSGVPDRFSGSISGNKAALTITGAQA

VYLQMNS1,KTEDTAVYYCAKRAGWGMGDYSGQGTQVTVS

EDEADYYCALDIGDITEFGGGTQLTVL S

ELOLLESGGGLVQPGGSLRLSCAASGPTFSSYAKSWVRQ 90 , 354.

QTVVTQEPSLSVSPGGIVTLTCGLS3GSVTA3NYPGKYQQT APGKGPFWVSR I SAGGGSTYYGDSVKGRFTISRDNAKNT

11 IB7 PGQAPRALIYSTNDRHSGVPSRFSGSILGNKAALTITGAQP

VYLQKSSLKPEDTAVYYCANRAGSGMGDYSGQGTQVTVS EDESDYYCAL IGDITEFGGGTHLTVL S

EVQLI,ESGGGLVQPGGSLRL3CAASGFTFSSYAMSBVRQ 91. 355.

QTVVTQEPSFSVSPGGIVTLTCGLS3GSVTA3NYPGKYQQT AJGKGPEWSRISAGGGSTYYGDSWGRFTISRDNSKNT

1 IIC11 PGQAPRALIYSTN11RHSGVPSRFSGSISGNKAALT1TGAQP

LYLQKiiSL PEDTAVYYCAiRAGWGMGDYWGQGTLVTVS

s EDESDYYCAL IGDITEFGGGTQLTVL

EVQLV^SGGGLA'QPGGSLRLSCAJVSGFTFSSYRMYSVRQ 92. 356.

QTVVTQEPSI ;VSPGGIVTLTCGLS3GSVTA3NYPGWYQQT AJGKGPEWSAISAGGGSTYYGDSWGRFTISRDNSKNT

1 IIC5 PGQAPRTLIYSTN11RHSGVPDRFSGSISGNKAALT1TGAQP

LYLOMMSLKPEDTAVYYCANRAGViG GDYKGQGTQVTVS

DDEADYYCALiLEGDITEFGGGTHLTVL

s

EVQLLESGGGLVQFGGSLRL3CAJiSGFTFSSYAM35ft¾Q 93. 357.

QTVVTQEPSFSVSPGGIVTLTCGLS3GSVTA3NYPGWPQQT APGKGPEWSRISSGGGSTYYGDSVKGRFTISRDNSKNT

1 IIC9 PGQAPRALIYSTNDRHSGVFDRFSGSISGNKAALTITGAQP

VYLOMMSLKPEDTAVYYCANRAGViG GDYKGQGTQVTVS

DDEADYYCALDIGDITEFGGGTKLTVL S

ELQLLESGGGLV0PGGSLRLSCAASGFIFS5YA YKVR0 94. 358 ,

QTVVTQEPSLSVSPGGTVTITrCGLSSGSVTASNYPGWYQQT PPGKGiLSWVSRISSGGGS YGDSVKGRFIdSRDNAKNT

11 ID7 PGQAPRALIYSTMDRRSGVPSRFSGSISGMKAALTITGAQA

LALOKNSLKTEDTAVYYCANRAGWGMGDYWGQG QV VS DDESDYYCALDIGDI EFGGGTHLTVL

s

EyQ /ESGGGLVOPGGSLRLSCAASGFTFSSYAKYWVRO 95. 359 ,

QT TQEPSFSVSPGGTVTLTCGLSSGSVTAS YPGl'iFQQT APGKGPEWVSRISAGGGS YGDSVKGRFTISRDNAKNT

11 ID9 PGQAPRALIYSTMDRRSGVPSRFSGSISGMKAALTITGAQA

VYLQMMSLKPEDTAVYYCAMRAGWGMGDYKGQGTQVTVS EDEADYYCALDIGDITEFGGGTKLTVL

S

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAliYWVRQ 96. 360.

QTVVTQEPSFSVSPGGTVTLTCGLSSGSVTASNYPGWFQQT

'GKGPEWVSRISAGGGSTYYGDSyKGRFTISRDMSKMT

111 E 11 PGQAPRALI YSTNDRHSGVPDRFSGS LGNKAALT TTGAQP

LYLQM[\ ^T 3LKPED!AVY YGA RAGWG GD YKGOG'ILVT VS

E E SD YYCALD I GD I TE FGGGTQLTVL

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAliYWVRQ 97. 361.

QTVVTQEPSFSVSFGGWTLTCGLSSGSVTASNYPGSYQQT APGKGPEWVSRISAGGGSTYYGDSVKGRFTISRDNSKNT

I 11E7 PGQAPRALI YSTNDRHSGVPDRFSGS SGNKAALT TTGAQP

LYLQKNSLKPEDlAVY CA RAGWGMGDYWGOG'rQV'rVS

DDE AD YYCALD IGDITEFGGGTHLTVL S

EVOLLESGGGLVQPGGSlRLSCAASGFTFSSYAMSliVRQ 98 , 362.

QTVVTQEPSFSVSFGGWTLTCGLSSGSVTASNYPGSYQQT APG GPEWVSR : SAGGGSTY YGDSVK.GRFT I SRDNAKNT

11 IE 9 GO AP RAL I YST DRH S GVP S RF S S I S TIT GAQR

VYLQKNSLRAiDlAVY CA RAGWGMGDYWGOG'rQV'rVS

EDEADYYCALDIGDI'TEFGGGTKLTVL

S

EVOLVESGGGLVQPGGSlRLaCAASGF' SSYAMYWVRQ 99 , 363.

QTWTQEPSFSVSPGGTVTLTCGLSSGSVTAS YPGi'iYOQT : SSGGGSTY YGDSVK.GRFT I SRDNAKNT

11 IF 11 PGQAPRTLI YSTNDRHSGVPDRFSGS ISGNKAALT TTGAQP

VYLQMNSI,KTEDTAVYYCAKRAGWGMGDYSGQGTQVTVS

DDESDYYCALDIGDI'TEFGGGTQLTVL

S

EVOLLESGGGLVQPGGSIRLSCAASGPT SSYAKSWVRQ 100. 364.

QTVVTQEPSFSVSPGGTV'ILTCGLSSGSVTASNYPGKYQQT APGKGPEWVS I SAGGGSTY YG SVKGRFTISRDNAKNT

11 IF 7 PGQAPRALI YSTNDRHSGVPDRFSGS ISGNKAALT ITGAQA

VYLQKSSLKTEDTAVYYCANRAGSGMGDYSGQGTQVTVS DDESDYYCALD IGDITEFGGGT LTVL S

EVQLVESGGGLVQPGGSLRL3CAASGFTTSSYRKYBVRQ 101. 365.

QTVVTQEPSFSVSPGGTV'ILTCGLSSGSVTASNYPGKYQQT APGr GlESySAISAGGGSTYYGDSVKGRFTISRD SK T

111F9 PGQAPRAL I YSTNllRHSGVPDRFSGSILGNKAALT TTGAQP

VYLQKSSLRAEDTAVYYCANRAGSGMGDYSGQGTLVTVS

s EDEADYYCALD IGDITEFGGGTKLTVL

EyQLLESGGGLyQPGGSLRLSCAASGFTFSSYA^YSyp 102. 366. APGKGPESVSRISAGGGSTYYGDSVKGRFTISRD SK T

111G1 PGQAPRAL lYSTNll HSGVPSRFSGSILGNKAALTTTGAQA

VYLOM SlKPEDTAVYYCANRAGViG GDYSGQGTLVTVS

s DDEADYYCAI.D IGDITEFGGGTHLTVL

EVQLWSGGGLVQPGGSLRLSCAASGFTFSSYR SSVRQ 103. 367.

QTVVTQEPST'SVSPGGTVT 'ASNYPGWYQQT APGKGLEWVSAISAGGGSTYYGDSVKGRFTISRDNAKNT

111 11 PGQAPRALI YSTNDRHSGVFiH¾FSGS ISGNKAALT ITGAQA

VYLOM SlKTEDTAVYYCANRAGViG GDYSGQGTQVTVS

DDE SD YYCALD I GD I EFGGGTHLTVL S

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMYWVRQ 104. 368 ,

QTVVTQEPSLSVSPGGTVTLTCGLS3GSVTA3NYPGWYQQT PPGKGPEWVSRISAGGGST YGDSVKGRFTIKRDNSJKNT

111G7 AP RA LI YSTMDRRS GVP D RF S S I S GNKAA T I T AQP

VYLQKNSLKPEDTAVYYeANRAGWGMGDYWGQGTQVTVS

DDE SD YYCALD I GD I EFGGGTHLTVL

S

/FSGGGIVOPGGSLRLSCAASGE!FSSYRKYWVRO 105. 369.

QT TQEPSLSVSPGGTVTLTCGLSSGSVTAS YPGl'iYQQT APG GidSWVSAISSGGGa ^' r YGDaVKGRETISRDNAKN ^' r

111G9 AP RA LI YSTMDRRS GVP SRFSGSI L GNKAA T I T AQP

VYLQMNSLKPEDTAVYYCANRAGWGMGDYKGQGTQVTVS EDEADYYCALD IGDITEFGGGTHLTVL

S

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAliYWVRQ 106. 370.

QTVVTQEPSILSVSPGGTVTLTCGLSSGSVIASNYPGWYQQT PPGKGPEWVSKlSAGGGSTYYGDSyKGRFTISRDMSKMT

111 H7 PGQAPRALIYSTNDRHSGWPSRFSG3 I LGNKAALT TTGAQP

VYLQM SLKTEDTAVYYCANRAGWG GDYiiGOGTQVTyS

DDEADYYCALDIGDITEFGGGTHLTVL S

EVQLVESGGGLVQPGGSLRLSCSASGFTFSSYRMSWVRQ 107. 371.

QTVVTQEPSLSV3FGGTVTLTCGLSSGSVTASNYPGSFQQT APGKGLEWVSAiSAGGGSTYYGDSVKGRFTISRDNAK T

111 H9 PGQAPRALIYSTNDRHSGWPDRFSG3 I SGN AALT TTGAQA

VYLQKNSLKPEDTAVY CA RAGWGMGD WGQGTLVTVS DDEADYYCALDIGDITEFGGGTHLTVL S

EVOLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSiiVRQ 103. 372.

QTVVTQEPSLTV3FGGTVTLTCGLSSGSVTASNYPGSFQQK APG GPEWVSR : SSGGGSTY YGDSVK.GRFTISRDNSKNT

112A11 PGQAPRALIYSTNDRH3GVPARFSG3LLGGKAALTILGAQA

LYLQKNSIdiAEDTAVY CA RAGWGMGD WGQGTQVTVS

DDEAEYYCALDIGDITEFGGGTQLTVL S

EVOLVESGGGLVQPGGSLRLaCAASGF' FSS RMSWVRQ 109. 373.

QTWTQEPSLTVSPGGTVTLTCGLSSGSVTAS YPGi'iFQQi; APGKGLEWVSA : SAGGGSTY YGDSVK.GRFTISRDNAKNT

112A4 PGQAPRALIYSTNDRHSKVPARFSGSISGGKAALTILGAQP

LYLQMNST.P.ASDTAVY'/CAKRAG GMGD'WGQGTQVTVS

EDEAEYYCALDIGDITEFGGGTQLTVL S

EVOLVESGGGLVQPGGSLRLSCAASGPTFSS AKYWVRQ 110. 374.

QTVVTQEPSLSVSPGGIVTLTCGLS3GSVTA3NYPGKFQQK APGKGF'EWVSR I 3SG1GGSTYYGDSVKGRF ISRDNS NT

112A7 PGQAPRALIYSTNDRHSGVPARFSGSLSGGKAALTILGAQP

VYLQKSSLKPEDTAVYYCANRAGSGMGDYSGQGTQVTVS EDEADYYCALDIGDITEFGGGTHLTVL S

EVQLLESGGGLVQPGGSLRL3CAASGFTFSSYRMSWP.Q 111. 375.

QTVVTQEPSLTVSPGGIVTLTCGLS3GSVTA3NYPGKFQQK APGi;GLE SA SAGGGSTYYGDSVKC47FTISRDNAKNT

112B1 PGQAPRALlYSTNDRHSWVPARJ'SGSLLGG AALTII'GAQP

LYLQKiiSL PEDTAVYYCAS!RAGWGMGDYWGQGTLVTVS

3 EDEAEYYCALDIGDITEFGGGTHLTVL

EVQI^SGGGIA'QPGGSLRLSCAASGFTFSSYAJ^SSVRQ 112. 376.

QTVVTQEPSRIVSPGGIVTITL IGLSSGSVTASNYPGWFQQK APG¾GPE SRISAGGGSTYYGDSVKC47FTISRDNAKNT

112B11 PGQAPRALIYSTN11RHSWVPARFSGSLSGGKAALTLTGAQP

VYLOMNSLKPEDTAVYYCANRAGViG GDYKGQGTLVTVS

3 EDEAEYYCALDIGDITEFGGGTHLTVL

EVQLWSGGGLVQFGGSLRLSCAJiSGFTFSSYRLYWVRQ 113. 377.

QTVVTQEPSRIVSPGGIVTITL IGLSSGSVTASNYPGWFQQK PPGKGPEWVSAISAGGGSTYYGDSVKGRFT ISRDNAKNT

112C11 PGQAPRALIYSTNDRHSWVPSRFSGSLSGGKAALTITGAQP

VYLOMNSLKPEDTAVYYCANRAGViG GDYKGQGTQVTVS

EDEAEYYCALDIGDITEFGGGTQLTVL

3

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMYWVRQ 114. 378 ,

QTVVTQEPSLTVSPGGTVTLTCGLS3GSVTA3NYPGWFQQT PPGKGPEWVSRISAGGG3T YGD3VKGRELT ISRDNAKNT

112C7 PGQAPRALIYSTNDRHSGVPARFSGSLSGGKAALTITGAQP

LYLOKNSLKPEDTAVYYCANRAGWGMGDYWGQG QV VS EDEADYYCALDIGDITEFGGGTHLTVL S

EVQLJJESGGGLVOPGGSLRLSCAASGFTFSSYAKYWVRO 115. 379.

QivV^/TQEPSLTVSPGGTVTLTCGLSSGSVTAS YPGl'iFQQT APGKGPEWVSRISAGGG3T YGD3VKGRELT ISRDNAKNT

112C9 PGQAPRALIYSTMDRHSGVPARF3G3L3GNKAALTITGAQA

LYLQMNSLRAEDTAVYYCANRAGWGMGDYKGQGTLVTVS EDEAEYYCALDIGDITEFGGGTHLTVL

S

EVQLLESGGGLVQPGGSLRLSCSASGFTFSSYRMSWVRQ 116. 360.

QTVVLQEPSLSVSPGGTVTLTCGLSSGSVTASNYPGWFQQK PPGKGPEWVSAISSGGGSTYYGDSVKGRFTISRDNSKNT

112D11 FGQAPRALIYSTNDRHSGVPARFSG31LGGKAALTLTGAQP

LYLQM[\ ^T 3LKPEDT7YVY YGA RAGWG GDYKGQGTQVTVS

EDEftEYYCALDIGDITEFGGGTQLTVL

S

EVQLVESGGGLVQPGGSLRLSCSASGFTFSSYRMSWVRQ 117. 361.

QTVVTQEPSLTV3FGGTVTLTCGLSSGSVTASNYPGSFQQT APGKGPEWVSAiSSGGGSTYYGDSyKGRF'IISRDNAK T

112D7 PGQAPRALIYSTNDRHSWPSRFSGSLLGGKAALTT.TGAQP

LYLQKNSLKPEDTAVY CA RAGWGMGDYWGQGTLVTVS EDEAEYYCALDIGDITEFGGGTQLTVL S

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMY!SVRQ 113. 382.

QTVVTQEPSLTV3FGGTVTLTCGLSSGSVTASNYPGSFQQK PPGKGLEWVSR : SSGGGSTY YGDSVK.GRFT SRDNAKNT

112D9 PGQAFRALIYSTNDRHSGOTSRFSGSLLGGAALTLLGAQP

VYLQKNSLKPEDTAVY CA RAGWGMGDYWGQGTQVTVS EDEAEYYCALD IGDITEFGGGTQLTVL S

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQ 119. 383.

QTWTQEPSLTV3PGGTVTLTCGLSSGSVTAS YPGSF0QT APGKGPEWVSR : SAGGGSTY YGDSVK.GRFT SRDNAKNT

112E11 PGQAPRALIYSTNDRHSSVPARFSGSLSGGKAALTLTGAQP

1,YLQMNSI,KTEDTAVYYCAKRAGWGMGDYSGQGTQVTVS

EDEADYYCALDIGDITEFGGGTKLTVL

s

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYRMYWVRQ 120. 384.

QAVVTQEPSLSVSPGGTVTLTCGLSSGSVTAS YPGSFQQT PPGKGF'EWVSA I SSGGGSTYYGDSVKGRF'T SRD A NT

112E4 PGQAPRALIYSTNDRHSSVPARFSGSLLGGKAALTITGAQP

VYLQKSSIKTEDTAVYYCANRAGSGMGDYSGQGTQVTVS EDEAEYYCALDIGDITEFGGGTHLTVL

s

EVQLLESGGGLVQPGGSLRL3CAASGFTFSSYAMYWP.Q 121. 385.

QTVVTQEPSLSVSPGGTVTLTCGLSSGSVTAS YPGSFQQT AJGKGLEWSRISSGGGSTYYGDSWGRFTISRDNSKNT

112E7 PGQAPRALlYSTNDRHSGVPARFSGSILGGKAALTILGAQP

irYLQKSSlKPEDTAVYYCANRAGSGMGDYSGQGTQVTVS

s EDEAEYYCALDIGDITEFGGGTHLTVL

EVQLLESGGGLVQFGGSLRLSCAASGFTFSSYA SWVRQ 122. 386.

QTVVTQEPSI ;VSPGGIVTLTCGLS8GSVTA8NYPGWPQQT APGrGPESySR SSGGGSTYYGDSVKGRFTISRD AK T

112F 11 PGQAPRALIYSTNDRHSGVPSRFSGSILGGKAALTLTGAQP

VYLOM SLKTEDTAVYYCANRAGViG GDYKGQGTLVTVS

s EDEAEYYCALDIGDITEFGGGTQLTVL

EVQLLESGGGLVQFGGSLRLSCAASGFTFSSYA SWVRQ 123. 387.

QTVVTQEPSLSVSPGGTVrLT'CGLSSGSVT'ASNYPGWFQQT APGKGLEWVSRISAGGGSTYYGDSVKGRFT 1SRDNSKNT

112G11 PGQAPRALIYSTNDRHSGVFARFSGSISGGKAA1TLLGAQP

VYLQFj SLKPEDTAVYYCAKRAGWGMGDYWGQGTQVTVS

EDEAEYYCALDIGDITEFGGGTQLTVL S

EVQLLESGGGLVQPGGSLRLSCfiASGFTFSSYAMYWVRQ 124. 388,

QTV/TQEPSLSVSPGGTVTLTCGLS3GSVTA3NYPGWFQQX PPG G 5WVSRISAGGG8T YGD8VKGRELTdSRDNAKNT

112G4 PGQAPRALIYSTNDRHSGVPARFSGSISGGKAALTLLGAQA

VYLOKNSLRAEDTAVYYeANRAGWGMGEjYWGQGTLVTVS

EDEAEYYCALDIGDITEFGGGTQLTVL

8

EVQ!JJiSGGGLVQPGGSLRLSCAASGFIFSSYAKYWVRQ 125. 389 ,

QT TQEPSLSVSPGGTVTLTCGLSSGSVTAS YPGl'iFQQT SPGKGPEWVSRISSGGG3T YYGDSVKGRF I KRDNSKNT

112G7 PGQAPRALIYSTMDRRSGVP RFSGSLSGMKAALTITGAQP

LYLQM SLJ PEDTAVYYCANRAGWGMGDYKGQGTLVTVS EDEAEYYCALDIGDITEFGGGTQLTVL

S

ELQLLESGGGLVQPGGSLRLSCSASGFTFSSYR YWVRQ

.PEWVSAISAGGGSTYYGDSyKGRFTISRD AK T

YSTNDRHSGVPARFSGS I EDEftD

Table 16. VH and VL Amino Acid Sequences of Exemplary Germlined Variants of Fab Clone 68F2.

FAB VH SEQUENCE SEQ VL SEQUENCE SEQ !D CLONE ID NO NO

QVQLQESGPGLVKPSQTLSLTCTVSGGS ITTRYYAWSWI RQPPGKGLEW GVIDYEGDTyYSPSL SRVSISWDTSKK

QFSLQLSSVTAEDTAVYYCARDPDyVTGFHYDYWGOGTO EDEADYYCASYRNFNNAVFGRGTKLTVL

QVQLQE I .YYAWS l QSVLTQPPSVSGAPGQTVTISCAGAN DIGTYAYVSWYQQL RQPPGKGLEGWIGVI DYDGDTYYSPSLKSRTT SWDTSKNi 'FAPRLL

QFSLQLSSVTPEDTAVYYCARDPDVVTGFHYD WGQGTM EijEADYYCAS RNFNNAVF'GGGTKLTVL

VTVSS

QVQLQESGPGLVKPSQTLSLTCTVSGGS TSRYYAWSWI QSALTHPPLVSGAPGQTVTI SCAGANND IGTYAYVSWYQQL RQPPGKGLEGWIGVI DYDGDTYYSPSLKSRTT SWDTSKNi 'FAPKLL

QFSLKI.SSVTAADTAVYYCARDPDVVTGPaYDYWGQGTM EDEADYYCASYRNFNNAVFGRGTKLTVL

TVSS

EVQLQESGPGLVKPSQTLSLTCTVSGGS TSRYYAWSWI QSALTQPPLVSGAPGQRVTI SCAGANND IGTYAYVSWYQQL RQPPGKGLEWTGVTDYDGDTYYSPSLKSRT ISWDTSKNi PGT PKLLT YKVTTR SGVP' SRF'SGSKSGlftTASLTTSELQS QFSLQI.SSVTPEDTAVYYCARDPDVVTGFLIYDYWGKGTL EDEADYYCASYRNFNNAVFGGGTLLTVL

^VSS

EVQLQESGPGLVKPSQTLSLTCTVSGGS ΐ TSRYYAWSWI I

RQPPGKGLEWIGVIDYDGBTYYSPSLKSRTTISWDTS N I YKVT RA GVP DRFSGSKSGN AS i GL

QFSLKLSSVTPADTAVYYCARDPDVVTGFHYDYWGQGTL EDEADYYCASYRNF NAVEGGGTKLTVL VTVSS

QVQLQESGPGLVKPSQTLSLTCTVSGGS ITSRYYAWSWI QSALTQPPLVSGAPGQTVTISCAGANNDIGTYAYVSWYQQL RQPPGKGLEWIGVIDYDGBTYYSPSLKSRVSISWDTS N Y'KVT

QFSLQLSSVTAADTAVYYCARDPDWTGFHYDYWGQGT EDEADYYCASYRNFNNAVEGGGTKLTVL VTVSS

EVQLQESGPGLVKPSQTLSLTCTV'SGGSITTRYYAWSWI QSVLTQPPLVSGAPGQTVTISCAGANNDIGTYAYVSWYQQL RQPPGKGLE IGVID YDGDT .TS S DTSKN PGTAPKLLIYKVTTPASGVPDRFSGSKSGNTASLAITGLOA

QFSLKLS3VTAEDTAVYYCARDPDWTGFHYDYSGQGTT EFEAFYYCASYR F AVFGRGTKLTVL

VTVSS

128E7 EVQLQESGFGLVKPSQTLSLTCTVSGGSTTSRYYAKSWI ! 35. QSVLTQF'PJVSGAPGQTVTTS GA DT TYAYVSViYQQLi 399.

RQPPGKGLEWIGVIDYDGDTYYSPSLKSP.TTISVDTSKN PGTAPKLLlYKVTTP^SGVPURFSGSKSGATASLTiTGLQS QFSLFILSSVTAEDTAVYYCARDPDWTGFHYDYSGQGTQ EDEADYYCASYR F AVFGRGTHLTVL VTVSS

128F3 EVQLQESGPGLVKPSQTLSLTCTVSGGSITSRYYAWSWI 136. QSVLTQPPLVSGAPGQTVTISCAGANNDIGTYAYVSWYQQL 400.

RQPPGKGLEWIGVIDYDGDTYYSPSLKSP.TSISVDTSKN PGTAPKLLIYKVTTRASGiPSRFSGSKSGNSASLTiSGLQA QFSLQLSSVTAEDTAVYYCARDPDWTGFHYDYWGQGTL EDEADYYCASYRNFNNAVFGGGTKLTVL VTVSS

123F7 EVOLQESGPGLVKPSQTLSLTCTVSGGSITTRYYAiiSWI 137 , OSVLTQPPLVSGAPGQTVTISCAGA DIGTYAYVSKYQQL 401.

RQPPGKGLEWTGVTDYDGDTYYSPSLKSP.VTI SWDTSK[\ ^T PGTAPKLLIYKVTTPASG1PSRFSGSKSGNTASLT1TGLCA QF'SLQFSSVTPED'raVYYCARDPDVyTGFHYDYWGQGT ^' r EDEADYYCASYRNF NAVFGRGTKLTVL

VTVSS

123F6 QVQLQESGPGLVKPSQTLSLTCTVSGGSITTRYYAWSWI 138 , OSVLTQPPLVSGTPGQRVTISCAGA DIGTYAYVSKYQQL 402.

RQPPGKGLEWIGVID DGDTYYSPSLKSRTT.L SWDT8KN PGTAPKLLTYKVTTRASG; PDRFSGSKSGNTASLT FIGLCA QF' SLQ FS S VTTdiD'raVYYCARDPDVyTGFHYDYWGQGTL EDEADYYCASYRNF NAVFGRGTHLTVL

VTVSS

128G3 EVCljpJLSGPGLVKPSQTLSiFCCTVSGGSTTTRYYAWSWI 139 , OSAFTQPPSVSGAPGQTVTl SCAGANND : GTYAYVSWYQQL 403.

RQPFGKGLE IGVIDYDGDTYYSPSLKSRTTTSVDTSKN PGTAPKLLTYKVTTRASGVPDRFSGSKSGNTASL iSGLCA QFSLKLSSVTPEDTAVYYCARIJPDVVTGFFYDYWGQGTM EDE DYYCASYRNFKNAVFGGGTaLTVL

VTVSS

; 28H7 EVQLQESGPGIA/KPSQTPSLTCTVSGGSITTRYYASSWI 140. QSALTQPPLVSGSPGQSVTTSCAGANNDIGTYAYVSBYQQF 404.

RQPFGKGLE IGVIDYDGDTYYSPSLKSRTTTSWDTSKN P'GTAPKTiLTYKVTTRASG TPiTRFSGSKSG T SFT I SGLQS QFSLKLSSVTAEBTAVYYCARDPDyV GFHYDYWGQGTQ EDEADYYCA _T SYRMFNMA-/FGGGTKLTVL

VTVSS

; 29A10 EVQLQESGPGIA/KPSQTPSLTCTVSGGSITSRYYASSWI 141. QSALTQPPLVSGSPGQTVTT SCAGANND I GTYAYVSBYQQF 405.

RQPFGKGLEWIGVIDYDGDTYYSPSLKSRTTISWDTSKN PGTAPKLf-'iiYKVTTRASGIPSRFSGSKSG TASLTTSG QS QFSLKLSSVTAABTAVYYCARDPDyV GFHYDYWGQGT EDEADYYCA _T SYRMFNMA-/FGTGTKLTVL

VTVSS

129A3 EVQLQESGPGLVKPSQTLSLTCTVSGGSITTRYYASS I 142. QSALTQFPSVSGSPGQTVTISCAGAN DIGTYAYVSWYQQL 406.

RQPFGKGLEWIGVIDYDGDTYYSP3LK3RT3ISWDT3KN PGTAPKLf-'iiYKVTTRASGIPDRFSGSKSG TASLTTSG QS QFSLKLSSVTAADTAVYYCARDPDVVTGFHYDYWGPGTO EDEADYYCASYR FN AFVF'GR.GTRLTVL

VTVSS

129A5 EVQLQESGPGLVKPSQTLSLTCTVSGGSITSRYYASS I 143. QSALTQFPSVSGSPGQTVTISCAGAN DIGTYAYVSWYQQL 407.

RQPPGKGLEiilGVlDYDGDTYYSPSLKSRTSISVDTSKN PGTAPKLL1YKVTTRASGIPSRFSGSKSG TASLTISGLQS QFSLQLSSVTAEDTAVYYCARDPDVVTGFT'YD WGOGTM EDEADYYCASYRNFNNAVF'GTGTKLTVL

VTVSS

129A9 QVQLQESGPGLVKPSQTLSLTCTVSGGSITTPA'YAWSWI 144. QSALTQPPLVSGiPGQSVTISCAGANNDIGTYAYVSKYQOL 403.

RQPPGKGLEWMGV; DYDGDTYYSPSFKSRVSISWDTSKNi PGTAPKFL; YKVTTRaSGVPDRF'SGSKSGNTASLTISGLQS

QFSLOPSSVTAEDTAVYYCARBPBWTGFHYBYSGQGTL EBEABYYCASYR F AVFGTGTKLTVL

VTVSS

12933 EVQLQESGFGLVKPSQTLSLTCTVSGGSTTTRYYAK3WI ; 45. QSftLTQPPSVSGSPGQTVTISCftGA BIGTYAYVS YQQL 409.

RQPPGKGLEWIGVIBYBGBTYYSPSLKSR\rTISVBTSKN PGTAPKLMTYKVTTR.ASGiPSRFSGSKSGNTA _L Sl,TiSGLCA QFSLKLSSVTPEDTAVYYCARBPBWTGFHYBYSGQGTM EDEADYYCASYR F AVFGGGTKLTVL VTVSS

129B7 EVQLQESGPGLVKPSQTLSLTCTVSGGSITTRYYAWSWI 46. QSALTQPPLVSGSPGQSVTISCAGANNBIGTYAYVSWYQQL 410.

RQPPGKGLEWIGVIDYDGBTYYSPSLKSRTTISWDTS N PGTAPKLMIYK\ ^r TTRASGVPBRFSGSKSGNTASL iSGLQS QFSLKLSSVTAEDTAVYYCARBPBVVTGFHYBYWGQGTQ EBEABYYCASYRNFNNAVFGTGTKLTVL VTVSS

129B6 EV0LQESGPGLVK?SQ ¹ ΓLSLTC ¹ ΓVSGGSITSRYYA.KSWI 147 , OSALTQPPLVSGSPGQTVTISCAGA BlGTYAYVSiiYQQL 411.

RQPPGKGLEWTGVTDYDGDTYYSPSLKSP.TT I SWDTSKN PGTAPKLLIYKVTTPASGVPBRFSGSKSGSTASLTiSGLOS QFSLQLSSVTAElTl'AVYYCARllPDVVTGFHYDYWGQGTQ EBEAB YCASYRNF NAVFGRGT LTVL

VTVSS

129C10 QVOLQESGPGLVKPSQTLSLTCTVSGGSITSRYYAWSWI 148 , OSA TQPPSVSGSPGQSVTISCAGA BlGTYAYVSiiYQQP 412.

RQPPGKGLEWIGyiD BGDT YSPSLKSRTTl SWDT8KN PGTAP LLlYKVTTRASGVPSRFSGSKSGNiTASLT ISGLQS QFSLKLSSVTAElTl'AVYYCARllPDVVTGFHYDYWGQGTQ EBEAB YCASYRNF NAVFGTGT LTVL

VTVSS

123011 QVOLQESGPGTA/ PSQTLSLTCTVSGGSTTTRY AWSWI 149 , OSALTQPP.LVSGSPGQTVTT SCAGANN!) 1 GTYAYVSW QQ.L 413.

RQPPGKGLE IGVIBYBGDTYYSPSLKSRV TSVBTSKN AGTAP LMTYKVTTRASG; PSRFSGSKSGNTASLT ISGLQA OISLJ^LSSVTPEBTAVYYCARIJPDVVTGFLYDYWGQGTQ EDE DYYCASYRNFKNAVFGTGTaLTVl,

VTVSS

; 29 11 EVQLQESGPGLVKPSQTPSLTCTVSGGSITSRYYABSWI 150. QSALTQPPLVSGSPGQSVTTSCAGANNBIGTYAYVSBYQQL 414.

RQPPGKGLE IGVIBYBGDTYYSPSLKSRVSTSWBTSKN P'GTAP liM Y V ^' TTRASGVPSRFSGISKSG T SLT I SGLQS QFSLKLSSVTAEBTAVYYCARDPDyV GFHYDYWGQGTL E3EA3YYCA _t SYRMFNMA-/FGTGTHLTVL

VTVSS

; 29 2 EVQLQESGPGIA/KPSQTPSLTCTVSGGSITPRYYVSTWI 151. QSALTQPPSVSGSPGQTVTT SCAGANNB I GTYAYVSBYQQL 415.

RHPPGKGLBWIGVIBY3GBTYYSPSLKSRTTIS BTSKN PGTAPKll-'iiYKVTTRASGVPSRFSGSISG TASLTISG .QA QFSLQLSSVTAEBTAVYYCAR7TBWTGFHYDYWGQGTQ E3EA3YYCA _t SYRMFNMA-/FGGGTHLTVL

TVSS

129D3 QVQLQESGPGLVKPSQTLSLTCTVSGGS ITSRYYASSWI 152. QSALTQPPSVSGTPGQSVTISCAGANNBIGTYAYVSWYQQL 416.

RQPFGKGLEWIGVIBY3GBTYYSP3LK3RV3ISWBT3KN PGTAPKll-'iiYKVTTRASGIPBRFSGSKSG TASLTISG .QA QFSLKLSSVTPABTAVYYCARBPBVVTGFHYBYWGOGT EBEABYYCASYR FN AA/F'GTGTKLTVL

VTVSS

129D5 EVQLQESGPGLVKPSQTLSLTCTVSGGSITSRYYASS I 153. QSALTQPPSVSG3PGQSVTISCAGAN BIGTYAYV3WYQQL 417.

RQPPGKGLEWIGVIDYDGDTYYSPSLKSRTTISWC'TSKN PGTAPKLLIYKVTTRASGVPBRFSGSKSG TASLTISGLQS QFSLQLS3VTAADTAVYYCARDPDVVTGFHY3 WG0GTL EOEAOYYCAS RNFNNGVF'GTGTKLTVL

VTVSS

129D3 EVQLQESGPGLVKPSOTLSLTCTVSGGSITSRYYAKSWI 154. QSALTQPPLVSGSPGQSVTISCAGANNBIGTYAYVSKYQOL 413.

RQPPGKGLEWIGVI DYDGBTYYSPSP SRTSISWBTSKN PGTAPKLKl YKVTTRASGVPORFSGSKSGNTASLTISGLQA

QFSLQLSSVTAEDTAVYTCARBPDWTGFHYDYSGQGTL EDEADYYCASYR F AVFGGGTHLTVL

VTVSS

129H6 EVQLQESGFGLVKPSQTLSLTCTVSGGSTTTRYYAKSWI ; 65. QS ITQF'PJVSGTPGQTVT! S GAN DT TYAYVSViYQQLi 429.

RQPPGKGLEWKGVIDYDGBTYYSPSLKSRTSISWDTS N PGTAPKLLlYKVTTRASGiPSRFSGSKSGNTASLTiSCiQS QFSLOPSSVTPEDTAVYYCARDPDWTGFHYDYSGQGTQ EDEADYYCASYR F AVFGRGTHLTVL VTVSS

129H7 QVQLQESGPGLVKPSQTLSLTCTVSGGSITSRYYAWSWI 166. QSA TOPPSVSGSPGQSVTISCAGAM DIGTYAYVSWYQQP 430.

RQPPGKGLEWIGVIDYDGDTYYSPSLKSP.TTISWDTSKN PGTAPKLLlYKVTTPASGVPSRFSGSKSGSTASLTiSGLQS QFSLKLSSVTAEDTAVYYCARDPDWTGFHYC'YWGQGTQ EBEABYYCASYRNFNNAVFGTGTKLTVL

VTVSS

129H6 EVQLQESGPGLVKPSQTLSLTCTVSGGSITTRYYAWSWI 167, 03ALTQPPLV3GSPGQSVTT SCAGA [\ ^T D 1GTYAYVSKYQQL 431.

RQPPGKGLEWTGVTDYDGDTYYSPSLKSP.VSI SWDTS [\ ^T PGTAPKLLIYKVTTPASGVPSRFSGSKSGSTASLTiSGLOS QFSLQLSSVTAEDTAVYYCARDPDVVTGFHYDYWGQGTL EDEADYYCASYRNF NAVFG GTOLTVL VTVSS

129H9 EVQLQESGPGLVKPSQTLSLTCTVSGGSITTRYYAWSWI 168, OSALTQPPSVSGSPGQTVTT SCAGA [\ ^T D 1GTYAYVSKYQQ? 432.

RQPPGKGLEV iGyTDYDGDT YSPSLESRTT.L SWDT8KN PGTAPKLMTYKVTTRASGVPSRFSGS 1 SGNTAS LT : SGLGA QFSLNLSSVTAEDTAVYYCARDPDVVTGFHYDYWGQGTL EDEADYYCASYRNF NAVFGTGTHLTVL VTVSS

126F4 EVOLpJPSGPGLVKPSQirLSLTCTVSGGSTTSRYYAWSWI 169 , OSALTQPPLVSGSPGQSVTT SCAGANND : GTYAYVSWYQQL 433.

RQPPGKGLESMGVIDYDGDTYYSPSI.KSRVTTSVPTSJ^N PGTAPKLLTYKVTTRASG; PDRFSGSKSGNTASLT : SGLGA QFSLJ^LSSVTPADTAVYYCARIJPDVVTGFLYDYWGQGTL EDERDYYCASYRNFKNAVFGGGTKLTVL,

VTVSS

; 27D11 EVQLQESGPGLVKPSQTLSLTCTVSGGSITSRYYABSWI 170. QSALTQPPSVSGSPGQTVTT SCAGANND I GTYAYVSBYQQL 434.

RQPPGKGLE IGVIDYDGDTYYSPSLKSRVSTSWDTSKN P'GTAP liMTY V ^' TTRASGVPSRFSGISKSG TASLT I SGliQS OFSLQLSSVTPEDTAVYYCATRDPDWTGFHYDYWGQGTK EDEADYYCASYRMFNMA-/FGGGTHLTVL

VTVSS

; 27H10 EVQLQESGPGLVKPSQTLSLTCTVSGGSITTRYYABSWI 171. QSVLTQPPLVSGAPGQRVTT SCAGANND I GTYRYVSBYQQL 435.

RQPFGKGLEWIGVIDYDGDTYYSP3LK3RT3ISWDT3KN PGTAPKl.LlYKVTTR.ASGVTDKFSGSKSG SASLTITG QA OFSLKLSSVTAEDTAVYYCARDPDWTGFHYDYWGQGTQ EDEADYYCASYRMFNMA-/FGGGTi;LTVL

TVSS

127H1 EVQLQESGPGLVKPSQTLSLTCTVSGGSITTRYYASS I 172. QSVLTQPPLVSGAPGQTVTISCAGAN DIGTYAYVSWYQQL 436.

RQPFGKGLEWIGVIDYDGDTYYSP3LK3RV3ISWDT3KN PGTAPKl.LlYKVTTR.ASGVTDKFSGSKSG TASLTITG QA QFSLQLSSVTAADTAVYYCARDPDVVTGFHYPYWGOGTO EDEADYYCASYR FN AVFGRGTKLTVL VTVSS

127G1 EVQLQESGPGLVKPSQTLSLTCTVSGGSITSRYYASS I 173. QSVLTQPPLVSGTPGQTVTISCAGAN DIGTYAYVSWYQQL 437.

RQPPGKGLEiilGVlDYDGDTYYSPSLKSRTTISVDTSKS PGTAPKLLIYKVTTRASGIPDRFSGSKSG TASLAISGLQA QFSLOLS3VTPEDTAVYYCARDPDVVTGFHYDYWG0GTT EDEAijYYCASYRNFNNAVF'GGGTKLTVL

VTVSS

126H5 QVQLQESGPGLVKPS0TLSLTCTVSGGSITSRYYAK3WI 174. QSALTQPPSVSGSPGQSVTISCAGANSDIGTYAYVSSYQOL 433.

RQPPGKGLEW I GVI DYDGDTYYSPSLKSRTS I SVD SKN PGTAPKLLI YKVTTRASGTPijRF'SGSKSGNTASLTISGLQA

QFSLKLSSVTPEDTAVYYCARDPDWTGFHYDYSGQGTT E3EA3YYCASYR F AVFGTGTHLTVL

VTVSS

127312 EVQLQJSSGPGLVKPSQTLSLTCTVSGGSITSRYYAKSWI ; 75. QSAl.TQPPSVSGSPGQTVTISCAGAN DTGTYAYVSSYQQL 439.

RQPPGKGLEWIGVIDYDGBTYYSPSLKSRVSISWDTS N PGTAPKLMIYKVTTPJiSGVPSRFSGSKSGNTASLTiSGLQS QFSLOPSSVTPEDTAVYYCARDPDWTGFHYDYSGQGTK EDEADYYCASYR F AVFGGGTHLTVL VTVSS

127F1 EVQLQESGPGLVKPSQTLSLTCTVSGGSITTRYYAWSWI 176. QSVLTQPPSVSGTPGQRVTISCAGANNBIGTYAYVSWYQQL 440.

RQPPGKGLEWIGVIDYDGBTYYSPSLKSRVSISWDTS N PGTAPKLLIYKVTTRASGVP3RFSGSKSGNSASLT TGLQS QFSLKLSSVTAABTAVYYCARBPBWTGFHYBYWGQGTL EBEABYYCASYRNFNNAVFGGGTHLTVL VTVSS

127D7 QVOLQESGPGLVKPSQTLSLTCTVSGGSITSRYYAWSWI 1 '77 _o OSALTQPPLVSGTPGQPVTISCAGA DIGTYAYVSKYQQL 441.

RQPPGKGLEWTGVTDYDGDTYYSPSLKSP.VT1 SW3TSK[\ ^T PGTAPKLLISKVTTPASGVP3RFSGSKSGTTASLTiTGL05 Ql'SLQPSSVTAAD'lAVYYCARDPDVyTGFHYDYWGQGTL E:3EA3YYCASYRNF NGVFGGGTHLTVL

VTVSS

127F5 QVOLQESGPGLVKPSQTLSLTCTVSGGSITSRYYAWSWI 178 , OSALTQPPLVSGTPGQTVTISCAGA DIGTYAYVSKYQQL 442.

RQPPGKGLEWlGyiD DGDT YSPSLKSRTT SWDT8KN PGTAPKLLTYKVTTRASGVPSRFSGSKSGNTASLT iSGLOS Ql'SLQPSSyTAED'lAVYYCARDPDVyTGFHYDYWGQGTL E:3EA3YYCASYRTF NAVFGSGTHLTVL

VTVSS

:27C6 QVQiJQE:SGPGLyKPSQTLS LTCTVSGGSIΪSRY AWSWI 179 , 0SA3TQPPLVSGSPGQTVT1 SCAGANND : GT YAYVSWYQQP 443.

RQPPGKGLE IGVIBYBGDTYYSPSLKSRV TSWBTSKN PGTAPKL LTYKVTTRASG; 3RFSGS1 SGNTAS LT : SGLOA QFSLKLSSVTAEDTAVYYCARDPDVVTGFiPYDYWGQGTQ E3E 3YYCASYRNFKNAVFGRGTKLTVL,

VTVSS

; 27F3 QVQLQESGPGLVKPSQTPSLTCTVSGGSITSRYYABSWI ISO . QSVLTQPPSVSGAPGQTVTl SCAGANND I GTYAYVSBYQQL 444.

RQPPGKGLE IGVIBYBGDTYYSPSLKSRTSTSVBTSKN P'GTAPKliL YKVTTRASG PSRELGSKSG T SLT TTGLQS OFSLKLSSVTPADTAVYYCARDPDWTGFHYDYWGQGTL EDEADYYCA _T 3YR FN AVFGGGTHLTVL

VTVSS

; 27G5 EVQLQESGPGLVKPSQTLSLTCTVSGGSITSRYYABSWI 1 S 1. QSALTQPPSVSGTPGQSVTTSCAGANNDIGTYAYVSSYQQP 445.

RQPPGKGLEWMGVIDYBGDTYYSPSLKSRTTIS BTSKN PGTAPKLLIYKVTTRASG IPSRFSGSKSG TASLTTSG QS OFSLQLSSVTAABTAVYYCARDPDWTGFHYDYWGQGTL EDEADYYCA _T 3YR FN AVFGSGTHLTVL

VTVSS

126H2 QVQLQESGPGLVKPSQTLSLTCTVSGGSITTRYYASS I 182. QSALTQPPLVSG3PGQSVTISCAGAN BIGTYAYV3WYQQL 446.

RQPFGKGLEWIGVIBYBGBTYYSPSLKSRTTISWBTSKN PGTAPKLLIYKVTTRASG IPDRFSGSKSG TASLTTSG QA QFSLQLSSVTPEDTAVYYCARDPDVVTGFHYDYWGOGTO EDEADYYCASYR FN AVFGGGTKLTVL VTVSS

127D5 EVQLQESGPGLVKPSQTLSLTCTVSGGSITSRYYASS I 183. QSALTQPPLVSG3PGQSVTISCAGAN BIGTYAYV3WYQQL 447.

RQPPGKGLEWIGVIBYBGBTYYSPSLKSRTSISWC'TSKN PGTAPKLMIYKVTTRASGIPBRFSGSKSGSTASLTISGLQS QFSLQLSSVTAEDTAVYYCARDPDVVlGFHYD WGOG' 3 EDEADYYCAS RNFNNAVFGT'GTKLTVL

VTVSS

12735 EVQ3QESGPGLVKPS0TLSLTCTVSGGSITSRYYASSWI 184. QSALTQPPLVSGSPGQSVTISCAGANSDIGTYAYVSSYQOL 443.

RQPPGKGLEWIGVI DYDGDTYYSPS3KSRTTI SWBTSKN P 77VPK3L I YKVTTR SGJ7P.0RFSGSK3Gf\LTASLT1SGLQS

QFSLKLSSVTAEDTAVYTCARDPDWTGFHYDYSGQGTT EDEADYYCASYRNF NAVFGSGTKLTVL

VTVSS

126E1 EVQLQESGFGIVKPSQTLSLTCTVSGGSTTSRYYAKSWI ; 85. QSALTQF'PLVSGAPGQTVT!S GAN DT TYAYVSViYQQP 449.

RQPPGKGLESIGVIDYDGDTYYSPSLKSRV ISWDTSK PGTAPKLLlYKVTTP^SGiPURFSGSKSGNTASLTiTGLQA QFSLOPSSyTAEDTAVYYCARDPDWTGFHYDYSGQGTQ EDEADYYCASYRNF NAVFGGGTHLTVL

VTVSS

126B5 QVQLQESGPGLVKPSQTLSLTCTVSGGSITTRYYAWSWI 186. QSALTQPPLVSGSPGQSVTISCAGANNDIGTYAYVSWYQQL 450.

RQPPGKGLESIGVIDYDGDTYYSPSLKSRV ISWDTSK PGTAPKLMIYKVTTPJiSGVPSRFSGSKSGNTASLTiSGLQA QFSLQLSSVTPEDTAVYYCARDPDWTGFHYC'NWGQGTL EBEABYYCASYRNFNNAVFGSGTHLTVL

VTVSS

127B6 EVQLQESGPGLVKPSQTLSLTCTVSGGSITTRYYAWSWI 187 , OSALTQPPSVSGSPGQTVTISCAGAN DlGTYAYVSiiYQQL 451.

RQPPGKGLEWTGVTD YDGDTYYSPSLKSP.VSI SWDTSKN PGTAPKLLIYKVTSPASGVPSRFSGSKSGSTASLSiSGLOA QF'SLKPSSVTAED'rAVYYCARDPDVyTGFHYDYWGQGTQ EDEADYYCASYRNFNNAVFGSGTKLTVL

VTVSS

127Ξ1 EVQLQESGPGLVKPSQTLSLTCTVSGGSITSRYYAWSWI 188 , OSVLTQPPLVSGAPGQTVTISCAGANNDiGTYAYVSliYQQL 452.

RQPPGKGLEWIGyiDYDGDTYYSPSLKSRTTl SWDT8KN PG A L L TYKVTTRAS I DRFSGS KSGNS S L Ϊ I TGLQ8 QE'SLQPSSVTPED'rAVYYCARDPDVyTGFHYDYWGQGTQ EDEADYYCASYRNFNNAVFGGGTKLTVL

VTVSS

I26G2 EVOLQESGPGLy PSQTLSLTCTVSGGSIT' YYAWSWI 189 , OSAPTQPPLVSGSPGQTVT.I SCAGANN!) I GTYAYVSWYQQL 453.

RQPPGKGLESIGVIDYDGDTYYSPSI.KSRVTTSVPTSJ^N PGTAPKLLTYKVTTRASGVPDRFSGSKSGNTASLT ISGLOA QFSLKLSSVTAftDTftVYYCARDPDWTGFHYDYWGQGTM EDE DYYCASYRNFKNAVFGTGT ILTVL,

VTVSS

; 26 2 EVQLQESGPGLVKPSQTPSLTCTVSGGSITSRYYABSWI 190. QSALTQPP1,VSGSPGQTVTT SCAGANND I GTYAYVSBYQQL 454.

RQPPGKGLE IGVIDYDGDTYYSPSLKSRTTTS DTSKN P'GTAPKTi L TYKV ^' TTRASGVPSRFSGISKSG T S LT I SGliQA QFSLKLSSVTAEBTAVYYCARDPDyV GFHYDYWGQGTQ EDEADYTCALSYRNFNNAVFGGGTHLTVL,

VTVSS

; 26G3 EVQLQESGPGIA/KPSQTPSLTCTVSGGSITTRYYASSWI 191. QSALTQPPSVSGAPGQRVTT SCAGANND I GTYAYVSBYQQL 455.

RQPPGKCJYEWIGVIDYBGDTYYSPSLKSRVTISSDTSKS PGTAPKLLIYKVTTRASGVPSRFSGSKSG SASLTTTGLQS OTSLQLSSVTPEDTAVYYCAJKDPDWTGFHYDYWGQGTL EBEABYYCA _T SYRMFNNA-/FGGGTHLTVL

TVSS

126D4 EVQLQESGPGLVKPSQTLSLTCTVSGGSITTRYYASS I 192. QFALTQPPLVSGTPGQSVTISCAGAN DIGTYAYVSWYQQL 456.

RQPPGKCJYEWIGVIDYBGDTYYSPSLKSRVTISVDTSKS PGTAPKLLIYKVTTRASGVPSRFSGSKSG TASLTTSGLQS QFSLKLS8VTPADTAVYYCARDPDVVTGFHYDYWG0GTT EDEADYYCASYRNFNNAA/FGTGTHLTV'L

VTVSS

127F2 QVQLQESGPGLVKPSQTLSLTCTVSGGSITSRYYASS I 193. QSALTQPPLVSGSPGQTVTISCAGAN DIGTYAYVSWYQQL 457.

RQPPGKGLEK1GV1DYDGDTYYSPSLISRVT1S DTS N PGT7YPKLM1YKVTTRASGVPSRF'SGSKSGMTASLTISGLQA QFSLKLSSVTPEDTAVYYCARDPDVVrGFHYDYWGOGTO EijEADYYCASYRNFNNAVT'GSGTKLTVL

VTVSS

127H2 EVQLQESGPGLVKPSOTLSLTCTVSGGSITTRYYASS I 194. QSALTQPPLVSGSPGQTVTISCAGANSDIGTYAYVSSYQOL 453.

RQPPGKGLEWMGVI DYDGDTYYSPSLKSRTTISWDTSKN PGTAPKLLI YKVTTRASGVPDRE'SGSKSGNTASLTISGLQS

QFSLOPSSVTPEDTATVYYCARDPDWTGFHYDYSGQGTQ EDEADYYCASYR F AVFGGGTHLTVL

VTVSS

126D5 QVQLQJSSGFGLVKPSQTLSL/TCTVSGGSITTRYYAKSWI 205. QS iTQF'PSVSGTPGQTVTIS AGAN DT TYAYVSViYQQP 469.

RQPPGKGLEV- GVIDYDGDTYYSPSLKSRVTISVTJTSK PGTAPKLLlYKVTTPASGVP RFSGSKSGSTASLTiSGLQS HFSLKLSSyTAEDTAVYYCATDPDWTGFHYDYSGQGTT EDEADYYCASYR F AVFGTGTKLTVL

VTVSS

127D8 EVQLQESGPGLVKPSQTLSLTCTVSGGSITSRYYAWSWI 206. QSALTOPPSVSGTPGQTVTISCAGAM DIGTYAYVSWYQQL 470.

RQPPGKGLEWIGVIDYDGDTYYSPSLKSRVSISVDTSKN PGTAPKLMIYK\TTRASGVP ^" VRFSGSKSGNTAS ,TiSGLQA QFSLKLSSVTAEDTAVYYCARDPDVVTGFHYDNViGQGTL EDEADYYCASYRNFNNAVFGGGTKLTVL

VTVSS

126Ξ4 EVQLQESGPGLVKPSQTLSLTCTVSGGSITSRYYAWSWI 207 , OSALTQPPLVSGSPGQTVTISCAGA DIGTYAYVSKYQQL 471.

RQPPGKGLEWMGVTDYDGDTYYSPSLKSPVTI SVPTSK1\ ^T PGTAPKLLIYKVTTPASGVPDRFSGSKSGSTASLTISGLOA QFSLQLSSVTAEDTAVYYCARDPDVVTGFHYDYWGQGTQ EDEAD YCASYRNF NAVFGTGTHLTVL VTVSS

126F2 QVOLQE GPGLVKPSQ'rLSLTC'rVSGGSITTRYYAKSWI 208 , OFALTQPPLVSGTPGQSVTISCAGA DIGTYAYVSKYQQL 472.

RQPPGKGLEWIGyiD DGDT YSPSLKSRTTl SWDT8KN PGTAP LLlYKVTTRASGVPSRFSGSKSGNTASLT iSGLOS QFSLKLSSVTAEDTAVYYCARDPDVVTGFHYDYWGQGTQ EDEAD YCASYRNF NAVFGTGTHLTVL VTVSS

132A7 EVOLQESGPGLV PSQTLSLTCTVSGGSTTSRY AWSWI 209 , OSViLCQPPLVSGTPGQRVTT SCAGANND : GT AYVSW QQL 473.

RQPPGKGLE IGVIDYDGDTYYSPSLKSRTTTS DTSKN PGTAPKL LIYKVTTRASG; PBRFSGSKSGNTASLT ISGLOS QFSLKLSSVTAEDTAVYYCARIJPDVVTGFHYDYWGQGTL EDEAiTYYCASYRNFNNAVFGGGTHLTVL

VTVSS

; 32E1 EVQLQESGPGLVKPSQTLSLTCTVSGGSITSRYYABSWI 210. QSVLTQPPLVSGAPGQRVTl SCAGANND I GTYAYVSBYQQL 474.

RQPPGKGLE IGVIDYDGDTYYSPSLKSRTTTSVDTSKN P'GTAPKJlLIYKV ^' TTRASGVPDRFSGISKSG TAS LT I SGLQA OFSLKLSSVTAEDTAVYYCARDPDWTGFHYDYWGQGTL EDEADYYCASYRMFNMA-/FGGGTHLTVT,

VTVSS

; 32E2 EVQLQESGPGLVKPSQTLSLTCTVSGGSITTRYYABSWI 211. QSALTQPPSVSGAPGQTVTl SCAGANND I GTYAYVSBYQQL 475.

RQPFGKGLEWIGVIDYDGDTYYSP3LK3RT3ISWDT3KN PGTAPKJJLIYIWTTRASGIPBKFSGSKSSNTASLTISGJJQA OFSLKLSSVTPEDTAVYYCATRDPDWTGFHYDYWGQGTL EDEADYYCASYRMFNMA-/FGGGTHLTVL,

TVSS

13237 EVQLQESGPGLVKPSQTLSLTCTVSGGSITSRYYASS I 212. QSVLTQPPSVSGTPGQT-/TISCAGA. SDIGTYAY\ ⁷ SSYQQL 476.

RQPFGKGLEWIGVIDYDGDTYYSPSLKSRTTISWDTSKN PGTAPKJJLIYIWTTRASGWSKFSGSKSGATASLTISGJJQS QFSLQLSSVTAADTAVYYCARDPDVVTGFHYDYWGOGTT EDEADYYCASYRNFNNAVFGGGTHLTVL VTVSS

132D3 EVQLQESGPGLVKPSQTLSLTCTVSGGSITSRYYASS I 213. QSALTQPPSVSGAPGQTVTISCAGAN DVGTYAYVSWYQQL 477.

RQPPGKGLEWIGVIDYEGDTYYSPSLKSRTTISWC'TSKN PGTAPKLLIYKVTTRASGVPDRFSGSKSG SASLTITGLQS QFSLQLSSVTAEDTAVYYCARDPDVVTGFHYD WGOGTL EDEADYYCAS RNFNNAVFGRGTHLTVL VTVSS

132Ξ7 EVQLQESGPGLVKPSOTLSLTCTVSGGSITSRYYAKSWI 214. QSVLTQPPRVSGAPGQTVTISCAGANSDIGTYAYVSSYQOL 473.

RQPPGKGLEWIGVI DYDGDTYYSPSLKSRVTISWDTSKNi PG'FAPKLL I YKVTTRASGVP.ORF'SGSKSGfSASLTTT'GLQA

QFSLKLS3VTPADTAVYYCARDPDWTGFHYDYSGQGTT EDEADYYCASYR F AVFGGGTKLTVL

VTVSS

132 1 EVQLEESGFGLVKPSQTLSLTCTVSGGSTTTRYYAKSWI 215. QSVLTQF'PLVSGAPGQRVT!S AGA DT TYAYVSViYQQL 479.

RQPPGKGLESIGVIDYDGDTYYSPSLKSRV ISWDTSK PGTAPKLLlYK\ ^r TTRASGVPSRFSGSKSGNTASL iTGLQA QFSLKLS3VTPEDTAVYYCARDPDWTGFHYDYSGQGTL EDEADYYCASYR F AVFGRGTHLTVL

VTVSS

132F2 EVQLQESGPGLVKPSQTLSLTCTVSGGSITSRYYAWSWI 216. QSVLTQPPSVSGAPGQRVTISCAGANNDIGTYAYVSWYQQL 480.

RQPPGKGLEWIGVIDYDGBTYYSPSLKSRTTISWDTS N PGTAPKLLlYKVTTPJiSGiPSRFSGSKSGNTASLTiTGLQS QFSLQLSSVTAEDTAVYYCARDPDWTGFHYC'YWGQGTT EDEADYYCASYRNFNNAVFGRGTHLTVL

VTVSS

132G1 EVOLQESGPGLVKPSQTLSLTCTVSGGSITTRYYAiiSWI 217, OSVLTQPPLVSGAPGQTVTISCAGA DlGTYAYVSiiYQQL 431.

RQPPGKGLEWTGVTDYDGDTYYSPSLKSP.TS I SWDTSK[\ ^T PGTAPKLLIYKVTTPASGIPNRFSGSKSGNTASLTITGLOA QE'SLKijSSVTAaD'raVYYCARDPDVyTGFHYDYWGQGT ^' r EDEADYYCASYRNF NAVFGGGTHLTVL

VTVSS

132G2 EVQLQESGPGLVKPSQTLSLTCTVSGGSITSRYYAWSWI 218, OSVLTQPPLVSGTPGQRVTISCAGA DlGTYAYVSiiYQQL 432.

RQPPGKGLEWIGyiD DGDTYYSPSLKSRTTl SWDT8KN PGTAP L LIYKVTTRASG; PDRFSGSKSGNTASLT ISGLOS QE'SLKLSSVTAED'raVYYCARDPDVyTGFHYDYWGQGTL EDEADYYCASYRNF NAVFGGGTHLTVL

VTVSS

:32G3 EVOLQESGPGLy PSQTLSLTCTVSGGSITS YYAWSWI 219 , OSVLTQPPLVSGaPGQTVTX SCAGANND : GTYAYVSWYQQL 433.

RQPPGKGLE IGVIDYDGDTYYSPSLKSRTTTSWDTSKN PGTAPKLLIYKVTTRASG; PDRFSGSKSGNTASLT ISGLOS QFSLOLSSVTAADTAVYYCARDPDVVTGFSYDYWGQGTT EDEADYYCASYRNFKNAVFGGGTKLTV1,

VTVSS

; 32G7 QVQLQESGPGLVKPSQTPSLTCTVSGGSITTRYYABSWI 220. QSALTQPPSVSGAPGQTVTT SCAGANND I GTYAYVSBYQQL 434.

RQPPGKGLE IGVIDYDGDTYYSPSLKSRTTTSWDTSKN F'GTAPKLLIYKVTTRASG TPDRFSGSKSG T SLT I SG1,QA OFSLQLSSVTPADTAVYYCARDPDWTGFHYDYWGQGTM EDEADYYCA _l SYRMFNMA-/FGRGTKLTVT,

VTVSS

; 33A3 EVQLQESGPGLVKPSQTPSLTCTVSGGSITTRYYABSWI 221. QSALTQPPT.VSGTPGQTVTT SCAGANND I GTYAYVSBYQQL 435.

RQPFGKGLEWIGVIDYDGDTYYSPSLKSRTTISWDTSKN PGTAPKLLIYKVTTRASGVPDRFSGSISG TASLTXSG QS OFSLQLSSVTPEDTAVYYCAJSDPDWTGFHYDYWGQGTL EDEADYYCA _L SYRMFNMG-/FGTGTHLTVT,

TVSS

133A7 EVELQESGPGLVKPSQTLSLTCTVSGGSITSRYYASS I 222. QSALTQPPLVSGSPGQTVTISCAGAN DIGTYAYVSWYQQL 486.

RQPFGKGLEWIGVIYYEGDTYYSP3LK3RT3ISWDT3KN PGTAPKLLIYKVTTRASGIPSRFSGSKSG TASLTXSG QS QFSLQLS8VTPEDTAVYYCA_RDPDVVTGF'fiYDYWGGGTL

VTVSS

133A9 EVQLQESGPGLVKPSQTLSLTCTVSGGSITTRYYASS I 223. QSALTQPPLVSG3PGQTVTISCAGAN DIGTYAYV3WYQQP 487.

RQPPGKGLEiilGVlDYEGDTYYSPSLKSRVTISVDTSKN PGTAPKLMIYKVTTRASGIPDRFSGSISGSTASLTISGLQS QFSLKLS3VTAEDTAVYYCARDPDVVTGFHYD WG0GTM EDEADYYCAS RNFNNAVFGTGTKLTVL

VTVSS

133D1 EVQLQESGPGLVKPSOTLSLTCTVSGGSITSRYYASS I 224. QSALTQPPLVSGSPGQTVTISCAGANSDIGTYAYVSSYQOL 483.

RQPPGKGLEWIGVI NYDGDTYYSPSLKSRTTISWDTSKNi PGTAPKLMI YKVTT'RaSGVPDRF'SGSKSGNTASL'riSGLQS

QFSLQLSSVTPEDTAVYYCARYPD TGFHYDYisGQGTQ EPEAPYYCASYR F AVFGRGTKLTVL

VTVSS

133D8 EVQLQESGFGLVKPSQTISI/TCTVSGGSTTSRYYAKSWI 225. QSAITOFTLVSGSPGQSVTTSGAGA DI TYAYVSViYQQL 489.

RQPPGKGLEWIGVIDYDGBTYYSPSLKSRTTISWDTS N PGTAPKLLlYKVTTPASGVPSRFSGSKSGSTASLTiSGLQA QFSLOPSSVTAEDTAVYYCARDPDWTGFHYDYisGQGTL EDEADYYCASYR F AVFGRGTKLTVL

VTVSS

133Ξ3 EVQLQESGPGLVKPSQTLSLTCTVSGGSITSRYYAWSWI 226. QSAITOPPSVSGSPGQSVTLSCAGAM DIGTYAYVSWYQQL 490.

RQPPGKGLEWIGVIDYDGBTYYSPSLKSRTSISWDTS N PGTAPKLMTYK\TTRASGVPPRFSGSKSGNTAS ,TiSGLQS QFSLHLSSVTAEDTAVYYCARDPDVVTGFHYDYViGQGTQ EDEADYYCASYRNFNNAVFGSGTKLTVL

VTVSS

133Ξ5 QVOLQESGPGLV PSQTLSLTCTVSGGSITSRYYABSWI 227 , OSALTQPPSV3GSPGQTVTT SGAGAN[\ ^T D1GTYAYVSKYQQL 491.

RQPPGKGLEW1GV1DYDGDTYYSPSLKSP.VT1 SWDTS [\ ^T PGTAPKLMIYKVTTPASGIPDRFSGSKSGSTASLTISGLOA QF'SLKPSSVTPED'raVYYCARDPDVyTGFHYDYWGQGTQ EDEAD YCASYRNF NAVFGRGT LTVL

VTVSS

133F2 EVOLQESGPGLV PSQTLSLTCTVSGGSITSRYYABSWI 228, OSALTQPPSVSGSPGQSVT1 SGAGAN[\ ^T D1GTYAYVSKYQQL 492.

RQPPGKGLEWlGyiD DGDT YSPSLKSRTTl SWDT8KN PGTAPQLLTYKVTTRASG; PDRFSGSKSGNTASLT iSGLOS QFSLQPSSVTPEDTAVYYCARDPDVVTGFHYDYWGQGTL EDEAD YCASYRNF NAVFGGGT LTVL VTVSS

133G8 EVOLQESGPGLV PSQTLSLTCTVSGGSITSRY AWSWI 229 , OSALTQPPSVSGTPGQSVTl SCAGANND : GT AYVSW QQL 493.

RQPPGKGLE IGVIDYDGDTYYSPSLKSRTTS DTSKN PGTAPKL LTYKVTTRASG; PDRFSGSKSGNTASLT iSGLQS OFSLQISSVTAEDTAVYYCARDPDVVTGFLYDYWGQGTQ EDE DYYCASYRNFKNAVFGGGTaLTVL

VTVSS

; 33H2 EVQLQESGPGLVKPSQTLSLTCTVSGGSITTRYYABSWI 230. QSA1TQPPLVSGSPGQTVTT SCAGANND I GTYAYVSSYQQP 494.

RQPPGKGLE IGVIDYDGDTYYSPSLKSRTTTS DTSKN F'GTAP LM Y VTTRASG TPDRFSGSKSG TASLT I SGliQA OFSLKLSSVTP DT VYYCARDPDWTGFHYDYWGQGTT EDEADYYCASYR FN AVFGSGTrLTVL

VTVSS

; 33H9 OVQLQESGPGLVKPSQTLSLTCTVSGGSITTRYYABSWI 231. QSALTQPPSVSGSPGQTVTT SCAGANND I GTYAYVSSYQQP 495.

RQPFGKGLEWIGVIDYDGDTYYSP3LK3RT3ISWDT3KN PGTAPKLLIYKVTTR _^ ASGVPDRFSGSKSG TASLTISGLQS OFSLKLSSVTAEDTAVYYCARDPDWTGFHYDYWGQGTQ EDEADYYGASYRNFNNAVFGTGTHLTVL TVSS

27Ξ2 EVQLQESGPGLVKPSQTLSLTCTVSGGSITTRYYASS I 232. QSALTQPPSVSGSPG0T-/TISCAGA SDIGTYAY\ ⁷ SSY0QP 496.

ROPPGKCJTEWIGVIDYDGDTYYSP3LK3RVTISVDT3KN PGTAPKl.LiYKVTTR.ASGVTDRFSGSKSG TASLTISG.L.QA QFSLQLSSVTAEDTAVYYCARDPDVVTGFHYDYWGOGTO EDEADYYCASYR FN AVFGGGTHLTVL VTVSS

Table 17 Sequence Variants of Fab 129D3 CDR Amino Acid Sequences and CDR Consensus Sequences Thereof

CDR SEQUENCE SEQ !D HQ

HCDR3 DPDWTGFHYDY 497.

YPDVVTGFHYDY 498.

DPDWTGFHYDN 499.

XiPDWTGFHYDX ₂ 500.

where :

XI- D or Y

X2= Y or N

HCDR2 V DY ^' DGDTYYSPSLKS 501.

VID YDGDTYYSPSLES 502.

VID YDADTYYSPSLKS 503.

VID YEGDTYYSPSLKS 504.

VIY YEGDTYYSPSLKS 505.

VINYDGDI'YYSPSLKS 506.

VIX; Y ^' X ₂ X;,DTY YSP SLX ₄ S 507.

Where :

XI- D, Y or N

X2 D or E

X3= A or G

X4= E or K

HCDRl SRYYAWS 508.

TRYYAWS 509.

PRYYVWT 510.

SSYYAWS 511.

X ₁ X ₂ YYX,WX ₄ 512.

Where :

Xl= T, S or P

X2- R or S

X3= A or V

X4= S or T

LCDR3 ASYRMFMNAV 513.

ASYRHYMNAV 51 .

ASYRRTIDNI 515.

ASYRSS NAV 516.

ASYRMRMNAV 517.

ASYRDFN AV 518.

ASYKTYN W 519.

ASYRYFN AV 520.

ASYRNFNNGV 521.

SSYRNFN AV 522.

ASYRTFN AV 523.

ASY ^" X; X2X3X4X5XβX7 524.

Where :

Xl= R or K

X2 M, E, R, S, D, T or Y

X3- F, Y, T, S or R

X4- N or I

X5= N or D

X6= V, N, G o A

X7- V or I

Table 18 Sequence Variants of Fab 1 1 1 A7 CDR Amino Acid Sequences and CDR Consensus Sequences Thereof

CDR SEQUENCE SEQ ID HO

HCDR3 P.A.GWGMGDY 543.

RAG .iG . ₂ G 544.

Where :

XI any amino acid, or no amino

acid

X2 ∞ any amino acid

HCDR2 RI SAGGGSTYYGDSVKG 545.

AI SAGGGSTYYGDSVKG 546.

RI SSGGGSTSYADSVKG 547.

RISSGGGSTNYADSVKG 548.

RI SSGGGSAYYADSVKG 549.

AI SSSGVSTYYTDSVKG 550.

AISSGGGSTYYGDSVKG 551.

RISSGGGSTYYGDSVKG 552.

P I SAGGGSTYYGDSVKG 553.

X _x ISX ₂ X ₃ GX ₄ SX ₅ X ₆ YX-,DSVKG 554.

Where :

Xl= A, P or R

X2- A. or S

X3= S or G

X4= G or V

X5= A or T

X6- Y, N or S

X7= G, A or T

KCDR1 SYA S 555.

TYAMS 556.

SYRMY 557.

SHRMY 558.

SYAMY 559.

SYRMS 560.

SYRLY 561.

X] ? '^4^;; 562.

Whe e :

XI- S or T

X2= H or Y

X3= A or R

X4= or L

X5- S or Y

LCDR3 A.LDIGDITE 563.

LCDR2 STNDR.HS 564.

LCDR1 GLSSGSV'TAS YPG 565. Table 19. Production levels and potencies (pM) of germlined 68F2 variants

Table 20 CMC Optimized Sequence Variants of Fab 111 A7

CIVSC Variant CDRH3 SEQUENCE SE VH SEQUENCE SEQ

Q !D ID HQ NO

111A7M _.. A RAGWGlG 566 . EVQLLESGGGLVQPGGSLRLSCAASGFTFS 569 .

S YAM S W V RQ AP GKG P E WV S R I S G G G S T Y Y GD SVKGRF T I S RDN SKNT L YLQMNS LRAE D TAVYYCANRAGWG!GDYWGQGTLVTVSS

111A7 _L RAG¾G|G 567 . EVQLLESGGGLVQPGGSLRLSCAASGFTFS 570 .

SYAMSWVRQAPGKGPEWVSRI SAGGGSTYY GD S KGRFTIS RDN SKNT L YLQMNS LRAE D TAVYYCANRAGWG|GDY¾GQGTLVTVSS

111A7 M__S RAGWGlG 568 . EVQLLESGGGLVQPGGSLRLSCAASGFTFS 571 .

SYAMSWVRQAPGKGPEWVSRI SAGGGSTYY GDSVKGRFTISRDNSKNTLYLOMNSLRAED TAVYYCANRAGWGIGDYWGQGTLVTVSS Table 21 IL-6 Bindin Kinetics of CMC Optimized Sequence Variants of Fab 1 1 1 A7

Table 22 Non-Compartmental PK Analysis of anti-IL-6 mAbs after Single Intravenous

MRT = Mean Residence Time; k = mean elimination rate constant; tl/2 :

elimination half-life; CI = elimination clearance Table 23 In Vitro IL-6 Neutralization Assay Using B9 Cells

Table 24 In Vitro IL-6 Neutralization Assay Using 7TD1 Cells

Potency

IC50 relative

(pM) to 88F2

VH_133E5#A2.1 0.10 6.69

VH_133A9(QSV)#A3.3 0,17 4.00

VH_133A9#A3.1 0.18 3.66

CNT0136LB 0.19 3.49

129D3U#15.1 0.23 2.91

129D3#A1.1 0.25 2.65

Alder_hulU#12.5 0.29 2.31

VH_133H2#A1.8 0.35 1.94

VH_132E7#A1.9 0.37 1.85

111B1_SDM #A6.7 0.38 1.79

VH_133H2#A9.11 0.47 1.44

11181..1 SDM M/L#A6.9 0.51 1.32

104C1_1 SDM M/L#A6.6 0.59 1.14

11 IB 1_S DM 2_M 100L#A9.5 0.67 1.01

68F2#A8.10 0.67 1.00

104C1_SDM2_M100A#A9.2 0,73 0.93

GL18LB 0.76 0.89

104C1_SDM2_M 100L#A9.3 0.98 0.69

111 A7 _„ S DM2__M 100A# A9.7 1,57 0.43

61H7#A8.11 3,48 0.19 "able 25, Groups and Treatment Regimes Employed in Psoriasis Xenograft Model.

Group Treatment group size Dose -route Treatment frequency

1 Betamethasone 3 Topical 2 x day. three weeks dipropiorsate

2 PBS 4 i.p. 200μί, 2 x weeks, 3 weeks

3 Remicade 7 i.p 200μ!, 2 x weeks, 3 weeks (lOmg/kg)

4 68F2 5 i.p. 200μί, 2 x weeks, 3 weeks

(IQmg/kg)

Table 26. In Vivo 1L-6 Neutralization in an SAA Mouse Model