EA044026B1 - ANTIBODIES AGAINST LAG-3 AND METHODS OF THEIR APPLICATION - Google Patents

Description

1. Related applications

This patent application claims priority to US Provisional Application No. 62/406,766, filed October 11, 2016; and 62/420280, filed November 10, 2016, each of which is incorporated herein by reference in its entirety.

2. Field of technology

This invention relates to antibodies that specifically bind to LAG-3 (eg, human LAG-3) and methods of using them.

3. State of the art

Lymphocyte activation gene 3 (LAG-3), also known as CD223, is a type I membrane protein of the immunoglobulin (Ig) superfamily that consists of four extracellular Ig domains and a cytoplasmic domain containing a conserved EP repeat motif and one conserved KIEELE motif (Triebel et al., (1990) J Exp Med, 171: 1393-405; Workman et al., (2002) J Immunol, 169: 5392-5). LAG-3 is expressed on activated effector T lymphocytes (Teff), activated regulatory T lymphocytes (Treg), activated B lymphocytes, a subset of resting natural killer (NK) cells, and resting plasmacytoid dendritic cells (PDC) (Huang et al., (2004) Immunity, 21: 503-13; Workman et al., (2009) J Immunol, 182: 1885-91; Kisielow et al., (2005) Eur J Immunol, 35: 2081-8; Baixeras et al., (1992 ) J Exp Med, 176: 327-37; Workman et al., (2002) Eur J Immunol, 32: 2255-63). Under conditions of chronic antigenic challenge, such as chronic pathogenic infections or in the tumor microenvironment (TME), LAG-3 expression is maintained on T regulatory cells type 1 (Tr1) and so-called exhausted antigen-specific T cells (Park et al., (2012) Cell Immunol, 278: 76-83; Gagliani et al., (2013) Nat Med, 19: 739-46; Blackburn et al., (2009) Nat Immunol, 10: 29-37).

LAG-3 functions in a way that negatively regulates activated T cells. The ligand for LAG-3 is MHC class II, expressed on antigen presenting cells (APCs) and activated T cells (Roche and Furuta (2015) Nat Rev Immunol, 15: 203-16). The interaction between LAG-3 and its ligand inhibits the proliferation and secretion of cytokines by CD4+ and CD8+ Teff cells (Macon-Lemaitre and Triebel (2005) Immunology, 115: 170-8; Huard et al., (1997) Proc Natl Acad Sci USA, 94 : 5744-9). LAG-3 in Tregs and PDCs contributes to the negative regulation of T cell function (Huang et al., (2004) Immunity, 21: 503-13; Workman et al., (2009) J Immunol, 182: 1885-91). Consistent with its role in maintaining immune homeostasis, LAG-3 deficiency induced lethal myocarditis in mice also genetically deficient for PD-1 (Okazaki et al., (2011) J Exp Med, 208: 395-407). Additionally, in vivo blockade with a mouse anti-LAG-3 monoclonal antibody, combined with PD-1 blockade, provided a synergistic effect to enhance antitumor immunity in syngeneic mouse tumor models (Woo et al., (2012) Cancer Res, 72: 917-27 ).

Given the role of LAG-3 in modulating immune responses, therapeutic agents designed to antagonize LAG-3 signaling hold great promise for the treatment of diseases that involve LAG-3-mediated immune suppression.

4. Essence of the invention

This invention provides antibodies that specifically bind to LAG-3 (eg, human LAG-3) and antagonize the function of LAG-3, eg, LAG-3-mediated immune suppression. Also provided are pharmaceutical compositions containing these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for producing these antibodies, and methods of treating a subject using these antibodies. The antibodies described herein are particularly useful for increasing T cell activation in response to an antigen (eg, a tumor antigen or an infectious disease antigen) and/or for reducing Treg-mediated immune suppression, and therefore for treating a cancer in a subject, or treating or preventing infectious disease in the subject.

Accordingly, in one aspect, the present invention provides an antibody or isolated antibody comprising a heavy chain variable region comprising the complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising the complementarity determining regions CDRL1, CDRL2 and CDRL3, wherein:

(a) CDRH1 contains the amino acid sequence DX1YX ₂ X ₃ (SEQ ID NO: 140), where

X1 represents T or N,

X ₂ represents I or M, and

X ₃ represents H, Y or D;

(b) CDRH2 contains the amino acid sequence X1IDPANX ₂ X ₃ X4X5X ₆ X ₇ PX ₈ X ₉ QX ₁₀ (SEQ ID NO: 142), where

X1 represents E, R, S or K,

X2 represents D or G,

X3 represents N or H,

X ₄ represents T or S,

X ₅ represents K or H,

- 1 044026

X ₆ represents Y or F,

X7 represents D or A,

X ₈ represents K or R,

X ₉ represents F or L, and

X ₁₀ represents G or D;

(c) CDRH3 contains the amino acid sequence YX1X ₂ X ₃ YX ₄ VGGX ₅ DY (SEQ ID NO: 144), where

X ₁ represents Y, F or S,

X2 represents Y or D,

X ₃ represents K or R,

X4 represents D or E, and

X ₅ represents F or C;

(d) CDRL1 contains the amino acid sequence SVSSX1ISSSX2LX3 (SEQ ID NO: 147), where

X ₁ represents S or G,

X ₂ represents N or T, and

X ₃ represents H or Y;

(e) CDRL2 contains the amino acid sequence GTSNLAS (SEQ ID NO: 104); and (f) CDRL3 contains the amino acid sequence QQWX1X2YPX3T (SEQ ID NO: 149), where

X ₁ represents S, N or R,

X2 represents S, T or R, and

X ₃ represents F, L, H or W.

In another aspect, the present invention provides an antibody or isolated antibody that specifically binds to human LAG-3, comprising a heavy chain variable region comprising the complementarity determining regions of CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising the complementarity determining regions of CDRL1 , CDRL2 and CDRL3, in which:

(a) CDRH1 contains the amino acid sequence DX1YX2X3 (SEQ ID NO: 140), where

X ₁ represents T or N,

X ₂ represents I or M, and

X ₃ represents H, Y or D;

(b) CDRH2 contains the amino acid sequence X ₁ IDPANX ₂ X ₃ X ₄ X ₅ X ₆ X ₇ PX ₈ X ₉ QX ₁₀ (SEQ ID NO: 142), where

X1 represents E, R, S or K,

X2 represents D or G,

X3 represents N or H,

X4 represents T or S,

X ₅ represents K or H,

X ₆ represents Y or F,

X ₇ represents D or A,

X ₈ represents K or R,

X ₉ represents F or L, and

X10 represents G or D;

(c) CDRH3 contains the amino acid sequence YX ₁ X ₂ X ₃ YX ₄ VGGX ₅ DY (SEQ ID NO: 144), where

X1 represents Y, F or S,

X2 represents Y or D,

X3 represents K or R,

X ₄ represents D or E, and

X ₅ represents F or C;

(d) CDRL1 contains the amino acid sequence SVSSX1ISSSX2LX3 (SEQ ID NO: 147), where

X ₁ represents S or G,

X ₂ represents N or T, and

X3 represents H or Y;

(e) CDRL2 contains the amino acid sequence GTSNLAS (SEQ ID NO: 104); and (f) CDRL3 contains the amino acid sequence QQWX1X2YPX3T (SEQ ID NO: 149), where

X1 represents S, N or R,

X2 represents S, T or R, and

X3 represents F, L, H or W.

In certain embodiments, CDRH1 comprises the amino acid sequence DX1YX2X3 (SEQ ID NO: 141), where X1 is T or N; X ₂ represents I or M; a X ₃

- 2044026 is H or Y. In certain embodiments, CDRH2 comprises the amino acid sequence X ₁ IDPANX ₂ X ₃ X ₄ KX5X ₆ PX ₇ FQX ₈ (SEQ ID NO: 143), where X ₁ is E, R or S; X ₂ represents D or G; X ₃ represents N or H; X ₄ represents T or S; X ₅ represents Y or F; X ₆ represents D or A; X ₇ represents K or R; and X ₈ is G or D. In certain embodiments, CDRH3 comprises the amino acid sequence YX ₁ X ₂ X ₃ YDVGGX ₄ DY (SEQ ID NO: 145), where X ₁ is Y, F or S; X2 represents Y or D; X ₃ represents K or R; and X ₄ is F or C. In certain embodiments, CDRH3 comprises the amino acid sequence YYYX ₁ YX2VGGFDY (SEQ ID NO: 146), where X ₁ is K or R; and X ₂ is D or E. In certain embodiments, CDRL1 contains the amino acid sequence SVSSSISSSNLX ₁ (SEQ ID NO: 148), where X ₁ is H or Y. In certain embodiments, CDRL3 contains the amino acid sequence QQWX ₁ SYPX2T (SEQ ID NO: 150), where X ₁ represents S, N or R; and X ₂ represents F, L or H.

In certain embodiments:

(a) CDRH1 contains the amino acid sequence DTYIH (SEQ ID NO: 79);

(b) CDRH2 contains the amino acid sequence EIDPANDNTKYDPKFQG (SEQ ID NO: 90);

(c) CDRH3 contains the amino acid sequence YYYX ₁ YX2VGGFDY (SEQ ID NO: 146), where X ₁ represents K or R; and X ₂ represents D or E;

(d) CDRL1 contains the amino acid sequence SVSSSISSSNLH (SEQ ID NO: 100);

(e) CDRL2 contains the amino acid sequence GTSNLAS (SEQ ID NO: 104); and (f) CDRL3 contains the amino acid sequence QQWSSYPFT (SEQ ID NO: 105).

In certain embodiments, CDRH1, CDRH2, and CDRH3 comprise the amino acid sequences set forth in SEQ ID NO: 79, 90, and 98, respectively. In certain embodiments, the heavy chain variable region comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, and 99% identical to the amino acid sequence of SEQ ID NO: 65 or 220. In certain embodiments, the variable region heavy chain comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 65 or 220. In certain embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 220. In certain embodiments, the heavy chain variable region comprises amino acid sequence of SEQ ID NO: 65. In certain embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 220. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 226. In certain embodiments, the antibody comprises a heavy chain chain containing the amino acid sequence of SEQ ID NO: 169.

In certain embodiments, CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences set forth in SEQ ID NO: 100, 104, and 105, respectively. In certain embodiments, the light chain variable region comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, and 99% identical to the amino acid sequence of SEQ ID NO: 73 or 221. In certain embodiments, the variable region the light chain comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 73 or 221. In certain embodiments, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 221. In certain embodiments, the light chain variable region comprises amino acid sequence of SEQ ID NO: 73. In certain embodiments, the antibody contains a light chain comprising the amino acid sequence of SEQ ID NO: 228. In certain embodiments, the antibody contains a light chain containing the amino acid sequence of SEQ ID NO: 187.

In certain embodiments, CDRH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 78-82. In certain embodiments, CDRH2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 83-93. In certain embodiments, CDRH3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 94-99. In certain embodiments, CDRL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 100-103. In certain embodiments, CDRL3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 105-112.

In certain embodiments, CDRH1, CDRH2, and CDRH3 comprise the amino acid sequences CDRH1, CDRH2, and CDRH3, respectively, set forth in SEQ ID NOs: 78, 83, and 94; 78, 85 and 95; 78, 86 and 96; 78, 86 and 97; 78, 91 and 94; 78, 92 and 96; 79, 84 and 95; 79, 88 and 95; 79, 89 and 95; 79, 90 and 95; 79, 90 and 98; 79, 90 and 99; 80, 85 and 96; 81, 87 and 96 or 82, 93 and 95.

In certain embodiments, CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences CDRL1, CDRL2, and CDRL3, respectively, set forth in SEQ ID NOs: 100, 104, and

105; 100, 104 and 106; 100, 104 and 107; 100, 104 and 109; 100, 104 and 110; 101, 104 and 108; 102, 104 and 105; 102, 104 and 112 or 103, 104 and 111.

In certain embodiments, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences set forth in SEQ ID NO: 78, 83, 94, 100, 104, and 105; 78, 85, 95, 100, 104 and 105; 78, 86, 96, 100, 104 and 105; 78, 86, 96, 100, 104 and 109; 78, 86, 96, 100, 104 and 110;

78, 86, 96, 101, 104 and 108; 78, 86, 96, 103, 104 and 111; 78, 86, 97, 102, 104 and 112; 78, 91, 94, 100, 104 and 107;

78, 92, 96, 100, 104 and 105; 78, 92, 96, 100, 104 and 109; 79, 84, 95, 100, 104 and 105; 79, 84, 95, 100, 104 and 106;

79, 84, 95, 102, 104 and 105; 79, 88, 95, 100, 104 and 105; 79, 89, 95, 100, 104 and 105; 79, 90, 95, 100, 104 and 105;

79, 90, 98, 100, 104 and 105; 79, 90, 99, 100, 104 and 105; 80, 85, 96, 100, 104 and 105; 81, 87, 96, 100, 104 and 105;

81, 87, 96, 100, 104 and 107 or 82, 93, 95, 100, 104 and 105 respectively.

In another aspect, the present invention provides an antibody or isolated antibody comprising a heavy chain variable region comprising the complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising the complementarity determining regions CDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2 , CDRH3, CDRL1, CDRL2 and CDRL3 contain the amino acid sequences specified in SEQ ID NO: 79, 90, 95, 100, 104 and 105, respectively.

In another aspect, the present invention provides an antibody or isolated antibody that specifically binds to human LAG-3, comprising a heavy chain variable region comprising the complementarity determining regions of CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising the complementarity determining regions of CDRL1 , CDRL2 and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 contain the amino acid sequences shown in SEQ ID NO: 79, 90, 95, 100, 104 and 105, respectively.

In another aspect, the present invention provides an antibody or isolated antibody comprising a heavy chain variable region comprising the complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising the complementarity determining regions CDRL1, CDRL2 and CDRL3, where CDRH1, CDRH2 , CDRH3, CDRL1, CDRL2 and CDRL3 contain the amino acid sequences shown in SEQ ID NO: 79, 90, 98, 100, 104 and 105, respectively.

In another aspect, the present invention provides an antibody or isolated antibody that specifically binds to human LAG-3, comprising a heavy chain variable region comprising the complementarity determining regions of CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising the complementarity determining regions of CDRL1 , CDRL2 and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 contain the amino acid sequences shown in SEQ ID NO: 79, 90, 98, 100, 104 and 105, respectively.

In certain embodiments, the heavy chain variable region comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, and 99% identical to the amino acid sequence of SEQ ID NO: 65 or 220. In certain embodiments, the variable region heavy chain comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 65 or 220. In certain embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 220. In certain embodiments, the heavy chain variable region comprises amino acid sequence of SEQ ID NO: 65. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 226. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 169.

In certain embodiments, the light chain variable region comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, and 99% identical to the amino acid sequence of SEQ ID NO: 73 or 221. In certain embodiments, the variable region light chain comprises an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 73 or 221. In certain embodiments, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 221. In certain embodiments, the light chain variable region comprises amino acid sequence of SEQ ID NO: 73. In certain embodiments, the antibody contains a light chain containing the amino acid sequence of SEQ ID NO: 228. In certain embodiments, the antibody contains a light chain containing the amino acid sequence of SEQ ID NO: 228. In certain embodiments, the antibody contains light chain containing the amino acid sequence of SEQ ID NO: 187.

In certain embodiments, the heavy chain variable region contains an amino acid sequence that is at least 75, 80, 85, 90, 95, 97, 98, and 99% identical to the amino acid sequence of SEQ ID NO: 65 or 220, and the light chain variable region contains an amino acid sequence that is at least 75, 80, 85, 90, 95, 97, 98 and 99%

- 4 044026 is identical to the amino acid sequence of SEQ ID NO: 73 or 221. In certain embodiments, the heavy chain variable region contains an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 65 or 220, and the light chain variable region contains an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 73 or 221. In certain embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 220. In certain embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 65. In certain embodiments, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 73 or 221. In certain embodiments, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 73.

In another aspect, the present invention provides an isolated antibody that specifically binds to human LAG-3, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 65 or 220. In certain embodiments, the antibody comprises a light chain variable region comprising the amino acid sequence which is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, and 99% identical to the amino acid sequence of SEQ ID NO: 73 or 221. In certain embodiments, the antibody comprises a light chain variable region comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 73 or 221.

In another aspect, the present invention provides an isolated antibody that specifically binds to human LAG-3, comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 73 or 221. In certain embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, and 99% identical to the amino acid sequence of SEQ ID NO: 65 or 220. In certain embodiments, the antibody comprises a heavy chain variable region comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 65 or 220.

In certain embodiments, the antibody comprises a heavy chain variable region comprising framework regions of a heavy chain variable region sequence of SEQ ID NO: 151 or 222. In certain embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 151 or 222. B In certain embodiments, the antibody comprises a heavy chain variable region comprising the framework regions of a heavy chain variable region sequence of SEQ ID NO: 218 or 223. In certain embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 218 or 223. In certain embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 218 or 223. In certain embodiments In embodiments, the antibody comprises a heavy chain variable region comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 56-72 and 220. In certain embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 56-72 and 220. In certain embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 220. In certain embodiments, the variable the heavy chain region comprises the amino acid sequence of SEQ ID NO: 65. In certain embodiments, the antibody comprises a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 168-186 and 225-227. In certain embodiments, the antibody comprises a heavy chain variable region having human-derived framework regions. In certain embodiments, the antibody comprises a heavy chain variable framework that is or is derived from an amino acid sequence encoded by a human gene, wherein said amino acid sequence is selected from the group consisting of IGHV1-46*01 (SEQ ID NO: 153), IGHV1-69 -2*01 (SEQ ID NO: 154), IGHV1-3*01 (SEQ ID NO: 155), IGHV1-24*01 (SEQ ID NO: 156), IGHV1-2*01 (SEQ ID NO: 157) , IGHV1-45*01 (SEQ ID NO: 158) and IGHV1-18*01 (SEQ ID NO: 159). In certain embodiments, the antibody comprises a heavy chain variable framework region that is derived from the amino acid sequence of IGHV1-46*01 (SEQ ID NO: 153), wherein at least one amino acid is in the amino acid sequence of IGHV1-46*01 (SEQ ID NO: 153 ) is replaced by an amino acid at a similar position in the corresponding non-human heavy chain variable framework. In certain embodiments, the amino acid substitution is at an amino acid position selected from the group consisting of 4, 5, 12, 23, 27, 28, 29, 30, 48, 69, 71, 75, 76, 80, 81, and 94, wherein amino acid position is indicated according to the Kabat numbering system. In certain embodiments, the amino acid substitution is selected from the group consisting of 4M, 5K, 12V, 23T, 27F, 28N, 29I, 30K, 48I, 69I, 71A, 75S, 76N, 80L, 81Q, and 94T, wherein the position of the amino acid substitution is indicated in according to the Kabat numbering system. In certain embodiments, the amino acid substitution is at an amino acid position

- 5 044026 selected from the group consisting of 4, 27, 28, 29, 30, 69, 71 and 94, the amino acid position being indicated in accordance with the Kabat numbering system. In certain embodiments, the amino acid substitution is selected from the group consisting of 4M, 27F, 28N, 29I, 30K, 69I, 71A, and 94T, with the position of the amino acid substitution indicated in accordance with the Kabat numbering system.

In certain embodiments, the antibody comprises a light chain variable region comprising framework regions of a light chain variable region sequence of SEQ ID NO: 152 or 224. In certain embodiments, the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 152 or 224. B in certain embodiments, the antibody comprises a light chain variable region comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73-77 and 221 In certain embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 73-77 and 221. In certain embodiments, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 221. In certain embodiments, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 73-77 and 221. the light chain variable region comprises the amino acid sequence of SEQ ID NO: 73. In certain embodiments, the antibody comprises a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 187-191. In certain embodiments, the antibody comprises a light chain variable region having human-derived framework regions. In certain embodiments, the antibody comprises a light chain variable framework that is or is derived from an amino acid sequence encoded by a human gene, wherein said amino acid sequence is selected from the group consisting of IGKV3-20*01 (SEQ ID NO: 160), IGKV3D-15 *01 (SEQ ID NO: 161), IGKV3-15*01 (SEQ ID NO: 161), IGKV3D-20*01 (SEQ ID NO: 162), IGKV3D-7*01 (SEQ ID NO: 163), IGKV1 -9*01 (SEQ ID NO: 164) and IGKV3-11*01 (SEQ ID NO: 165). In certain embodiments, the antibody comprises a light chain variable framework region that is derived from the amino acid sequence IGKV3-20*01 (SEQ ID NO: 160). In certain embodiments, the antibody comprises a light chain variable framework region that is derived from the amino acid sequence of IGKV3-20*01 (SEQ ID NO: 160), wherein at least one amino acid is in the amino acid sequence of IGKV3-20*01 (SEQ ID NO: 160 ) is replaced by an amino acid at a similar position in the corresponding non-human light chain variable framework. In certain embodiments, the amino acid substitution is at an amino acid position selected from the group consisting of 3, 22, 36, 43, 47, 58, 70, and 71, wherein the amino acid position is indicated in accordance with the Kabat numbering system. In certain embodiments, the amino acid substitution is selected from the group consisting of 3L, 22T, 36F, 43S, 47W, 58V, 70S, and 71Y, with the position of the amino acid substitution indicated in accordance with the Kabat numbering system.

In another aspect, the present invention provides an antibody or isolated antibody comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 56-72 and 220. In certain embodiments, the heavy chain variable region comprises an amino acid sequence SEQ ID NO: 220. In certain embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 65. In certain embodiments, the antibody comprises a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 168-186 and 225-227. In certain embodiments, the antibody contains a heavy chain containing the amino acid sequence of SEQ ID NO: 225. In certain embodiments, the antibody contains a heavy chain containing the amino acid sequence of SEQ ID NO: 168. In certain embodiments, the antibody contains a heavy chain containing the amino acid sequence of SEQ ID NO: 168. NO: 226. In certain embodiments, the antibody contains a heavy chain containing the amino acid sequence of SEQ ID NO: 169. In certain embodiments, the antibody contains a heavy chain containing the amino acid sequence of SEQ ID NO: 227. In certain embodiments, the antibody contains a heavy chain containing amino acid sequence SEQ ID NO: 170.

In another aspect, the present invention provides an antibody or isolated antibody that specifically binds to human LAG-3, the antibody comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 56-72 and 220. In certain embodiments, the heavy chain variable region comprises the amino acid sequence SEQ ID NO: 220. In certain embodiments, the heavy chain variable region comprises the amino acid sequence SEQ ID NO: 65. In certain embodiments, the antibody comprises a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 168-186 and 225-227. In certain embodiments, the antibody contains a heavy chain containing the amino acid sequence of SEQ ID NO: 225. In certain embodiments, the antibody contains a heavy chain containing the amino acid sequence of SEQ ID NO: 168. In certain embodiments, the antibody contains a heavy chain containing amino acid sequence of SEQ ID NO: 226. In certain embodiments, the antibody contains a heavy chain comprising the amino acid sequence of SEQ ID NO: 169. In certain embodiments, the antibody contains a heavy chain containing the amino acid sequence of SEQ ID NO: 227. In certain embodiments, the antibody contains a heavy chain containing the amino acid sequence of SEQ ID NO: 170.

In another aspect, the present invention provides an antibody or isolated antibody comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 73-77 and 221. In certain embodiments, the light chain variable region comprises an amino acid sequence SEQ ID NO: 221. In certain embodiments, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 73. In certain embodiments, the antibody comprises a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 187-191 and 228. In certain embodiments, the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 228. In certain embodiments, the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 187.

In another aspect, the present invention provides an antibody or isolated antibody that specifically binds to human LAG-3, the antibody comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 73-77 and 221. B In certain embodiments, the light chain variable region comprises the amino acid sequence SEQ ID NO: 221. In certain embodiments, the light chain variable region comprises the amino acid sequence SEQ ID NO: 73. In certain embodiments, the antibody comprises a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 187-191 and 228. In certain embodiments, the antibody contains a light chain comprising the amino acid sequence of SEQ ID NO: 228. In certain embodiments, the antibody contains a light chain comprising the amino acid sequence of SEQ ID NO: 187.

In another aspect, the present invention provides an antibody or isolated antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region, respectively, comprising the amino acid sequences set forth in SEQ ID NOs: 56 and 73; 56 and 74; 56 and 75; 56 and 76; 56 and 77; 57 and 73; 57 and 74; 57 and 75; 57 and 76; 57 and 77; 58 and 73; 58 and 74; 58 and 75; 58 and 76; 58 and 77; 59 and 73; 59 and 74; 59 and 75; 59 and 76; 59 and 77; 60 and 73; 60 and 74; 60 and 75; 60 and 76; 60 and 77; 61 and 77; 62 and 77; 63 and 73; 64 and 73; 65 and 73; 220 and 73; 65 and 221; 220 and 221; 66 and 73; 67 and 73; 68 and 73; 69 and 73; 70 and 73; 71 and 73 or 72 and 73. In some embodiments, the heavy chain variable region and the light chain variable region, respectively, comprise the amino acid sequences set forth in SEQ ID NOs: 65 and 73. In some embodiments, the heavy chain variable region and the light chain variable region the chains accordingly contain the amino acid sequences set forth in SEQ ID NOs: 220 and 73; 65 and 221 or 220 and 221.

In another aspect, this disclosure relates to an antibody or isolated antibody comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region, respectively, consist of the amino acid sequences set forth in SEQ ID NO: 56 and 73; 56 and 74; 56 and 75; 56 and 76; 56 and 77; 57 and 73; 57 and 74; 57 and 75; 57 and 76; 57 and 77; 58 and 73; 58 and 74; 58 and 75; 58 and 76; 58 and 77; 59 and 73; 59 and 74; 59 and 75; 59 and 76; 59 and 77; 60 and 73; 60 and 74; 60 and 75; 60 and 76; 60 and 77; 61 and 77; 62 and 77; 63 and 73; 64 and 73; 65 and 73; 220 and 73; 65 and 221; 220 and 221; 66 and 73; 67 and 73; 68 and 73; 69 and 73; 70 and 73; 71 and 73 or 72 and 73. In some embodiments, the amino acid sequences of the variable heavy chain region and the variable light chain region, respectively, consist of the amino acid sequences set forth in SEQ ID NOs: 65 and 73. In some embodiments, the amino acid sequences of the variable heavy chain region chain and light chain variable region, respectively, consist of the amino acid sequences specified in SEQ ID NO: 220 and 73; 65 and 221 or 220 and 221.

In another aspect, the present invention provides an antibody or isolated antibody that specifically binds to human LAG-3, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region respectively comprise amino acid sequences, presented in SEQ ID NO: 56 and 73; 56 and 74; 56 and 75; 56 and 76; 56 and 77; 57 and 73; 57 and 74; 57 and 75; 57 and 76; 57 and 77; 58 and 73; 58 and 74; 58 and 75; 58 and 76; 58 and 77; 59 and 73; 59 and 74; 59 and 75; 59 and 76; 59 and 77; 60 and 73; 60 and 74; 60 and 75; 60 and 76; 60 and 77; 61 and 77; 62 and 77; 63 and 73; 64 and 73; 65 and 73; 220 and 73; 65 and 221; 220 and 221; 66 and 73; 67 and 73; 68 and 73; 69 and 73; 70 and 73; 71 and 73 or 72 and 73. In some embodiments, the heavy chain variable region and the light chain variable region, respectively, comprise the amino acid sequences set forth in SEQ ID NOs: 65 and 73. In some embodiments, the heavy chain variable region and the light chain variable region, respectively, contain the amino acid sequences shown in SEQ ID NO: 220 and 73; 65 and 221 or 220 and 221.

In another aspect, this disclosure relates to an antibody or isolated antibody that specifically binds to human LAG-3 comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region, respectively, consist of the amino acid sequences presented in SEQ ID NO: 56 and 73; 56 and 74; 56 and 75; 56 and 76; 56 and 77; 57 and 73; 57 and 74; 57 and 75; 57 and 76; 57 and 77; 58 and 73; 58 and 74; 58 and 75; 58 and 76; 58 and 77; 59 and 73; 59 and 74; 59 and 75; 59 and 76; 59 and 77; 60 and 73; 60 and 74; 60 and 75; 60 and 76; 60 and 77; 61 and 77; 62 and 77; 63 and 73; 64 and 73; 65 and 73; 220 and 73; 65 and 221; 220 and 221; 66 and 73; 67 and 73; 68 and 73; 69 and 73; 70 and 73; 71 and 73 or 72 and 73. In some embodiments, the amino acid sequences of the variable heavy chain region and the variable light chain region, respectively, consist of the amino acid sequences set forth in SEQ ID NOs: 65 and 73. In some embodiments, the amino acid sequences of the variable heavy chain region chain and light chain variable region, respectively, consist of the amino acid sequences specified in SEQ ID NO: 220 and 73; 65 and 221 or 220 and 221.

In certain embodiments of any of the above aspects, where applicable, X in SEQ ID NO: 220 is Q. In certain embodiments of any of the above aspects, where applicable, X in SEQ ID NO: 220 is pyroglutamate. In certain embodiments of any of the above aspects, where applicable, X in SEQ ID NO: 221 is E. In certain embodiments of any of the above aspects, where applicable, X in SEQ ID NO: 221 is pyroglutamate. In certain embodiments of any of the above aspects, where applicable, X in SEQ ID NO: 220 is Q and X in SEQ ID NO: 221 is E. In certain embodiments of any of the above aspects, where applicable, X in SEQ ID NO: 220 is Q and X in SEQ ID NO: 221 is pyroglutamate. In certain embodiments of any of the above aspects, where applicable, X in SEQ ID NO: 220 is pyroglutamate and X in SEQ ID NO: 221 is E. In certain embodiments of any of the above aspects, where applicable, X in SEQ ID NO: 220 is pyroglutamate, and X in SEQ ID NO: 221 is pyroglutamate.

In another aspect, the present invention provides an antibody or isolated antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 168 or 225 and a light chain containing the amino acid sequence of SEQ ID NO: 187 or 228.

In another aspect, the present invention provides an antibody or isolated antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 168 and a light chain containing the amino acid sequence of SEQ ID NO: 187.

In another aspect, this disclosure provides an antibody or isolated antibody that specifically binds to human LAG-3, the antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 168 or 225 and a light chain containing the amino acid sequence of SEQ ID NO: 187 or 228.

In another aspect, this disclosure provides an antibody or isolated antibody that specifically binds to human LAG-3, the antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 168 and a light chain containing the amino acid sequence of SEQ ID NO: 187.

In another aspect, the present invention provides an antibody or isolated antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 169 or 226, and a light chain containing the amino acid sequence of SEQ ID NO: 187 or 228.

In another aspect, the present invention provides an antibody or isolated antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 169 and a light chain comprising the amino acid sequence of SEQ ID NO: 187.

In another aspect, this disclosure provides an antibody or isolated antibody that specifically binds to human LAG-3, the antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 169 or 226 and a light chain containing the amino acid sequence of SEQ ID NO: 187 or 228.

In another aspect, this disclosure provides an antibody or isolated antibody that specifically binds to human LAG-3, the antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 169 and a light chain containing the amino acid sequence of SEQ ID NO: 187.

In another aspect, the present invention provides an antibody or isolated antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 170 or 227, and a light chain containing the amino acid sequence of SEQ ID NO: 187 or 228.

In another aspect, the present invention provides an antibody or isolated antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 170 and a light chain comprising the amino acid sequence of SEQ ID NO: 187.

In another aspect, this disclosure provides an antibody or isolated antibody that specifically binds to human LAG-3, the antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 170 or 227 and a light chain comprising the amino acid sequence of SEQ ID NO: 187 or 228.

In another aspect, this disclosure provides an antibody or isolated antibody that specifically binds to human LAG-3, the antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 170 and a light chain containing the amino acid sequence of SEQ ID NO: 187.

In another aspect, the present invention provides an antibody or isolated antibody comprising a heavy chain and a light chain, wherein the heavy chain amino acid sequence consists of the amino acid sequence set forth in SEQ ID NO: 168 or 225, and the light chain amino acid sequence consists of the amino acid sequence set forth in SEQ ID NO: 187 or 228.

In another aspect, this disclosure relates to an antibody or isolated antibody comprising a heavy chain and a light chain, wherein the amino acid sequences of the heavy chain and light chain, respectively, consist of the amino acid sequences set forth in SEQ ID NOs: 168 and 187.

In another aspect, the present invention provides an antibody or isolated antibody that specifically binds to human LAG-3 comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 168 or 225, and the amino acid sequence the light chain sequence consists of the amino acid sequence shown in SEQ ID NO: 187 or 228.

In another aspect, this disclosure relates to an antibody or isolated antibody that specifically binds to human LAG-3 comprising a heavy chain and a light chain, wherein the amino acid sequences of the heavy chain and light chain, respectively, consist of the amino acid sequences set forth in SEQ ID NO: 168 and 187.

In another aspect, the present invention provides an antibody or isolated antibody comprising a heavy chain and a light chain, wherein the heavy chain amino acid sequence consists of the amino acid sequence set forth in SEQ ID NO: 169 or 226, and the light chain amino acid sequence consists of the amino acid sequence set forth in in SEQ ID NO: 187 or 228.

In another aspect, this disclosure relates to an antibody or isolated antibody comprising a heavy chain and a light chain, wherein the amino acid sequences of the heavy chain and light chain, respectively, consist of the amino acid sequences set forth in SEQ ID NOs: 169 and 187.

In another aspect, the present invention provides an antibody or isolated antibody that specifically binds to human LAG-3 comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 169 or 226, and the amino acid sequence the light chain sequence consists of the amino acid sequence shown in SEQ ID NO: 187 or 228.

In another aspect, this disclosure provides an antibody or isolated antibody that specifically binds to human LAG-3 comprising a heavy chain and a light chain, wherein the amino acid sequences of the heavy chain and light chain, respectively, consist of the amino acid sequences set forth in SEQ ID NO: 169 and 187.

In another aspect, the present invention provides an antibody or isolated antibody comprising a heavy chain and a light chain, wherein the heavy chain amino acid sequence consists of the amino acid sequence set forth in SEQ ID NO: 170 or 227, and the light chain amino acid sequence consists of the amino acid sequence set forth in in SEQ ID NO: 187 or 228.

In another aspect, this disclosure relates to an antibody or isolated antibody comprising a heavy chain and a light chain, wherein the amino acid sequences of the heavy chain and light chain, respectively, consist of the amino acid sequences set forth in SEQ ID NOs: 170 and 187.

In another aspect, the present invention provides an antibody or isolated antibody that specifically binds to human LAG-3 comprising a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 170 or 227, and the amino acid sequence the light chain sequence consists of the amino acid sequence shown in SEQ ID NO: 187 or 228.

In another aspect, this disclosure provides an antibody or isolated antibody that specifically binds to human LAG-3 comprising a heavy chain and a light chain, wherein the amino acid sequences of the heavy chain and light chain, respectively, consist of the amino acid sequences set forth in SEQ ID NO: 170 and 187.

In certain embodiments of any of the above aspects, where applicable, X in SEQ ID NO: 225 is Q. In certain embodiments of any of the above aspects, where applicable, X in SEQ ID NO: 225 is pyroglutamate. In certain embodiments of any of the above aspects, where applicable, X in SEQ ID NO: 228 is E. In certain embodiments of any of the above aspects, where applicable, X in SEQ ID NO: 228 is pyroglutamate. In certain embodiments of any of the above aspects, where applicable, X in SEQ ID NO: 225 is Q and X in SEQ ID NO: 228 is E. In certain embodiments of any of the above aspects, where applicable, X in SEQ ID NO: 225 is Q and X in SEQ ID NO: 228 is pyroglutamate. In certain embodiments of any of the above aspects, where applicable, X in SEQ ID NO: 225 is pyroglutamate and X in SEQ ID NO: 228 is E. In certain embodiments of any of the above aspects, where applicable, X in SEQ ID NO: 225 is pyroglutamate, and X in SEQ ID NO: 228 is pyroglutamate.

In certain embodiments of any of the above aspects, where applicable, X in SEQ ID NO: 226 is Q. In certain embodiments of any of the above aspects, where applicable, X in SEQ ID NO: 226 is pyroglutamate. In certain embodiments of any of the above aspects, where applicable, X in SEQ ID NO: 228 is E. In certain embodiments of any of the above aspects, where applicable, X in SEQ ID NO: 228 is pyroglutamate. In certain embodiments of any of the above aspects, where applicable, X in SEQ ID NO: 226 is Q and X in SEQ ID NO: 228 is E. In certain embodiments of any of the above aspects, where applicable, X in SEQ ID NO: 226 is Q and X in SEQ ID NO: 228 is pyroglutamate. In certain embodiments of any of the above aspects, where applicable, X in SEQ ID NO: 226 is pyroglutamate and X in SEQ ID NO: 228 is E. In certain embodiments of any of the above aspects, where applicable, X in SEQ ID NO: 226 is pyroglutamate, and X in SEQ ID NO: 228 is pyroglutamate.

In certain embodiments of any of the above aspects, where applicable, X in SEQ ID NO: 227 is Q. In certain embodiments of any of the above aspects, where applicable, X in SEQ ID NO: 227 is pyroglutamate. In certain embodiments of any of the above aspects, where applicable, X in SEQ ID NO: 228 is E. In certain embodiments of any of the above aspects, where applicable, X in SEQ ID NO: 228 is pyroglutamate. In certain embodiments of any of the above aspects, where applicable, X in SEQ ID NO: 227 is Q and X in SEQ ID NO: 228 is E. In certain embodiments of any of the above aspects, where applicable, X in SEQ ID NO: 227 is Q and X in SEQ ID NO: 228 is pyroglutamate. In certain embodiments of any of the above aspects, where applicable, X in SEQ ID NO: 227 is pyroglutamate and X in SEQ ID NO: 228 is E. In certain embodiments of any of the above aspects, where applicable, X in SEQ ID NO: 227 is pyroglutamate, and X in SEQ ID NO: 228 is pyroglutamate.

In certain embodiments, the antibody comprises a heavy chain constant region selected from the group consisting of human IgG1, _IgG2 , _IgG3 , _IgG4 , _IgA1 , and human _IgA2 . In certain embodiments, the heavy chain constant region is IgG1. In some embodiments, the IgG1 amino acid sequence contains the N297A mutation, numbered according to the EU numbering system. In certain embodiments, the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 194. In some embodiments, the IgG1 amino acid sequence contains the N297Q mutation numbered in accordance with the EU numbering system. In certain embodiments, IgG1 is afucosylated IgG1. In certain embodiments, the heavy chain constant region is IgG ₄ . In some embodiments, the IgG ₄ amino acid sequence contains the S228P mutation, numbered in accordance with the EU numbering system. In certain embodiments, the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 196.

In certain embodiments, the antibody comprises a light chain constant region selected from the group consisting of human IgGK and IgGλ. In certain embodiments, the light chain constant region is IgGK. In certain embodiments, the antibody comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 198. In certain embodiments, the antibody comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 219. In certain embodiments, the light chain constant region is IgGλ.

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In another aspect, this disclosure relates to an antibody or isolated antibody that competes for binding to human LAG-3 with an antibody as described herein. In some embodiments, the present invention provides an antibody or isolated antibody that competes for binding to human LAG-3 with an antibody comprising the heavy and light chain variable region amino acid sequences set forth in SEQ ID NOs: 15 and 16, respectively. In some embodiments, the present invention provides an antibody or isolated antibody that competes for binding to human LAG-3 with an antibody comprising the heavy and light chain variable region amino acid sequences set forth in SEQ ID NOs: 65 and 73; 220 and 73; 65 and 221 or 220 and 221 respectively.

In another aspect, this disclosure relates to an antibody or isolated antibody that binds to the same human LAG-3 epitope as the antibody disclosed herein. In certain embodiments, the present invention provides an antibody or isolated antibody that binds to the same human LAG-3 epitope as the antibody comprising the heavy and light chain variable region amino acid sequences set forth in SEQ ID NOs: 15 and 16, respectively. In certain embodiments, the present invention provides an antibody or isolated antibody that binds to the same human LAG-3 epitope as the antibody comprising the heavy and light chain variable region amino acid sequences set forth in SEQ ID NOs: 65 and 73; 220 and 73; 65 and 221 or 220 and 221 respectively.

In another aspect, the present invention provides an antibody or isolated antibody that binds, for example, specifically binds to an epitope of human LAG-3. In certain embodiments, the antibody binds to an epitope located in the human LAG-3 region consisting of the amino acid sequence SEQ ID NO: 216. In certain embodiments, the antibody binds to an epitope located in the human LAG-3 region consisting of the amino acid sequence SEQ ID NO: 215. In certain embodiments, the antibody binds to an epitope located in the human LAG-3 region consisting of the amino acid sequence of SEQ ID NO: 214. In certain embodiments, the antibody binds to an epitope located in the human LAG-3 region consisting of amino acid sequence SEQ ID NO: 213. In certain embodiments, the antibody binds to an epitope located in the human LAG-3 region consisting of the amino acid sequence SEQ ID NO: 212. In certain embodiments, the antibody binds to an epitope located in the LAG-3 region human, consisting of the amino acid sequence SEQ ID NO: 211.

In another aspect, this disclosure provides an antibody or isolated antibody that specifically binds to the same epitope of human LAG-3 as any antibody of the present invention. In certain embodiments, the antibody binds to an epitope located in the human LAG-3 region consisting of the amino acid sequence SEQ ID NO: 216. In certain embodiments, the antibody binds to an epitope located in the human LAG-3 region consisting of the amino acid sequence SEQ ID NO: 215. In certain embodiments, the antibody binds to an epitope located in the human LAG-3 region consisting of the amino acid sequence of SEQ ID NO: 214. In certain embodiments, the antibody binds to an epitope located in the human LAG-3 region consisting of amino acid sequence SEQ ID NO: 213. In certain embodiments, the antibody binds to an epitope located in the human LAG-3 region consisting of the amino acid sequence SEQ ID NO: 212. In certain embodiments, the antibody binds to an epitope located in the LAG-3 region human, consisting of the amino acid sequence SEQ ID NO: 211.

In another aspect, the present invention provides an antibody that, when bound to human LAG-3 protein or a fragment thereof comprising the amino acid sequence of SEQ ID NO: 217, reduces hydrogen/deuterium exchange in the region consisting of the amino acid sequence set forth in SEQ ID NO: 216 regarding hydrogen/deuterium exchange in the region consisting of the amino acid sequence presented in SEQ ID NO: 216 in the absence of an antibody, as determined by hydrogen/deuterium analysis. In another aspect, the present invention provides an antibody that, when bound to human LAG-3 protein or a fragment thereof comprising the amino acid sequence of SEQ ID NO: 217, reduces hydrogen/deuterium exchange in the region consisting of the amino acid sequence set forth in SEQ ID NO: 215 regarding hydrogen/deuterium exchange in the region consisting of the amino acid sequence presented in SEQ ID NO: 215 in the absence of an antibody, as determined by hydrogen/deuterium analysis. In another aspect, the present invention provides an antibody that, when bound to human LAG-3 protein or a fragment thereof comprising the amino acid sequence of SEQ ID NO: 217, reduces hydrogen/deuterium exchange in the region consisting of the amino acid sequence set forth in SEQ ID NO: 214 regarding hydrogen/deuterium exchange in the region consisting of the amino acid sequence presented in SEQ ID NO: 214 in the absence of an antibody, as determined by hydrogen/deuterium analysis. In another aspect, the present invention provides an antibody that, when bound to human LAG-3 protein or

- 11 044026 fragment containing the amino acid sequence of SEQ ID NO: 217, reduces the hydrogen/deuterium exchange in the region consisting of the amino acid sequence presented in SEQ ID NO: 213 relative to the hydrogen/deuterium exchange in the region consisting of the amino acid sequence presented in SEQ ID NO: 213 in the absence of antibody, as determined by hydrogen/deuterium analysis. In another aspect, the present invention provides an antibody that, when bound to human LAG-3 protein or a fragment thereof comprising the amino acid sequence of SEQ ID NO: 217, reduces hydrogen/deuterium exchange in the region consisting of the amino acid sequence set forth in SEQ ID NO: 212 regarding hydrogen/deuterium exchange in the region consisting of the amino acid sequence presented in SEQ ID NO: 212 in the absence of an antibody, as determined by hydrogen/deuterium analysis. In another aspect, the present invention provides an antibody that, when bound to human LAG-3 protein or a fragment thereof comprising the amino acid sequence of SEQ ID NO: 217, reduces hydrogen/deuterium exchange in the region consisting of the amino acid sequence set forth in SEQ ID NO: 211 regarding hydrogen/deuterium exchange in the region consisting of the amino acid sequence presented in SEQ ID NO: 211 in the absence of an antibody, as determined by hydrogen/deuterium analysis. In some embodiments, the reduction in hydrogen/deuterium exchange is measured using hydrogen-deuterium exchange (HDX), for example, as described herein in the Examples.

In another aspect, this disclosure provides an antibody or isolated antibody that specifically binds to the same epitope of human LAG-3 as any antibody of the present invention. In certain embodiments, the antibody, when bound to human LAG-3 protein or a fragment thereof comprising the amino acid sequence of SEQ ID NO: 217, reduces hydrogen/deuterium exchange in a region consisting of the amino acid sequence set forth in SEQ ID NO: 216 relative to hydrogen/deuterium exchange. deuterium in the region consisting of the amino acid sequence presented in SEQ ID NO: 216 in the absence of antibody, as determined by hydrogen/deuterium analysis. In certain embodiments, the antibody, when bound to human LAG-3 protein or a fragment thereof comprising the amino acid sequence of SEQ ID NO: 217, reduces the hydrogen/deuterium exchange in the region consisting of the amino acid sequence set forth in SEQ ID NO: 215 relative to the hydrogen/deuterium exchange. deuterium in the region consisting of the amino acid sequence presented in SEQ ID NO: 215 in the absence of antibody, as determined by hydrogen/deuterium analysis. In certain embodiments, the antibody, when bound to human LAG-3 protein or a fragment thereof comprising the amino acid sequence of SEQ ID NO: 217, reduces the hydrogen/deuterium exchange in the region consisting of the amino acid sequence set forth in SEQ ID NO: 214 relative to the hydrogen/deuterium exchange. deuterium in the region consisting of the amino acid sequence presented in SEQ ID NO: 214 in the absence of antibody, as determined by hydrogen/deuterium analysis. In certain embodiments, the antibody, when bound to human LAG-3 protein or a fragment thereof comprising the amino acid sequence of SEQ ID NO: 217, reduces the hydrogen/deuterium exchange in the region consisting of the amino acid sequence set forth in SEQ ID NO: 213 relative to the hydrogen/deuterium exchange. deuterium in the region consisting of the amino acid sequence presented in SEQ ID NO: 213 in the absence of antibody, as determined by hydrogen/deuterium analysis. In certain embodiments, the antibody, when bound to human LAG-3 protein or a fragment thereof comprising the amino acid sequence of SEQ ID NO: 217, reduces the hydrogen/deuterium exchange in the region consisting of the amino acid sequence set forth in SEQ ID NO: 212 relative to the hydrogen/deuterium exchange. deuterium in the region consisting of the amino acid sequence presented in SEQ ID NO: 212 in the absence of antibody, as determined by hydrogen/deuterium analysis. In certain embodiments, the antibody, when bound to human LAG-3 protein or a fragment thereof comprising the amino acid sequence of SEQ ID NO: 217, reduces hydrogen/deuterium exchange in a region consisting of the amino acid sequence set forth in SEQ ID NO: 211 relative to hydrogen/deuterium exchange. deuterium in the region consisting of the amino acid sequence presented in SEQ ID NO: 211 in the absence of antibody, as determined by hydrogen/deuterium analysis. In some embodiments, the reduction in hydrogen/deuterium exchange is measured using hydrogen-deuterium exchange (HDX), for example, as described herein in the Examples.

In certain embodiments, the antibody is a humanized antibody. In certain embodiments, the antibody is a murine antibody. In certain embodiments, the antibody is a chimeric antibody. In certain embodiments, the antibody is antagonistic for human LAG-3. In certain embodiments, the antibody deactivates, reduces, or inhibits human LAG-3 activity. In certain embodiments, the antibody inhibits the binding of human LAG-3 to MHC class II. In certain embodiments, the antibody induces the production of IL-2 by peripheral blood mononuclear cells (PBMCs) stimulated with staphylococcal enterotoxin A (SEA). In certain embodiments, the antibody induces the production of TNFa by tumor infiltrating lymphocytes (TILs) stimulated with anti-CD3 and anti-CD28 antibodies.

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In another aspect, this disclosure relates to an antibody or isolated antibody, as described herein, conjugated to a cytotoxic agent.

In another aspect, this disclosure relates to an antibody or isolated antibody, as described herein, conjugated to a cytotoxic agent.

In another aspect, this disclosure relates to an antibody or isolated antibody, as described herein, conjugated to a toxin.

In another aspect, this disclosure relates to an antibody or isolated antibody, as described herein, conjugated to a radionuclide.

In another aspect, this disclosure relates to an antibody or isolated antibody, as described herein, conjugated to a detectable label.

In another aspect, this disclosure relates to an antibody or isolated antibody as described herein, wherein the N-terminal amino acid residue of the heavy chain variable region is a pyroglutamate (e.g., as a result of post-translational cyclization of the free amino group of the N-terminal E or Q variable region residue heavy chain). In another aspect, this disclosure relates to an antibody or isolated antibody as described herein, wherein the N-terminal amino acid residue of the heavy chain is a pyroglutamate (eg, as a result of post-translational cyclization of the free amino group of the N-terminal E or Q residue of the heavy chain).

In another aspect, this disclosure relates to an antibody or isolated antibody as described herein, wherein the N-terminal amino acid residue of the light chain variable region is a pyroglutamate (e.g., as a result of post-translational cyclization of the free amino group of the N-terminal E or Q variable region residue light chain). In another aspect, this disclosure relates to an antibody or isolated antibody as described herein, wherein the N-terminal amino acid residue of the light chain is a pyroglutamate (eg, as a result of post-translational cyclization of the free amino group of the N-terminal E or Q residue of the light chain).

In another aspect, this disclosure relates to an antibody or isolated antibody, as described herein, in which the heavy chain is aglycosylated.

In another aspect, this disclosure relates to a pharmaceutical composition comprising an antibody disclosed herein and a pharmaceutically acceptable carrier or excipient.

In another aspect, this disclosure relates to a polynucleotide or isolated polynucleotide encoding the heavy and/or light chain of an antibody as described herein. In another aspect, this disclosure relates to a vector containing a polynucleotide. In another aspect, the present invention relates to a recombinant host cell containing a polynucleotide. In another aspect, the present invention relates to a recombinant host cell containing a polynucleotide. In another aspect, this disclosure relates to a method for producing an antibody as disclosed herein, the method comprising culturing a host cell such that the polynucleotide is expressed and the antibody is produced. In one embodiment, the method is an in vitro method.

In one embodiment, the present invention provides an antibody of the invention, or a pharmaceutical composition of the invention, or a polynucleotide of the invention, or a vector of the invention, or a recombinant host cell of the invention for use as a drug.

In one embodiment, the present invention provides an antibody of the invention, or a pharmaceutical composition of the invention, or a polynucleotide of the invention, or a vector of the invention, or a recombinant host cell of the invention for use in diagnostics.

In another aspect, this disclosure relates to a method of increasing T cell activation in response to an antigen in a subject, the method comprising administering to the subject an effective amount of an antibody or pharmaceutical composition as described herein. In another aspect, the present invention provides a method of treating cancer in a subject, comprising administering to the subject an effective amount of an antibody or pharmaceutical composition as described herein. In certain embodiments of the above methods, the antibody or pharmaceutical composition is administered subcutaneously. In certain embodiments of the above methods, the antibody or pharmaceutical composition is administered intravenously. In certain embodiments of the above methods, the antibody or pharmaceutical composition is administered intratumorally. In certain embodiments of the above methods, the antibody or pharmaceutical composition is delivered to a lymph node draining a tumor. In certain embodiments of the above methods, the antibody or pharmaceutical composition is administered intra-arterially. In certain embodiments of the above methods, the antibody or pharmaceutical composition is administered intranasally.

In one aspect, the invention provides an antibody, polynucleotide, vector, recombinant host cell, and/or pharmaceutical composition of the invention for use in a method of increasing T cell activation in response to an antigen.

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In one aspect, the invention provides an antibody, polynucleotide, vector, recombinant host cell, and/or pharmaceutical composition of the invention for use in a method of increasing T cell activation in response to an antigen in a subject.

In one aspect, the invention provides an antibody, polynucleotide, vector, recombinant host cell, and/or pharmaceutical composition of the invention for use in a method of increasing T cell activation in response to an antigen in a subject, comprising administering to the subject an effective amount of the antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition according to the invention.

In one aspect, the present invention provides an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition of the present invention for use in a method of treating cancer.

In one aspect, the present invention provides an antibody, polynucleotide, vector, recombinant host cell, and/or pharmaceutical composition of the present invention for use in a method of treating cancer in a subject.

In one aspect, the present invention provides an antibody, polynucleotide, vector, recombinant host cell, and/or pharmaceutical composition of the present invention for use in a method of treating cancer in a subject, comprising administering to the subject an effective amount of the antibody, polynucleotide, vector, recombinant host cell, and /or pharmaceutical composition according to the invention.

In one embodiment, the antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition for use in this invention, the antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition is administered subcutaneously or intravenously. In one embodiment, the antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition for use in this invention, the antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition is administered intratumorally or intraarterially. In one embodiment, the antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition for use in this invention, the antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition is administered intranasally.

In some embodiments, the above methods further comprise administering an additional therapeutic agent to the subject. Therefore, in one embodiment of an antibody, polynucleotide, vector, recombinant host cell, and/or pharmaceutical composition for use in a method of the present invention, the method further comprises administering an additional therapeutic agent to the subject.

In one aspect, the present invention relates to (a) an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition of the present invention and (b) an additional therapeutic agent for use as a drug.

In one aspect, the present invention provides (a) an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition of the present invention and (b) an additional therapeutic agent for use in a method of treating cancer.

In one aspect, the present invention provides a pharmaceutical composition, kit or set of parts comprising (a) an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition of the present invention and (b) an additional therapeutic agent.

In certain embodiments, the additional therapeutic agent is a chemotherapeutic agent. In certain embodiments, the additional therapeutic agent is a radiotherapy agent.

In certain embodiments, the additional therapeutic agent is a checkpoint-targeting agent. In certain embodiments, the checkpoint-targeting agent is selected from the group consisting of an anti-PD-1 antagonist antibody, an anti-PD-L1 antagonist antibody, an anti-PD-L2 antagonist antibody, an anti-CTLA-4 antagonist antibody, an anti-TIM-antagonist antibody, 3, anti-LAG-3 antagonist antibody, anti-CEACAM1 antagonist antibody, anti-GITR agonist antibody, anti-OX40 agonist antibody, anti-TIGIT antagonist antibody, anti-CD137 agonist antibody, anti-VISTA antagonist antibody, anti-CD73 antagonist antibody, and anti-CD96 antagonist antibody. In certain embodiments, the additional therapeutic agent is an anti-PD-1 antibody. In certain embodiments, the anti-PD-1 antibody is pembrolizumab. In certain embodiments, the anti-PD-1 antibody is nivolumab. In certain embodiments, the additional therapeutic agent is an anti-PD-L1 antibody. In certain embodiments, the additional therapeutic agent is an anti-CTLA-4 antibody.

In certain embodiments, the additional therapeutic agent comprises a small molecule. In certain embodiments, the additional therapeutic agent is a small molecule inhibitor of the PD-1 pathway. In certain embodiments, the additional therapeutic agent is a small molecule inhibitor of PD-1 or PD-L1.

In certain embodiments, the additional therapeutic agent is an indoleamine 2,3-dioxygenase (IDO) inhibitor. In certain embodiments, the inhibitor is selected from the group consisting of epacadostat BMS-986205 (also known as F001287, see Example 19 of WO 2016/073770, which is incorporated herein by reference in its entirety), indoximod, and NLG919. In certain embodiments, the inhibitor is epacadostat. In certain embodiments, the inhibitor is BMS-986205. In certain embodiments, the inhibitor is indoximod. In certain embodiments, the inhibitor is NLG919.

In certain embodiments, the additional therapeutic agent is an ARG, LSD1, CD112, CD112R, or VEGF inhibitor. In certain embodiments, the additional therapeutic agent is a stimulator of interferon genes (STING) agonist. In certain embodiments, the additional therapeutic agent is a CD80-FC protein.

In certain embodiments, the additional therapeutic agent is a vaccine. In certain embodiments, the vaccine comprises a heat shock protein peptide complex (HSPPC) comprising a heat shock protein complexed with an antigenic peptide. In certain embodiments, the heat shock protein is hsc70 and forms a complex with a tumor-associated antigenic peptide. In certain embodiments, the heat shock protein is gp96 and forms a complex with a tumor-associated antigenic peptide, wherein the HSPPC is derived from a tumor obtained from the subject. In certain embodiments, the heat shock protein is gp96 and forms a complex with a tumor-associated antigenic peptide, wherein the HSPPC is derived from a tumor obtained from the subject. In certain embodiments, the additional therapeutic agent comprises a TCR (Tissue Cell Receptor). In certain embodiments, the additional therapeutic agent is a soluble TCR. In certain embodiments, the additional therapeutic agent is a cell expressing a TCR. In certain embodiments, the additional therapeutic agent is a cell expressing a chimeric antigen receptor. In certain embodiments, the additional therapeutic agent is an antibody that specifically binds to the MHC peptide complex. In certain embodiments, the additional therapeutic agent is an adjuvant. In one aspect, the present invention relates to (a) an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition of the present invention and (b) a vaccine for use as a medicine, for example, for use in a method of treating cancer, in This vaccine contains a heat shock protein peptide complex (HSPPC), which contains a heat shock protein complexed with an antigenic peptide. In one aspect, the present invention relates to a pharmaceutical composition, kit or set of parts comprising (a) an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition of the present invention and (b) a vaccine, optionally the vaccine comprising a peptide complex heat shock protein (HSPPC), containing heat shock protein complexed with an antigenic peptide.

5. Brief description of graphic materials

Fig. 1A, 1B and 1C are a set of histograms showing the binding of anti-LAG-3 Fab to wild-type Jurkat cells or Jurkat cells engineered to express human LAG-3 as determined by flow cytometry. Anti-LAG-3 Fabs tested in this study include P01A12, P01C09, P05E01, P13A04, P13A06, P13B01, P13B02, P13B03, P13B11, P13C06, P13C08, P13C10, P13D04, P13D05, P13E02, P13 F01, P13F02, P13F06, P13F09, P13G01, P13G04, P13G05, P13H05, P14A04, P14B07, P14C04, P14F01, P14F06, P14G01, P14G03, P15B06, P15C02, P15E06, P15F06, P15G05, P16D04 and P16 H05.

Fig. 2A and 2B are graphs showing the results of assays that test the ability of anti-LAG-3 Fabs or negative control Fabs not specific for LAG-3 to block the binding of cross-linked recombinant LAG-3-6His to Raji cells expressing the MHC class II. Fig. 2A is a bar graph showing the percentage of blocking mediated by negative control Fab or Fab against LAG-3 P13B02, P13C08, P13C10, P13E02, P13F02, P01A12, P13B01, P05E01 or P01C09. Fig. 2B is a line graph showing the percentage of LAG-3 binding in the presence of a titration dose of anti-LAG-3 Fab P01A12, P13A06, P13B01, P13B02, P13C06, P13C08, P13C10, P13E02 or P14C04, or a negative control Fab.

Fig. 3 is a line graph similar to the graph shown in FIG. 2B, which shows the percentage of LAG-3 binding with dose titration of full-length chimeric anti-LAG-3 antibody P13A06, P13B01, P13B02, P13C06, P13C08, or P13E02 or an isotype control antibody.

Fig. 4 is a graph showing IL-2 production in human peripheral blood mononuclear cells (PBMCs) when stimulated with Staphylococcus enterotoxin A (SEA) in the absence of any antibody or in the presence of an isotype control antibody or anti-LAG-3 chimeric antibody P13B02.

Fig. 5A and 5B are sequence alignments of the humanized variable regions with the corresponding mouse and human germline sequences. In fig. 5A shows a sequence alignment comparing the humanized heavy chain variable regions H0-H4 (SEQ ID NO: 56-60, respectively), the mouse antibody P13B02 heavy chain variable region (SEQ ID NO: 15) and the human germline sequence IGHV1-46*01 ( SEQ ID NO: 153) and IGHJ1*01 (SEQ ID NO: 200). In fig. 5B shows a sequence alignment comparing the humanized light chain variable regions L0-L4 (SEQ ID NO: 73-77, respectively), the mouse antibody P13B02 light chain variable region (SEQ ID NO: 16) and the human germline sequence IGKV3-20*01 ( SEQ ID NO: 160) and IGKJ1*01 (SEQ ID NO: 201). The dots represent residues identical to the corresponding residues in H0 (Fig. 5A) or L0 (Fig. 5B). Dashes indicate the absence of amino acid residues compared to H0 (Fig. 5A) or L0 (Fig. 5B).

Fig. 6A and 6B are graphs showing the binding of anti-LAG-3 antibodies to Staphylococcal enterotoxin A (SEA)-activated human T cells as measured by flow cytometry. Fig. 6A is a set of histograms testing the chimeric antibody P13B02 (IgG1) and the humanized antibodies P13B02-06 (IgG1), P13B02-07 (IgG1), P13B02-16 (IgG1), P13B02-25 (IgG1), P13B02-26 (IgG1) , P13B02-27 (IgG1), P13B02-30 (IgG1 Gm17 N297A) and P13B02-30 (IgG4). Fig. 6B is a graph showing the binding of anti-LAG-3 antibody P13B0216 (IgG1) or isotype control antibody to activated primary human CD4+ T cells. Mean fluorescence intensity (MFI) values are presented as a function of antibody concentration.

Fig. 7A and 7B are line graphs similar to those shown in FIG. 2B, which shows the percentage of LAG-3 binding against various doses of isotype control antibody, chimeric antibody P13B02 (IgG1), humanized antibody P13B02-06 (IgG1), P13B02-07 (IgG1), P13B02-16 (IgG1), P13B02-26 (IgG1) or P13B02-27 (IgG1) (Fig. 7A) or humanized antibody P13B02-30 (IgGV G1m17 N297A) (Fig. 7B).

Fig. 8A and 8B are graphs showing IL-2 production induced by anti-LAG-3 antibody or isotype control antibody in human peripheral blood mononuclear cells (PBMCs) upon stimulation with human enterotoxin A (SEA). In fig. 8A, the anti-LAG-3 antibody tested was P13B02-30 (IgG1). In fig. 8B anti-LAG-3 P13B02-16 (IgG1) antibody or isotype control antibody was tested in the presence or absence of anti-PD-1 antibody, pembrolizumab (Pembro) or nivolumab (Nivo), anti-PD-L1 antibody #1, 2 or 3 or anti-CTLA-4 antibodies ipilimumab (Ipi).

Fig. 9A and 9B are graphs showing TNFa production by primary tumor infiltrating lymphocytes (TILs) induced by anti-LAG-3 antibody P13B02-30 (IgG1 G1m3 N297A) or isotype control antibody, alone or in combination with anti-PD-1 antibody pembrolizumab (Pembro ). TILs were isolated from renal cell carcinoma (Fig. 9A) or colorectal cancer (Fig. 9B) tumors and activated with anti-CD3/CD28 microbeads.

Fig. 10A and 10B are graphs showing that anti-LAG-3 antibody P13B0230 (IgG1 G1m3 N297A) enhanced T cell activation in a LAG-3 mediated cell suppression assay. Jurkat:-NFAT-luciferase-LAG-3 cells were incubated in the presence of sextulette titers of either anti-LAG-3 antibody (black dots) or isotype control antibody (white dots), a fixed concentration of Raji cells, and a fixed concentration of staphylococcal enterotoxin E (SEE). . In the first experiment, antibody concentrations of 0.2-50 μg/ml were tested (Fig. 10A). In the second experiment, antibody concentrations of 0.1-100 μg/ml were tested (Fig. 10B). Unrelated light units of the luciferase reporter.

6. Detailed description

This invention provides antibodies that specifically bind to LAG-3 (eg, human LAG-3) and antagonize the function of LAG-3, eg, LAG-3-mediated immune suppression. Also provided are pharmaceutical compositions containing these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for producing these antibodies, and methods of treating a subject using these antibodies. The antibodies described herein are particularly useful for increasing T cell activation in response to an antigen (eg, a tumor antigen or an infectious disease antigen) and, therefore, for treating cancer in a subject or treating or preventing an infectious disease in a subject. All instances of isolated antibodies described herein are further considered to be antibodies that may, but are not necessarily, isolated. All instances of isolated polynucleotides described herein are further considered to be polynucleotides that may, but are not necessarily, isolated. All instances of antibodies described herein are further considered to be antibodies that may, but need not, be isolated. All instances of polynucleotides described herein are further considered to be polynucleotides that may, but are not necessarily, isolated.

6.1. Definitions.

As used herein, the terms about and approximately, when used to change a numeric value or numeric range, indicate variations of 5 to 10% greater (e.g., up to 5 to 10% greater) and 5 to 10% less (e.g., 5-10% less) of the value or range remain within the intended value of the given value or range.

As used herein, the term LAG-3 refers to lymphocyte activation gene 3 (also known as CD223). As used herein, the term human LAG-3 refers to the human LAG-3 protein encoded by the wild-type human LAG-3 gene, for example, GenBank™ accession number NM_002286.5. An example of an immature human LAG-3 amino acid sequence is shown as SEQ ID NO: 166. Exemplary human LAG-3 mature amino acid sequences are shown as SEQ ID NO: 167 and SEQ ID NO: 210.

As used herein, the terms antibody and antibodies include full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules containing antibody CDRs, VH regions, or VL regions. Examples of antibodies include monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, polyspecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, mouse antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies containing two heavy chain molecules and two light chain molecules chains, antibody light chain monomer, antibody heavy chain monomer, antibody light chain dimer, antibody heavy chain dimer, antibody heavy chain light chain antibody pair, intrabodies, heteroconjugate antibodies, antibody-drug conjugates, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fv (scFv), camel antibodies, affibodies, Fab fragments, F(ab') ₂ fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-I antibodies) and antigen-binding fragments of any of the above. In certain embodiments, the antibodies described herein are polyclonal antibody populations. Antibodies can be of any type (eg, IgG, IgE, IgM, IgD, IgA or IgY), any class (eg, IgG1, IgG2, IgG ₃ , IgG ₄ , IgA1 or IgA ₂ ) or any subclass (eg, IgG _2a or IgG ₂ b) immunoglobulin molecules. In certain embodiments, the antibodies described herein are IgG antibodies or a class (eg, human IgG1 or _IgG4 ) or subclass thereof. In a specific embodiment, the antibody is a humanized monoclonal antibody. In another specific embodiment, the antibody is a human monoclonal antibody.

As used herein, the terms VH region and VL region refer to the heavy and light chain variable regions of a single antibody, respectively, comprising FRs (framework regions) 1, 2, 3 and 4 and CDRs (complementarity determining regions) 1, 2 and 3 ( see Kabat et al., (1991) Sequences of Proteins of Immunological Interest (NIH Publication No. 91-3242, Bethesda), which is incorporated herein by reference in its entirety).

As used herein, the term CDR or complementarity determining region refers to contiguous antigenic junction sites found in the variable region of both heavy and light chain polypeptides. These specific areas have been described in Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest. (1991), in Chothia et al., J. Mol. Biol. 196:901-917 (1987) and in MacCallum et al., J. Mol. Biol. 262:732-745 (1996), all of which are incorporated herein by reference in their entirety, the definitions including overlapping or subsets of amino acid residues when compared to each other. In certain embodiments, the term CDR is a CDR as defined in Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest. (1991). In certain embodiments, the term CDR is a CDR as defined in Chothia et al., J. Mol. Biol. 196:901-917 (1987). In certain embodiments, the term CDR is a CDR as defined in MacCallum et al., J. Mol. Biol. 262:732-745 (1996) and Martin A. Protein Sequence and Structure Analysis of Antibody Variable Domains, in Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001).

As used herein, the term framework amino acid residues (FR) refers to amino acids in the framework region of an immunoglobulin chain. The term framework region or FR region as used herein includes amino acid residues that are part of the variable region but are not part of the CDR (eg, using the Kabat or Chothia definition of CDR).

As used herein, the terms variable region and variable domain are

- 17 044026 are used interchangeably and are common in the art. A variable region generally refers to a portion of an antibody, typically a portion of the light or heavy chain, typically the amino terminus 110 to 125 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which vary widely in sequence among antibodies and are used in the binding and specificity of a particular antibody for its particular antigen. Variation in the sequence is concentrated in those regions called complementarity determining regions (CDRs), while the more highly conserved regions in the variable domain are called framework regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the light and heavy chain CDRs are primarily responsible for antibody-antigen interaction and specificity. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region includes rodent or mouse CDRs and human framework regions (FRs). In specific embodiments, the variable region is a primate (eg, non-human primate) variable region. In certain embodiments, the variable region includes rodent or mouse CDRs and primate (eg, non-human primate) framework regions (FRs).

The terms VL and VL domain are used interchangeably to refer to the variable region of the light chain of an antibody.

The terms VH and VH domain are used interchangeably to refer to the variable region of the heavy chain of an antibody.

As used herein, the terms constant region and constant domain are used interchangeably and are common in the art. A constant region is a portion of an antibody, such as the carboxyl-terminal portion of the light and/or heavy chain, that is not directly involved in antibody binding to antigen, but that may exhibit various effector functions, such as interaction with an Fc receptor (e.g., Fc gamma receptor ). The constant region of an immunoglobulin molecule usually has a more conserved amino acid sequence relative to the variable domain of the immunoglobulin.

As used herein, the term heavy chain, when used in reference to an antibody, can refer to any single type, such as alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant domain, which leads to the antibody classes IgA, IgD, IgE, IgG and IgM, respectively, including IgG subclasses, for example, IgG1, _IgG2 , _IgG3 and _IgG4 .

As used herein, the term light chain, when used in relation to an antibody, can refer to any other type, such as kappa (k) or lambda (λ), based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art.

As used herein, the term EU numbering system refers to the EU numbering convention for antibody constant regions as described in Edelman, G.M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Dept. Health and Human Services, 5th edition, 1991, each of which is incorporated herein by reference in its entirety.

The term binding affinity generally refers to the strength of the total sum of non-covalent interactions between one binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise specified, binding affinity as used herein refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can usually be represented by a dissociation constant (K _D ). Affinity can be measured and/or expressed in several ways known in the art, including, but not limited to, the equilibrium dissociation constant (K _D ) and the equilibrium association constant (K _A ). K _D is calculated as the quotient of koff/kon, while K _A is calculated as the quotient of k _on /ko _f f. k _on refers to the rate constant of association, eg antibody to antigen, and kf refers to the rate constant of dissociation, eg antibody to antigen. kon and ko _ff can be determined by methods known to one skilled in the art, such as BIAcore® or KinExA. As used herein, the term lower affinity refers to higher K _D .

As used herein, the terms specifically binds, specifically recognizes, immunospecifically binds, and immunospecifically recognizes are similar terms in the context of antibodies and refer to molecules that bind to an antigen (eg, epitope or immune complex) as understood by one of ordinary skill in the art. . For example, a molecule that specifically binds an antigen may bind to other peptides or polypeptides, typically with lower affinity, as determined, for example, by an immunoassay, BIAcore®, KinExA 3000 instrument (Sapidyne Instruments, Boise, Idaho),

- 18 044026 or other analysis known in the art. In a specific embodiment, molecules that specifically bind to an antigen bind to the antigen with a KA that is at least 2 log (i.e., by a factor of 10), 2.5 log, 3 log, 4 log, or greater than the KA, when molecules bind nonspecifically to another antigen.

In another specific embodiment, molecules that specifically bind to an antigen do not cross-react with other proteins under similar binding conditions. In another specific embodiment, molecules that specifically bind to LAG-3 do not cross-react with other He-LAG-3 proteins. In a specific embodiment, provided herein is an antibody that binds to LAG-3 (eg, human LAG-3) with higher affinity than another unrelated antigen. In certain embodiments, provided herein is an antibody that binds to LAG-3 (e.g., human LAG-3) 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% or higher affinity than for another, unbound antigen, as measured, for example, by radioimmunoassay, surface plasmon resonance or kinetic exclusion assay. In a specific embodiment, the extent of binding of an anti-LAG-3 antibody described herein to an unrelated non-LAG-3 protein is less than 10, 15, or 20% of the binding of the antibody to a LAG-3 protein, as measured, for example, by radioimmunoassay .

As used herein, the term afucosylation or afucosylation in the context of Fc refers to a significant lack of fucose covalently attached, directly or indirectly, to residue 297 of the Fc region of human IgG1 numbered according to the EU numbering system, or the corresponding residue in immunoglobulins other than human IgG1 or IgG1 . Thus, in a composition containing a plurality of afucosylated antibodies, at least 70% of the antibodies will not be fucosylated, directly or indirectly (eg, via sugar intermediates) at residue 297 of the antibody Fc region, and in some embodiments at least 80, 85 , 90, 95 or 99% will not be fucosylated, directly or indirectly, at residue 297 of the Fc region.

As used herein, the term epitope is a term in the art and refers to a localized region of an antigen to which an antibody can specifically bind. An epitope may be, for example, contiguous amino acids of a polypeptide (linear or continuous epitope) or an epitope, for example, may be combined from two or more non-contiguous regions of a polypeptide or polypeptides (conformational, non-linear, discontinuous or non-contiguous epitope). In some embodiments, the epitope to which the antibody binds can be determined, for example, by NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen/deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography mass spectrometry , electrospray mass spectrometry), oligopeptide scanning-based array analysis (e.g., peptide constraint using CLIPS (chemical linkage of peptides to scaffolds) to map discontinuous or conformational epitopes, and/or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography crystallization can be performed using any of the methods known in the art (eg, Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303, all of which are incorporated herein by reference in their entirety). Antibody:antigen crystals can be studied using well-known x-ray diffraction techniques and can be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, for example, Meth Enzymol (1985) vol. 114 & 115, eds Wyckoff HW et al.,; U.S. 2004/0014194) and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276a: 361-423, ed Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10): 1316-1323, all of which are incorporated herein by reference in their entirety. Mutagenesis mapping studies can be performed using any method known to one skilled in the art. See, for example, Champe M et al., (1995) J Biol Chem 270: 13881394 and Cunningham BC & Wells JA (1989) Science 244: 1081-1085, each of which is incorporated herein by reference in its entirety for descriptions of mutagenesis techniques, including alanine scanning mutagenesis techniques. CLIPS (Chemical Linking of Peptides to Scaffolds) is a technology for presenting one or more peptides in a structurally constrained configuration that behave as functional mimics of complex protein domains. See, for example, US Patent Publications No. US 2008/0139407 A1 and US 2007/099240 A1, and US Patent No. 7,972,993, each of which is incorporated herein by reference in its entirety. In a specific embodiment, the antibody epitope is determined using alanine scanning mutagenesis studies.

In a specific embodiment, an antibody epitope is determined using hydrogen/deuterium exchange in combination with mass spectrometry. In a specific embodiment, the antibody epitope is determined using CLIPS epitope mapping technology from Pepscan Therapeutics.

- 19 044026

As used herein, the term epitope located in a human LAG-3 region consisting of a particular amino acid sequence or set of amino acid residues refers to an epitope containing one or more amino acid residues of the specified region, wherein the specified region contains the first specified amino acid residue and the last specified amino acid residue of the human LAG-3 region. In certain embodiments, the epitope comprises each of the amino acid residues located in the specified region. In certain embodiments, one or more additional amino acid residues of human LAG-3 outside the specified region bind to the antibody together with an epitope located in the specified region.

As used herein, the terms T cell receptor and TCR are used interchangeably and refer to full-length αβ or γδ heterodimeric TCRs, antigen-binding fragments of full-length TCRs, and molecules containing TCR CDRs or variable regions. Examples of TCRs include, but are not limited to, full-length TCRs, antigen-binding fragments of full-length TCRs, soluble TCRs lacking transmembrane and cytoplasmic regions, single-chain TCRs containing TCR variable regions attached by a flexible linker, TCR chains linked by an engineered disulfide bond, monospecific TCRs, multispecific TCRs (including bispecific TCRs), fusion TCRs, human TCRs, humanized TCRs, chimeric TCRs, recombinantly derived TCRs and synthetic TCRs. The term covers a wild-type TCR and a genetically engineered TCR (eg, a chimeric TCR comprising a chimeric TCR chain that contains a first portion from a TCR of the first species and a second portion from a TCR of the second species).

As used herein, the terms major histocompatibility complex and MHC are used interchangeably and refer to the MHC class I molecule and/or the MHC class II molecule.

As used herein, the term MHC peptide complex refers to a MHC molecule (MHC class I or MHC class II) with a peptide bound in the MHC peptide-binding pocket known in the art.

As used herein, the term treat, treatment, and treatment process refers to the therapeutic or prophylactic measures described herein. Methods of treatment include administering an antibody to a subject having a disease or disorder, or predisposed to such a disease or disorder, to prevent, treat, delay, reduce the severity or ameliorate one or more symptoms of the disease or disorder or recurrent disease or disorder, or to prolong survival of a subject beyond what would be expected in the absence of such treatment.

As used herein, the term effective amount, in the context of administering a drug to a subject, refers to the amount of drug that achieves the desired prophylactic or therapeutic effect.

As used herein, the term subject includes any human or non-human animal. In one embodiment, the subject is a human or non-human mammal. In one embodiment, the subject is a human.

Determining the percentage identity between two sequences (eg, amino acid sequences or nucleic acid sequences) can be performed using a mathematical algorithm. A specific, non-limiting example of a mathematical algorithm used to compare two sequences is the algorithm of Karlin S & Altschul SF (1990) PNAS 87: 2264-2268, modified as in Karlin S & Altschul SF (1993) PNAS 90: 5873-5877, each of which incorporated herein by reference in its entirety. Such an algorithm is included in the programs NBLAST and XBLAST Altschul SF et al., (1990) J Mol Biol 215: 403, which is incorporated herein by reference in its entirety. A BLAST nucleotide search can be performed with a set of NBLAST nucleotide program parameters, for example, score=100, word length=12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. A BLAST protein search can be performed with XBLAST program parameters set to, for example, score 50, word length=3, to obtain amino acid sequences homologous to a protein molecule described herein. To obtain a gap alignment for comparison, Gapped BLAST can be used as described in Altschul SF et al., (1997) Nuc Acids Res 25: 3389-3402, which is incorporated herein by reference in its entirety. Alternatively, PSI BLAST can be used to perform an iterative search that discovers distant relationships between molecules (Id). When using the BLAST, Gapped BLAST, and PSI Blast programs, you can use the default settings of the respective programs (eg, XBLAST and NBLAST) (see, for example, the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov). Another specific, non-limiting example of a mathematical algorithm used for sequence comparison is that of Myers and Miller, 1988, CABIOS 4: 11-17, which is incorporated herein by reference in its entirety. Such an algorithm is included in the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, you can use the PAM120 residue weight table, a gap extension penalty of 12, and a gap penalty of 4.

The percentage identity between two sequences can be determined using techniques similar to those described above, with or without consideration of gaps. When calculating percent identity, only exact matches are usually taken into account.

6.2. Antibodies against LAG-3.

In one aspect, the present invention provides antibodies that specifically bind to LAG-3 (eg, human LAG-3) and antagonize the function of LAG-3. The amino acid sequences of typical antibodies are given in Table 1 herein. 1-7.

One skilled in the art will appreciate that the N-terminal amino acid residue E or Q can, under certain conditions, spontaneously convert to pyroglutamate by post-translational cyclization of the free amino group to form a lactam. Accordingly, in certain embodiments, the invention provides antibodies comprising an antibody heavy chain variable region or light chain variable region disclosed herein (e.g., SEQ ID NOs: 5672 and 73-77, respectively) or a full-length heavy chain or light chain disclosed herein (e.g., SEQ ID NOs: 168-186 and 187-191, respectively), wherein the N-terminal amino acid residue of E or Q is converted to pyroglutamate (e.g., by post-translational cyclization of the free amino group of the N-terminal residue of E or Q Q).

Table 1

Amino acid sequences of typical anti-LAG-3 antibodies

SEQ ID NO: Description Amino acid sequence 1 VH Р01С09 QVQLKQSGAELVKPGASVKLSCTASGFNIKDTYMYWVK QRPEQGLEWIGRIDPANGNTKYDPKLQGKATITADTSS NTVYLQLSSLTSEDTAVFYCVIYSYRYDVGGFDYWGQG TTLTVS VL Р01С09, VL EIVLTQSPALMAASPGEKVTITCSSVSSSISSSNLHWYQ 2 P13F01, VL P13G05, VL P05E03 QKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSLTI SSMEAEDAATYYCQQWSSYPFTFGSGTKLEIK 3 VH P05E01 DVQLVE S GAE LVKPGASVKL S C TAS GFTIKDTYIHWVK

- 21 044026

4 VL P05E01 QRPEQGLEWIGEIDPANGNTKYDPKFQGKATITADTSS NTAYLQLSSLTSEDTAVYYCATYYYRYDVGGFDYWGQG TTLTVS DIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWFQ QKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSLTI SNMEAEDAATYYCQQWNSYPLTFGAGTKLELK EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMYWVK 5 VH P01A12 QRPEQGLDWIGRIDPANGNTKFDPKFQGKATITADTSS NTAYLQLSSLTSEDTAVYYCSTYYYRYDVGGFDYWGQG TTLTVS DIVLTQSPALMAASPGEKVTITCSSVSSSISSSNLHWYQ 6 VL P01A12 QKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSLTI SSMEAEDAATYYCQQWSSYPFTFGSGTKLEIK EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYIYWVK 7 VH P13B01 QRPERGLEWIGRIDPANGNTKFDPKFQGTATITADTSS NTAYLQLSSLTSEDTAVYYCTTYFYRYDVGGFDYWGQG TTLTVS 8 VL P13B01, P14C04, VL VL P14B07 ENVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQ QKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSLTI SSMEAEDAATYYCQQWSSYPFTFGSGTKLEIK 9 VH P13C10, P15C02, VH VH P13G05, P13F06, VH VH P16H01 VH P16D04, VH P14F01, QVQ L Q Q S GAE LVKPGASVELSCTASGFNIRDT YMYWVK QRPEQGLEWIGRIDPANGNTKFDPKFQDRATMTADTSS NTAYLQLSSLTSEDTAVYYCTTYFYRYDVGGFDYWGQG TTLTVS 10 VL P13C10, P13E02, VL VL P13B03, P13H05, VL VL P13F02, VL P13G04 ENVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWFQ QKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSLTI SSMEAEDAATYYCQQWSSYPFTFGSGTKLEIK QVQLKQS GAE LVKPGASVKL S C TAS G FNIKDNYIHWVK eleven VH P13C08 QRPEQGLEWIGSIDPANGNTKYDPKFQGKASITADTSS NTAYLQLSSLTSEDTAVYYCASYFYRYDVGGFDYWGQG TTLTVS 12 VL P13C08 DVVMTQTPALMAASPGEKVTITCSSVSSSISSSNLHWFQ QKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSLTI

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SSMEAEDAATYYCQQWSSYPFTFGSGTKLEIK 13 VH P13E02 QVQLQQPGAELVKPGASVKLSCTVSGFNIKDTYIHWVK QRPEQGLEWIGEIDPANGNSKYAPRFQGKATITADT S S NTAYLQLSSLTSEDTAVYYCATYYYRYDVGGFDYWGQG TTLTVS 14 VH P13F02 QVQLQQPGAELVKPGASVKLSCTASGFNIKDTYIHWVK QRPEQGLDWVGEIDPANGHTKYDPKFQGKATITADT S S NTAYLQLSSLTSEDTAVYYCATYYYRYDVGGFDYWGQG TTLTVS 15 VH P13B02 QVQMKQ S GAE LVHP GASVKL S C TAS GFNIKDTYIHWVK QRPEQGLEWIGEIDPANDNTKYDPKFQGKATITADTSS NTAYLQLSSLTSEDTAVYYCATYYYRYDVGGFDYWGQG TTLTVS 16 VL P13B02 EILLTQSPALMAASPGEKVTITCSVSSSISSSNLHWFQ QKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSLTI SSMEAEDAATYYCQQWSSYPFTFGSGTKLEIK 17 VH PlZAO6 QVQLKQSGAELVKPGASVKLSCTASGFNIKDTYMYWVK QRPEQGLEWIGRIDPANGNTKYDPKFQGKATITADTSS NTVYLQLSSLTSEDTAVFYCVIYSYRYDVGGFDYWGQG TTLTVS 18 VL PlZAO6 ENVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQ QKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSLTI SSMEAEDAATYYCQQWSSYPHTFGGGTKLEIK 19 VH P14C04 QVQLQQPGAELVKPGASVKLSCTASGFNIKDTYMYWVK QRPEQGLDWIGRIDPANGNTHFDPKFQGKATITADTSS NTAYLQLSSLTSEDTAVYYCSTYFYRYDVGGFDYWGQG TTLTVS 20 VH P14A04, VH P13F01, VH P15E06 QVQ L Q Q P GAE LVKPGASVELSCTASGFNIRDT YMYWVK QRPEQGLEWIGRIDPANGNTKFDPKFQDRATMTADTSS NTAYLQLSSLTSEDTAVYYCTTYFYRYDVGGFDYWGQG TTLTVS 21 VL P14A04, VL P14G01 DIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWFQ QKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSLTI SSMEAEDAATYYCQQWSSYPFTFGSGTKLEIK 22 VH P15F06 QVQLKQSGAELVKPGASVELSCTASGFNIRDTYMYWVK

-23044026

QRPEQGLEWIGRIDPANGNTKFDPKFQDRATMTADTSS NTAYLQLSSLTSEDTAVYYCTTYFYRYDVGGFDYWGQG TTLTVS 23 VL P15F06 ENVLTQSPALMAASPGEKVTITCSVSSSISSSTLHWFQ QKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSLTI SSMEAEDAATYYCQQWSSYPWTFGGGTKLEIK 24 VH P13B03 EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYIHWVK QRPEQGLEWIGEIDPANGNTKYDPKFQGKATITADTSS NTAYLQLSSLTSEDTAVYYCATYYYRYDVGGFDYWGQG TTLTVS 25 VL P15C02 DWMTQT PALMAAS PGEKVTITCS VS S SIS S SNLHWYQ QKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSLTI SSMEAEDAATYYCQQWSSYPFTFGSGTKLEIK 26 VL P16D04, VL P16H01 ENVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQ QKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSLTI SSMEAEDAATYYCQQWSSYPLTFGAGTKLELK 27 VH P13A04 QVQ L Q Q P GAE LVKPGASVELSCTASGFNIRDT YMYWVK QRPEQGLEWIGRIDPANGNTKFDPKFQDRATMTADTSS NTAYLQLSSLTSADTAVYYCTTYFYRYDVGGFDYWGQG TTLTVS 28 VL P13A04 DIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQ QKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSLTI SNMEAEDAATYYCQQWSSYPFTFGSGTKLEIK 29 VH P16H05 QVQLQQPGAELVKPGASVKLSCTASGFNIKDNYIHWVK QRPEQGLEWIGSIDPANGNTKYDPKFQGKASITADTSS NTAYLQLSSLTSEDTAVYYCASYFYRYDVGGFDYWGQG TTLTVS thirty VL P16H05 EIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQ QKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSLTI SSMEAEDAATYYCQQWSSYPHTFGGGTKLEIK 31 VH P13F09 QVQLQQS GAELVKPGASVKLS CTAS GENT KDNYIHWVK QRPEQGLEWIGSIDPANGNTKYDPKFQGKASITADTSS NTAYLQLSSLTSEGTAVYYCASYFYRYDVGGFDYWGQG TTLTVS 32 VL P13F09, VL EIVLTQSPALMAASPGEKVTITCSSVSSSISSSNLHWFQ

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P14F06 QKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSLTI SSMEAEDAATYYCQQWSSYPFTFGSGTKLEIK 33 VH P13G01, VH P15G05 EVQLQQSGAELVKPGASVELSCTASGFNIRDTYMYWVK QRPEQGLEWIGRIDPANGNTKFDPKFQDRATMTADTSS NTAYLQLSSLTSEDTAVYYCTTYFYRYDVGGFDYWGQG TTLTVS 34 VL P13G01 QIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQ QKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSLTI SSMEAEDAATYYCQQWSSYPFTFGSGTKLEIK 35 VH P13H05 QVQLQQPGAELVKPGASVKLSCTASGFNIKDTYIHWVK QRPEQGLEWIGEIDPANDNTKYDPKFQGKATITADTSS NTAYLQLSSLTSEDTAVYYCATYYYRYDVGGFDYWGQG TTLTVS 36 VH P13D04 QVQLQQSGAELVKPGASVKLSCTASGFNIKDNYMDWVK QRPEQGLEWIGKIDPANGNTKYDPKFQGKATITADTSS NTAYLQLSSLTSEDTAVYYCATYYYRYDVGGFDYWGQG TTLTVS 37 VL P13D04 DIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWFQ QKSGTPPKLWIYGTSNLASGVPVRFSGSGSGTSYSLTI SSMEAEDAATYYCQQWSSYPFTFGSGTKLEIK 38 VH P14G01, VH P05E03 QVQLKE S GAE LVKP GASVE L S C TAS G FNIRDTYMYWVK QRPEQGLEWIGRIDPANGNTKFDPKFQDRATMTADTSS NTAYLQLSSLTSEDTAVYYCTTYFYRYDVGGFDYWGQG TTLTVS 39 VH P14G03 QVQMKQ S GAE LVKP GASVKL S C TAS GFNIKDTYIHWVK QRPGQGLEWIGEIDPANGNTKYDPKFQGKATITADTSS NTAYLQLSSLTSEDTAVYYCATYYYRYDVGGFDYWGQG TTLTVS 40 VL P14G03 DIVLTQSPALMAASPGEKVTITCSVSSSISSSNLYWFQ QKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSLTI SSMEAEDAATYYCQQWSSYPFTFGSGTKLEIK 41 VL P13F06 EILLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQ QKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSLTI SSMEAEDAATYYCQQWSSYPFTFGSGTKLEIK 42 VH P13B11 QVQLQQPGAELVKPGASVKLSCTASGFNIKDNYMDWVK

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QRPEQGLEWIGKIDPANGNTKYDPKFQGKATITADTSS NTAYLQLSSLTSEDTAVYYCATYYYRYDVGGFDYWGQG TTLTVS 43 VL P13B11 QIVLTQSPALMAASPGEKVTITCSSVSSSISSSNLHWFQ QKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSLTI SSMEAEDAATYYCQQWSSYPFTFGSGTKLEIK 44 VL P14F01 ENVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQ QKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSLTI SSMEAEDAATYYCQQWSTYPFTFGSGTKLEIK 45 VH P14F06 QVQMKQ S GAE LVHP GASVKL S C TAS GENT KDNYIHWVK QRPEQGLEWIGSIDPANGNTKYDPKFQGKASITADTSS NTAYLQLSSLTSEDTAVYYCASYFYRYDVGGFDYWGQG TTLTVS 46 VH P13D05 QVQ L Q Q P GAE LVKPGASVELSCTASGFNIRDT YMYWVK QRPEQGLGWIGRIDPANGNTKFDPKFQDRATMTADTSS NTAYLQLSSLTSEDTAVYYCTTYFYRYDVGGFDYWGQG TTLTVS 47 VL P13D05 EIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWFR QKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSLTI SSMEAEDAATYYCQQWSSYPFTFGSGTKLEIK 48 VH P13G04 EVKLME S GAE LVKP GASAE L S C TAS G FNIRDTYMYWVK QRPEQGLEWIGRIDPANGNTKFDPKFQDRATMTADTSS NTAYLQLSSLTSEDTAVYYCTTYFYRYDVGGFDYWGQG TTLTVS 49 VL P15E06 ENVLTQSPALMAASPGEKVTITCSVSSGISSSNLHWYQ QKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSLTI SSMEAEDAATYYCQQWSRYPWTFGGGTKLEIK 50 VL P15G05 EIVLTQSPALMAASPGEKVTITCSVSSSISSSNLHWYQ QKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSLTI SSMEAEDAATYYCQQWSSYPLTFGAGTKLELK 51 VH P15B06 EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMYWVK QRPEQGLDWIGRIDPANGNTHFDPKFQGKATITADTSS NTAYLQLSSLTSEDTAVYYCSTYFYRYDVGGFDYWGQG TTLTVS 52 VL P15B06 QILLTQSPALMAASPGEKVTITCSSVSSSISSSNLHWYQ

-26044026

53 VH P14B07 QKSGTSPKLWIYGTSNLASGVPVRFSGSSGTSYSLTI SSMEAEDAATYYCQQWSSYPLTFGAGTKLELK QVQLQQPGAELVKPGASVKLSCTASGFNIKDNYIHWVK QRPEQGLEWIGSIDPANGNTKYDPKFQGKASITADTSS NTAYLQLSSLTSEDTTVYYCASYFYRYDVGGFDYWGQG TTLTVS QVQMKQ S GAE LVKPGASVELSCTASGFNITDT YMYWVK 54 VH P13C06 QRPEQGLEWIGRIDPANGNTKFDPKFQDRATMTADTSS NTAYLQLSSLTSEDTAVYYCTTYFDKYDVGGCDYWGQG TTLTVS EIVLTQSPALMAASPGEKVTITCSSVSSSISSSNLYWFQ 55 VL P13C06 HKSGTSPKLWIYGTSNLASGVPVRFSGSGSGTSYSLTI SSMDAENAATYYCQQWRSYPFTFGSGTKLEIK QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVR 56 HO QAPGQGLEWMGEIDPANDNTKYDPKFQGRVTMTRDTST STVYMELSSLRSEDTAVYYCARYYYRYDVGGFDYWGQG TLVTVSS QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVR 57 Hl QAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT S T STVYMELSSLRSEDTAVYYCATYYYRYDVGGFDYWGQG TLVTVSS QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVR 58 H2 QAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT S S NTVYMELSSLRSEDTAVYYCATYYYRYDVGGFDYWGQG TLVTVSS QVQMKQ S GAEVKKP GASVKVS C TAS GFNIKDTYIHWVR 59 H3 QAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT S S NTVYMELSSLRSEDTAVYYCATYYYRYDVGGFDYWGQG TLVTVSS QVQMKQ S GAEVVKP GASVKVSC TAS GFNIKDTYIHWVR 60 H4 QAPGQGLEWIGEIDPANDNTKYDPKFQGRVTITADTSS NTVYLQLSSLRSEDTAVYYCATYYYRYDVGGFDYWGQG TLVTVSS 61 H4_ _R98K QVQMKQ S GAEVVKP GASVKVS C TAS GFNIKDTYIHWVR QAPGQGLEWIGEIDPANDNTKYDPKFQGRVTITADTSS

- 27 044026

NTVYLQLSSLRSEDTAVYYCATYYYKYDVGGFDYWGQG TLVTVSS QVQMKQ S GAEWKPGASVKVSC TAS GFNIKDTYIHWVR 62 H4 _D100E QAPGQGLEWIGEIDPANDNTKYDPKFQGRVTITADTSS NTVYLQLSSLRSEDTAVYYCATYYYRYEVGGFDYWGQG TLVTVSS QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVR 63 Hl _R98K QAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT S T STVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQG TLVTVSS QVQ LVQ S GAEVKK P GAS VKVS CTASGFNIKDTYIHWVR 64 Hl _R98K_ K23I QAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT S T STVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQG TLVTVSS QVQMVQ S GAEVKK P GAS VKVS C KAS G ΕΝIKDTYIHWVR 65 Hl _R98K_ L4M QAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT S T STVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQG TLVTVSS XVQMVQ S GAEVKK P GAS VKVS C KAS G ΕΝIKDTYIHWVR QAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT S T 220 Hl_ _R98K_ _L4M STVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQG TLVTVSS, where X=glutamine (Q) or pyroglutamate (PE) QVQMVQ S GAEVKKP GASVKVS C TAS GFNIKDTYIHWVR 66 Hl _R98K_ L4M_ K23T QAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT S T STVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQG TLVTVSS QVQMKQ S GAEVKKP GASVKVS CKAS GFNIKDTYIHWVR 67 Hl _R98K_ L4M_ V5K QAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT S T STVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQG TLVTVSS 68 Hl 3T _R98K_ L4M_ V5K_K2 QVQMKQ S GAEVKKP GASVKVS C TAS GFNIKDTYIHWVR QAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT S T STVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQG

- 28 044026

TLVTVSS 69 H1_R98K_V5K QVQLKQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVR QAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT S T STVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQG TLVTVSS 70 H1_R98K_V5K_K23T QVQLKQSGAEVKKPGASVKVSCTASGFNIKDTYIHWVR QAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT S T STVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQG TLVTVSS 71 H2_R98K QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVR QAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT S S NTVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQG TLVTVSS 72 H3_R98K QVQMKQ S GAEVKKP GASVKVS C TAS GFNIKDTYIHWVR QAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT S S NTVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQG TLVTVSS 73 L0 EIVLTQSPGTLSLSPGERATLSCSVSSSISSSNLHWYQ QKPGQAPRLLIYGTSNLASGIPDRFSGSGSGTDFTLTI SRLEPEDFAVYYCQQWSSYPFTFGQGTKVEIK 221 L.O. XIVLTQSPGTLSLSPGERATLSCSVSSSISSSNLHWYQ QKPGQAPRLLIYGTSNLASGIPDRFSGSGSGTDFTLTI SRLEPEDFAVYYCQQWSSYPFTFGQGTKVEIK, where X=glutamate (E) or pyroglutamate (pE) 74 LI EIVLTQSPGTLSLSPGERATLSCSVSSSISSSNLHWYQ QKPGQAPRLLIYGTSNLASGIPDRFSGSGSGTSYTLTI SRLEPEDFAVYYCQQWSSYPFTFGQGTKVEIK 75 L2 EILLTQSPGTLSLSPGERATLSCSVSSSISSSNLHWFQ QKPGQAPRLLIYGTSNLASGIPDRFSGSGSGTSYTLTI SRLEPEDFAVYYCQQWSSYPFTFGQGTKVEIK 76 L3 EILLTQSPGTLSLSPGERATLSCSVSSSISSSNLHWFQ QKPGQAPRLWIYGTSNLASGVPDRFSGSGSGTSYTLTI SRLEPEDFAVYYCQQWSSYPFTFGQGTKVEIK 77 L4 EILLTQSPGTLSLSPGERATLTCSVSSSISSSNLHWFQ QKPGQSPRLWIYGTSNLASGVPDRFSGSGSGTSYTLTI

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SRLEPEDFAVYYCQQWSSYPFTFGQGTKVEIK 78 CDRH1 DTYMY 79 CDRH1 DTYIH 80 CDRH1 DTYIY 81 CDRH1 DNYIH 82 CDRH1 DNYMD 83 CDRH2 RIDPANGNTKYDPKLQG 84 CDRH2 EIDPANGNTKYDPKFQG 85 CDRH2 RIDPANGNTKFDPKFQG 86 CDRH2 RIDPANGNTKFDPKFQD 87 CDRH2 SIDPANGNTKYDPKFQG 88 CDRH2 EIDPANGNSKYAPRFQG 89 CDRH2 EIDPANGHTKYDPKFQG 90 CDRH2 EIDPANDNTKYDPKFQG 91 CDRH2 RIDPANGNTKYDPKFQG 92 CDRH2 RIDPANGNTHFDPKFQG 93 CDRH2 KIDPANGNTKYDPKFQG 94 CDRH3 YSYRYDVGGFDY 95 CDRH3 YYYRYDVGGFDY 96 CDRH3 YFYRYDVGGFDY 97 CDRH3 YFDKYDVGGCDY 98 CDRH3 YYYKYDVGGFDY 99 CDRH3 YYYRYEVGGFDY 100 CDRL1 SVSSSISSSNLH 101 CDRL1 SVSSSISSSTLH 102 CDRL1 SVSSSSISSNLY 103 CDRL1 SVSSGISSSNLH 104 CDRL2 GTSNLAS 105 CDRL3 QQWSSYPFT 106 CDRL3 QQWNSYPLT 107 CDRL3 QQWSSYPHT 108 CDRL3 QQWSSYPWT 109 CDRL3 QQWSSYPLT 110 CDRL3 QQWSTYPFT

-30044026

111 CDRL3 QQWSRYPWT 112 CDRL3 QQWRSYPFT 113 FR1 VH QVQLVQS GAEVKKPGASVKVS CKAS G FNIК 114 FR1 VH QVQMKQSGAEVKKPGASVKVSCTASGFNIK 115 FR1 VH QVQMKQS GAE WKPGAS VKVS C TAS G FN IК 116 FR1 VH QVQLVQSGAEVKKPGASVKVSCTASGFNIK 117 FR1 VH QVQMVQS GAEVKKPGASVKVS CKAS G FNIК 118 FR1 VH QVQMVQS GAEVKKPGASVKVS C TAS G FNIК 119 FR1 VH QVQMKQS GAEVKKPGASVKVS CKAS G FNIК 120 FR1 VH QVQLKQS GAEVKKPGASVKVS CKAS G FNIК 121 FR1 VH QVQLKQSGAEVKKPGASVKVSCTASGFNIK 122 FR2 VH WVRQAPGQGLEWMG 123 FR2 VH WVRQAPGQGLEWIG 124 FR3 VH RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR 125 FR3 VH RVTITADTSTSTVYMELSSLRSEDTAVYYCAT 126 FR3 VH RVTITADTSSNTVYMELSSLRSEDTAVYYCAT 127 FR3 VH RVTITADTSSNTVYLQLSSLRSEDTAVYYCAT 128 FR4 VH WGQGTLVTVSS 129 FR1 VL EIVLTQSPGTLSLSPGERATLSC 130 FR1 VL EILLTQSPGTLSLSPGERATLSC 131 FR1 VL EILLTQSPGTLSLSPGERATLTC 132 FR2 VL WYQQKPGQAPRLLIY 133 FR2 VL WFQQKPGQAPRLLIY 134 FR2 VL WFQQKPGQAPRLWIY 135 FR2 VL WFQQKPGQSPRLWIY 136 FR3 VL GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC 137 FR3 VL GIPDRFSGSGSGTSYTLTISRLEPEDFAVYYC 138 FR3 VL GVPDRFSGSGSGTSYTLTISRLEPEDFAVYYC 139 FR4 VL FGQGTKVEIK 140 consensus CDRH1 DXiYXsXs, where: Xi represents T or N; X ₂ represents I or M; and X ₃ represents Η, Υ or D 141 consensus CDRH1 ОХ^ХгХз, where:

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Xi represents T or N; X ₂ represents I or M; and X ₃ represents H or Y 142 CDRH2 consensus XilDPTW^XsXiXsXeXvPXsXgQXio, where: Xi represents E, R, S or K; X ₂ represents D or G; X ₃ represents N or H; X ₄ represents T or S; X ₅ represents K or H; X ₆ represents Y or F; X ₇ represents D or A; X ₈ represents K or R; X ₉ represents F or L, and Xu represents G or D 143 CDRH2 consensus XiIDPANX ₂ X ₃ X ₄ KX ₅ X ₆ PX ₇ FQX ₈ where: Xi is E, R or S; X ₂ represents D or G; X ₃ represents N or H; X ₄ represents T or S; X ₅ represents Y or E; X ₆ represents D or A; X ₇ represents K or R and X ₈ represents G or D 144 consensus CDRH3 YX ₁ X ₂ X ₃ YX ₄ VGGX ₅ DY where: Xi is Y, F or S; X ₂ represents Y or D; X ₃ represents K or R; X ₄ represents D or E and X ₅ represents F or C 145 consensus CDRH3 YX ₁ X ₂ X ₃ YDVGGX ₄ DY where: Xi is Y, F or S; X ₂ represents Y or D; X ₃ is K or R and X ₄ is E or C 146 consensus CDRH3 YYYXiYXWGGFDY, where:

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Xi represents K or R, and X ₂ represents D or E, and 147 consensus CDRL1 SVSSX!lSSSX ₂ LX3 where: Xi represents S or G; X ₂ is N or T and X ₃ is H or Y 148 consensus CDRL1 SVSSSISSSNLXi, where: Xi represents H or Y 149 CDRL3 consensus QQWXiX ₂ YPX ₃ T where: Xi represents S, N or R; X ₂ is S, T or R and X ₃ is F, L, H or W 150 CDRL3 consensus QQWXiSYPX ₂ T where: Xi is S, N or R and X ₂ is F, L or H 151 G u m a n o v a n e d Consensus VH QVQXiX ₂ QSGAEVX ₃ KPGASVKVSCKASGFNIKDTYIHW vrqapgqglewx ₅ geidpandntkydpkfqgrvtx ₆ tx ₇ DTSX ₈ X ₉ TVYXioXiiLSSLRSEDTAVYYCAXi ₂ YYYXi ₃ Y X14VGGFDYWGQGTLVTVSS where: Xi represents M or L; X ₂ represents V or K; Xs represents K or V; X ₄ represents K or T; X5 represents M or I; X ₆ represents I or M; X ₇ represents A or R; X ₈ represents T or S, X ₉ represents S or N; Xy represents M or L; Chi represents E or Q; X ₁₂ represents T or R; X13 represents K or R, and Xi represents D or E 218 Humanized consensus VH QVQX4X2QSGAEVX3KPGASVKVSCX4ASGFNIKDTYIH WVRQAPGQGLEWX ₅ GEIDPANDNTKYDPKFQGRVTX ₆ T

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XvDTSXgXgTVYXioXiiLSSLRSEDTAVYYCAX^YYYXi ₃ YXi ₄ VGGFDYWGQGTLVTVSS where: Xi represents M or L; X ₂ represents V or K; X ₃ represents K or V; X ₄ represents K or T; X ₅ represents M or I; X ₆ represents I or M; X ₇ represents A or R; X ₈ represents T or S, X ₉ represents S or N; Xy represents M or L; Chi represents E or Q; X12 represents T or R; X13 represents K or R, and X ₄₄ represents D or E 222 Humanized consensus VH XiVQX ₂ X ₃ QSGAEVX ₄ KPGASVKVSCKASGFNIKDTYIH wvrqapgqglewx ₅ geidpandntkydpkfqgrvtx ₆ t XvDTSXgXgTVYXioXnLSSLRSEDTAVYYCAXi^YYXi ₃ YX1 ₄ VGGFDYWGQGTLVTVSS, where: Xi represents Q or p E (pyroglutamate) X ₂ is M or L; X ₃ represents V or K; X ₄ represents K or V; X ₅ represents M or I; X ₆ represents I or M; X ₇ represents A or R; X ₈ represents T or S, X ₉ represents S or N; Xy represents M or L; Chi represents E or Q; X ₄₂ represents T or R; X13 represents K or R, and X ₄₄ represents D or E

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223 G u m a n o v a n e d Consensus VH X ₁ VQX ₂ X ₃ QSGAEVX ₄ KPGASVKVSCX ₅ ASGFNIKDTYI hwvrqapgqglewx ₆ geidpandntkydpkfqgrvtx ₇ TX ₈ DTSX ₉ X1OTVYX11X1 ₂ LSSLRSEDTAVYYCAX13YYY X1 ₄ YX1 ₅ VGGFDYWGQGTLVTV SS where: Xi represents Q or pE (pyroglutamate) X ₂ represents M or L; Xs represents V or K; X ₄ represents K or V; X ₅ represents K or T; X ₆ represents M or I; X ₇ represents I or M; X ₈ represents A or R; X ₉ represents T or S; Xy represents S or N; Chi represents M or L; X ₁₂ represents E or Q; Xis is T or R; X ₄₄ represents K or R, and Xu represents D or E 152 Humani zir o v aniy consensus VL EIXiLTQSPGTLSLSPGERATLX ₂ CSVSSSISSSNLHW x ₃ qqkpgqx ₄ prlx ₅ iygtsnlasgx ₆ pdrfsgsgsgtx ₇ X ₈ TLTISRLEPEDFAVYYCQQWSSYPFTFGQGTKVEI K, where: Xi represents V or L; X ₂ represents S or T; X ₃ represents Y or F; X ₄ represents A or S; X ₅ represents L or W; X ₆ represents I or V; X ₇ represents D or S; and X ₈ represents E or Y 224 Humani zir o v aniy consensus VL X ₁ IX ₂ LTQSPGTLSLSPGERATLX ₃ CSVSSSISSSNLHW x ₄ qqkpgqx ₅ prlx ₆ iygtsnlasgx ₇ pdrfsgsgsgtx ₈ X ₉ TLTISRLEPEDFAVYYCQQWSSYPFTFGQGTKVEI

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K, where:

Xi represents E or pE (pyroglutamate)

X ₂ represents V or L;

Xs represents S or T;

X ₄ represents Y or F;

X ₅ represents A or S;

X ₆ represents L or W;

X ₇ represents I or V;

X ₈ represents D or S; A

X ₉ represents F or Y

168 heavy chain full-length IgGi H1_R98K_L4M QVQMVQ S GAEVKK P GASVKVS CKAS G ΕΝIKDTYIHWVR QAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT S T STVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSW TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RWSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 225 heavy chain full-length IgGi H1_R98K_L4M XVQMVQ S GAEVKK P GAS VKVS C KAS G FNIKDTYIHWVR QAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT S T STVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSW TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RWSVLTVLHQDWLNGKE YKCKVSNKALPAP IEKT I SK AKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG,

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where X=glutamine (Q) or pyroglutamate (pE) 169 heavy chain N297A full size IgGi H1_R98K_L4M QVQMVQS GAEVKKPGASVKVS CKAS GFNIKDTYIHWVR QAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT S T STVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSW TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTY RWSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 226 heavy chain N297A full size IgGi H1_R98K_L4M S T PSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTY RWSVLTVLHQDWLNGKE YKCKVSNKALPAP IEKT I SK AKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, where X=glutamine (Q) or pyroglutamate (pE) 170 heavy chain S228P full-length IgG ₄ H1_R98K_L4M QVQMVQS GAEVKKPGASVKVS CKAS GFNIKDTYIHWVR QAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT S T STVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSW TVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWD

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VSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRW SVLTVLHQDWLNGKEYKCKVSNKGLPS SIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSL SLSLG 227 heavy chain S228P full-length IgG ₄ H1_R98K_L4M S T PSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWD VSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRW SVLTVLHQDWLNGKEYKCKVSNKGLPS SIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLG, where X=glutamine (Q) or pyroglutamate (pE) 171 heavy chain N297A full-length IgGi HO QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVR QAPGQGLEWMGEIDPANDNTKYDPKFQGRVTMTRDTST STVYMELSSLRSEDTAVYYCARYYYRYDVGGFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSW TVPSS SLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTY RWSVLTVLHQDWLNGKE YKCKVSNKALPAP IEKT ISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 172 heavy chain N297A full size IgGi Hl QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVR QAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT S T STVYMELSSLRSEDTAVYYCATYYYRYDVGGFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD

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YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSW TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTY RWSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTI SK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 173 heavy chain N297A of full-length IgGi H2 QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVR QAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT S S NTVYMELSSLRSEDTAVYYCATYYYRYDVGGFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSW TVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTY RWSVLTVLHQDWLNGKE YKCKVSNKALPAP IEKT I SK AKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 174 heavy chain N297A full size IgGi NZ QVQMKQS GAE VKKPGAS VKVS CTASGFNIKDTYIHWVR QAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT S S NTVYMELSSLRSEDTAVYYCATYYYRYDVGGFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSW TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTY RWSVLTVLHQDWLNGKE YKCKVSNKALPAP IEKT I SK AKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 175 heavy chain N297A of full-length IgGi H4 QVQMKQS GAE WK P GAS VKVS CTASGFNIKDTYIHWVR QAPGQGLEWIGEIDPANDNTKYDPKFQGRVTITADTS S NTVYLQLSSLRSEDTAVYYCATYYYRYDVGGFDYWGQG

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TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSW TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTY RWSVL TVLHQDWLNGKE YKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 176 heavy chain N297A of full-length IgGi H4_R98K QVQMKQ S GAE WK P GAS VKVS CTASGFNIKDTYIHWVR QAPGQGLEWIGEIDPANDNTKYDPKFQGRVTITADTS S NTVYLQLSSLRSEDTAVYYCATYYYKYDVGGFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSW TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTY RWSVLTVLHQDWLNGKE YKCKVSNKALPAP IEKT I SK AKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 177 heavy chain N297A full length IgGi H4_D100E QVQMKQ S GAE WK P GAS VKVS CTASGFNIKDTYIHWVR QAPGQGLEWIGEIDPANDNTKYDPKFQGRVTITADTS S NTVYLQLSSLRSEDTAVYYCATYYYRYEVGGFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSW TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTY RWSVLTVLHQDWLNGKE YKCKVSNKALPAP IEKT I SK AKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 178 heavy chain N297A full size QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVR QAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADTS T

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IgGi H1_R98K STVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSW TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYASTY RWSVLTVLHQDWLNGKE YKCKVSNKALPAP IEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 179 heavy chain N297A full-length IgGi H1_R98K_K23T QVQLVQSGAEVKKPGASVKVSCTASGFNIKDTYIHWVR QAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT S T STVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSW TV PSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTY RWSVLTVLHQDWLNGKE YKCKVSNKALPAP IEKT I SK AKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 180 heavy chain N297A of full-length IgGi Н1_R 9 8K_L 4M_K2 3 T QVQMVQ S GAE VKK P GAS VKVS CTASGFNIKDTYIHWVR QAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT S T STVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSW TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTY RWSVLTVLHQDWLNGKE YKCKVSNKALPAP IEKT I SK AKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 181 heavy chain N297A QVQMKQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVR

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182 183 full-length IgGi H1_R98K_L4M_V5K heavy chain N297A of full-length IgGi H1_R98K_L4M_V5K_K2 3D heavy chain N297A of full-length IgGi H1_R98K_V5K TC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTY RWSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD K SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG QVQMKQ S GAEVKKP GASVKVS C TAS GFNIKDTYIHWVR QAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT S T STVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSW TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTY RWSVLTVLHQDWLNGKE YKCKVSNKALPAP IEKT I SK AKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG QVQLKQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVR QAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT S T STVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSW TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTY RWSVLTVLHQDWLNGKE YKCKVSNKALPAP IEKT I SK AKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

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heavy chain N2 97A full size QVQLKQSGAEVKKPGASVKVSCTASGFNIKDTYIHWVR QAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT S T STVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSW TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH 184 185 IgGiHl_R98K_V5K_K2 ST heavy chain N297A of full-length IgGiH2_R98K heavy chain N297A TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTY RWSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVR QAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT S S NTVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSW TV PSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTY RWSVLTVLHQDWLNGKE YKCKVSNKALPAP IEKT I SK AKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG QVQMKQ S GAEVKKP GASVKVS C TAS GFNIKDTYIHWVR QAPGQGLEWMGEIDPANDNTKYDPKFQGRVTITADT S S NTVYMELSSLRSEDTAVYYCATYYYKYDVGGFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSW 186 full-length IgGiH3_R98K TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTY RWSVLTVLHQDWLNGKE YKCKVSNKALPAP IEKT I SK AKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK

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SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 187 full size light chain L0 K VYACEVTHQGLSSPVTKSFNRGEC 228 full size light chain L0 K VYACEVTHQGLSSPVTKSFNRGEC, where X=glutamate (E) or pyroglutamate (pE) 188 full size light chain L1 K VYACEVTHQGLSSPVTKSFNRGEC 189 full size light chain L2 K VYACEVTHQGLSSPVTKSFNRGEC 190 full size light chain L3 EILLTQSPGTLSLSPGERATLSCSVSSSISSSNLHWFQ QKPGQAPRLWIYGTSNLASGVPDRFSGSGSGTSYTLTI SRLEPEDFAVYYCQQWSSYPFTFGQGTKVEIKRTVAAP SVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC 191 full size light chain L4 EILLTQSPGTLSLSPGERATLTCSVSSSISSSNLHWFQ QKPGQSPRLWIYGTSNLASGVPDRFSGSGSGTSYTLTI

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192 193 208 Human IgGi Glm3 allotype (no terminal lysine) Human Glm3 IgGi allotype Human IgGi Glml7 allotype (no terminal lysine) SRLEPEDFAVYYCQQWSSYPFTFGQGTKVEIKRTVAAP SVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLT VLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPG ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLT VLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLT VLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPG 209 Human Glml7 IgGi allotype ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHE

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194 195 196 N297A IgGi (without C-terminal lysine) N297A IgGi S228P IgG ₄ (without C-terminal lysine) DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPG K ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYASTYRWSVLT VLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPG ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYASTYRWSVLT VLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSL GTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF LGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLH QDW LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFS CSVMHEALHNHYTQKSLSLSLG 197 S228P IgG ₄ ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSL GTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF

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LGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLH QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFS CSVMHEALHNHYTQKSLSLSLGK 198 Kt3 allotype of human kappa light chain constant region IGKC*01 RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 219 Kt3 allotype of human kappa light chain constant region IGKC*01 GTVAAPSVFI FPPSDEQLKSGTASWCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

table 2

Amino acid sequences of the heavy chain CDR of typical anti-LAG-3 antibodies*

VH CDRH1 (SEQ. ID NO:) CDRH2 (SEQ ID NO:) CDRH3 (SEQ ID NO:) VH P01C09 DTYMY (7 8) RIDPANGNTKYDPKLQG (83) YSYRYDVGGFDY (94) VH P05E01 DTYIH (79) EIDPANGNTKYDPKFQG (84) YYYRYDVGGFDY (95) VH P01A12 DTYMY (7 8) RIDPANGNTKFDPKFQG (85) YYYRYDVGGFDY (95) VH P13B01 DTYIY (80) RIDPANGNTKFDPKFQG (85) YFYRYDVGGFDY (96) VH P13C10 DTYMY (7 8) RIDPANGNTKFDPKFQD (86) YFYRYDVGGFDY (96) VH P13C08 DNYIH (81) SIDPANGNTKYDPKFQG (87) YFYRYDVGGFDY (96) VH P13E02 DTYIH (79) EIDPANGNSKYAPRFQG (88) YYYRYDVGGFDY (95) VH P13F02 DTYIH (79) EIDPANGHTKYDPKFQG YYYRYDVGGFDY

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(89) (95) VH P13B02 DTYIH (79) EIDPANDNTKYDPKFQG (90) YYYRYDVGGFDY (95) VH PlZAO 6 DTYMY (7 8) RIDPANGNTKYDPKFQG (91) YSYRYDVGGFDY (94) VH P14C04 DTYMY (7 8) RIDPANGNTHFDPKFQG (92) YFYRYDVGGFDY (96) VH P14A04 DTYMY (7 8) RIDPANGNTKFDPKFQD (86) YFYRYDVGGFDY (96) VH P15F06 DTYMY (78) RIDPANGNTKFDPKFQD (86) YFYRYDVGGFDY (96) VH P13B03 DTYIH (79) EIDPANGNTKYDPKFQG (84) YYYRYDVGGFDY (95) VH P15C02 DTYMY (78) RIDPANGNTKFDPKFQD (86) YFYRYDVGGFDY (96) VH P16D04 DTYMY (7 8) RIDPANGNTKFDPKFQD (86) YFYRYDVGGFDY (96) VH P13F01 DTYMY (7 8) RIDPANGNTKFDPKFQD (86) YFYRYDVGGFDY (96) VH P13A04 DTYMY (7 8) RIDPANGNTKFDPKFQD (86) YFYRYDVGGFDY (96) VH P16H05 DNYIH (81) SIDPANGNTKYDPKFQG (87) YFYRYDVGGFDY (96) VH P13F09 DNYIH (81) SIDPANGNTKYDPKFQG (87) YFYRYDVGGFDY (96) VH P13G01 DTYMY (7 8) RIDPANGNTKFDPKFQD (86) YFYRYDVGGFDY (96) VH P13H05 DTYIH (79) EIDPANDNTKYDPKFQG (90) YYYRYDVGGFDY (95) VH P13D04 DNYMD (82) KIDPANGNTKYDPKFQG (93) YYYRYDVGGFDY (95) VH P14G01 DTYMY (7 8) RIDPANGNTKFDPKFQD (86) YFYRYDVGGFDY (96) VH P14G03 DTYIH (79) EIDPANGNTKYDPKFQG (84) YYYRYDVGGFDY (95)

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VH P13G05 DTYMY (7 8) RIDPANGNTKFDPKFQD (86) YFYRYDVGGFDY (96) VH P13F06 DTYMY (7 8) RIDPANGNTKFDPKFQD (86) YFYRYDVGGFDY (96) VH P13B11 DNYMD (82) KIDPANGNTKYDPKFQG (93) YYYRYDVGGFDY (95) VH P14F01 DTYMY (7 8) RIDPANGNTKFDPKFQD (86) YFYRYDVGGFDY (96) VH P14F06 DNYIH (81) SIDPANGNTKYDPKFQG (87) YFYRYDVGGFDY (96) VH P13D05 DTYMY (7 8) RIDPANGNTKFDPKFQD (86) YFYRYDVGGFDY (96) VH P13G04 DTYMY (7 8) RIDPANGNTKFDPKFQD (86) YFYRYDVGGFDY (96) VH P15E06 DTYMY (7 8) RIDPANGNTKFDPKFQD (86) YFYRYDVGGFDY (96) VH P15G05 DTYMY (7 8) RIDPANGNTKFDPKFQD (86) YFYRYDVGGFDY (96) VH P15B06 DTYMY (7 8) RIDPANGNTHFDPKFQG ( 92 ) YFYRYDVGGFDY (96) VH P14B07 DNYIH (81) SIDPANGNTKYDPKFQG (87) YFYRYDVGGFDY (96) VH P05E03 DTYMY (7 8) RIDPANGNTKFDPKFQD (86) YFYRYDVGGFDY (96) VH P13C06 DTYMY (7 8) RIDPANGNTKFDPKFQD (86) YFDKYDVGGCDY (97) HO DTYIH (79) EIDPANDNTKYDPKFQG (90) YYYRYDVGGFDY (95) Hl DTYIH (79) EIDPANDNTKYDPKFQG (90) YYYRYDVGGFDY (95) H2 DTYIH (79) EIDPANDNTKYDPKFQG (90) YYYRYDVGGFDY (95) H3 DTYIH (79) EIDPANDNTKYDPKFQG (90) YYYRYDVGGFDY (95) H4 DTYIH (79) EIDPANDNTKYDPKFQG YYYRYDVGGFDY

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(90) (95) EIDPANDNTKYDPKFQG YYYKYDVGGFDY H4 R98K DTYIH (79) (90) (98) EIDPANDNTKYDPKFQG YYYRYEVGGFDY H4 D100E DTYIH (79) (90) (99) EIDPANDNTKYDPKFQG YYYKYDVGGFDY Hl R98K DTYIH (79) (90) (98) EIDPANDNTKYDPKFQG YYYKYDVGGFDY Hl R98K K23T DTYIH (79) (90) (98) EIDPANDNTKYDPKFQG YYYKYDVGGFDY Hl R98K L4M DTYIH (79) (90) (98) EIDPANDNTKYDPKFQG YYYKYDVGGFDY Hl R98K L4M K23T DTYIH (79) (90) (98) EIDPANDNTKYDPKFQG YYYKYDVGGFDY Hl R98K L4M V5K DTYIH (79) (90) (98) Hl R98K L4M V5K EIDPANDNTKYDPKFQG YYYKYDVGGFDY DTYIH (79) K23T (90) (98) EIDPANDNTKYDPKFQG YYYKYDVGGFDY Hl R98K V5K DTYIH (79) (90) (98) EIDPANDNTKYDPKFQG YYYKYDVGGFDY Hl R98K V5K K23T DTYIH (79) (90) (98) EIDPANDNTKYDPKFQG YYYKYDVGGFDY H2 R98K DTYIH (79) (90) (98) EIDPANDNTKYDPKFQG YYYKYDVGGFDY NC R98K DTYIH (79) (90) (98)

* Determined according to the Kabat numbering system.

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Table 3

Amino acid sequences of the light chain CDRs of typical anti-LAG-3 antibodies*

VL CDRL1 (SEQ ID NO:) CDRL2 (SEQ ID NO:) CDRL3 (SEQ ID NO:) VL Р01С09 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPFT (105) VL P05E01 SVSSSISSSNLH (100) GTSNLAS (104) QQWNSYPLT (106) VL Р01А12 SVSSSISSSNLH GTSNLAS (104) QQWSSYPFT (105) (100) VL Р13В01 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPFT (105) VL Р13С10 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPFT (105) VL Р13С08 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPFT (105) VL P13E02 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPFT (105) VL P13F02 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPFT (105) VL Р13В02 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPFT (105) VL P13A06 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPHT (107) VL Р14С04 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPFT (105) VL P14A04 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPFT (105) VL P15F06 SVSSSISSSTLH (101) GTSNLAS (104) QQWSSYPWT (108) VL Р13В03 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPFT (105) VL Р15С02 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPFT (105) VL P16D04 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPLT (109) VL P13F01 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPFT (105) VL P13A04 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPFT (105) VL Р16Н05 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPHT (107) VL P13F09 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPFT (105)

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VL P13G01 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPFT (105) VL Р13Н05 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPFT (105) VL P13D04 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPFT (105) VL P14G01 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPFT (105) VL P14G03 SVSSSSISSNLY (102) GTSNLAS (104) QQWSSYPFT (105) VL P13G05 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPFT (105) VL P13F06 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPFT (105) VL P13B11 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPFT (105) VL P14F01 SVSSSISSSNLH (100) GTSNLAS (104) QQWSTYPFT(110) VL P14F06 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPFT (105) VL P13D05 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPFT (105) VL P13G04 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPFT (105) VL P15E06 SVSSGISSSNLH (103) GTSNLAS (104) QQWSRYPWT(111) VL P15G05 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPLT (109) VL P15B06 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPLT (109) VL P14B07 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPFT (105) VL P05E03 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPFT (105) VL P13C06 SVSSSSISSNLY GTSNLAS (104) QQWRSYPFT (112) (102) L0 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPFT (105) LI SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPFT (105) L2 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPFT (105) L3 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPFT (105) L4 SVSSSISSSNLH (100) GTSNLAS (104) QQWSSYPFT (105)

* Determined according to the Kabat numbering system.

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Table 4

Framework (FR) heavy chain amino acid sequences of typical anti-LAG-3 antibodies*

VH FR1 VH (SEQ ID NO:) FR2 VH (SEQ ID NO: ) FR3 VH (SEQ ID NO:) FR4 VH (SEQ ID NO: ) But QVQLVQSGAEVKKP GASVKVSCKASGEN IK (113) WVRQAPG QGLEWMG (122) RVTMTRDTSTSTVYM ELSSLRSEDTAVYYC AR (124) WGQGTLV TVSS (128) H1 QVQLVQSGAEVKKP GASVKVSCKASGEN IK (113) WVRQAPG QGLEWMG (122) RVTITADTSTSTVYM ELSSLRSEDTAVYYC AT (125) WGQGTLV TVSS (128) H2 QVQLVQSGAEVKKP GASVKVSCKASGEN IK (113) WVRQAPG QGLEWMG (122) RVTITADTSSNTVYM ELSSLRSEDTAVYYC AT (126) WGQGTLV TVSS (128) NZ QVQMKQSGAEVKKP GASVKVSCTASGEN IK (114) WVRQAPG QGLEWMG (122) RVTITADTSSNTVYM ELSSLRSEDTAVYYC AT (126) WGQGTLV TVSS (128) H4 QVQMKQS GAE WKP GASVKVSCTASGEN IK (115) WVRQAPG QGLEWIG (123) RVTITADTSSNTVYL QLSSLRSEDTAVYYC AT (127) WGQGTLV TVSS (128) H4_R98K QVQMKQS GAE WKP GASVKVSCTASGEN IK (115) WVRQAPG QGLEWIG (123) RVTITADTSSNTVYL QLSSLRSEDTAVYYC AT (127) WGQGTLV TVSS (128)

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H4_D100E QVQMKQSGAEWKP GASVKVSCTASGEN IK (115) WVRQAPG QGLEWIG (123) RVTITADTSSNTVYL QLSSLRSEDTAVYYC AT (127) WGQGTLV TVSS (128) H1_R98K QVQLVQSGAEVKKP GASVKVSCKASGFN IK (113) WVRQAPG QGLEWMG (122) RVTITADTSTSTVYM ELSSLRSEDTAVYYC AT (125) WGQGTLV TVSS (128) H1_R98K_K23T QVQLVQSGAEVKKP GASVKVSCTASGEN IK (116) WVRQAPG QGLEWMG (122) RVTITADTSTSTVYM ELSSLRSEDTAVYYC AT (125) WGQGTLV TVSS (128) H1_R98K_L4M QVQMVQSGAEVKKP GASVKVSCKASGFN IK (117) WVRQAPG QGLEWMG (122) RVTITADTSTSTVYM ELSSLRSEDTAVYYC AT (125) WGQGTLV TVSS (128) H1_R98K_L4M_K23 T QVQMVQSGAEVKKP GASVKVSCTASGEN IK (118) WVRQAPG QGLEWMG (122) RVTITADTSTSTVYM ELSSLRSEDTAVYYC AT (125) WGQGTLV TVSS (128) H1_R98K_L4M_V5K QVQMKQSGAEVKKP GASVKVSCKASGFN IK (119) WVRQAPG QGLEWMG (122) RVTITADTSTSTVYM ELSSLRSEDTAVYYC AT (125) WGQGTLV TVSS (128) H1_R98K_L4M_V5K_K2 3T QVQMKQSGAEVKKP GASVKVSCTASGEN IK (114) WVRQAPG QGLEWMG (122) RVTITADTSTSTVYM ELSSLRSEDTAVYYC AT (125) WGQGTLV TVSS (128) H1_R98K_V5K QVQLKQSGAEVKKP GASVKVSCKASGFN IK (120) WVRQAPG QGLEWMG (122) RVTITADTSTSTVYM ELSSLRSEDTAVYYC AT (125) WGQGTLV TVSS (128) H1_R98K_V5K_K23 T QVQLKQSGAEVKKP GASVKVSCTASGEN IK (121) WVRQAPG QGLEWMG (122) RVTITADTSTSTVYM ELSSLRSEDTAVYYC AT (125) WGQGTLV TVSS (128) H2_R98K QVQLVQSGAEVKKP GASVKVSCKASGFN IK (113) WVRQAPG QGLEWMG (122) RVTITADTSSNTVYM ELSSLRSEDTAVYYC AT (126) WGQGTLV TVSS (128) H3_R98K QVQMKQSGAEVKKP GASVKVSCTASGEN IK (114) WVRQAPG QGLEWMG (122) RVTITADTSSNTVYM ELSSLRSEDTAVYYC AT (126) WGQGTLV TVSS (128)

* Heavy chain framework regions described in Table. 4 are determined based on the boundaries of the Kabat numbering system for the CDR. In other words, VH CDRs are defined using Kabat, and the framework regions are the amino acid residues surrounding the CDRs in the variable region in the format FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

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Table 5

Frame (F R)amino VL acid afterbirth FR1 VL (SEQ ID NO:) attractiveness FR2 VL (SEQ ID NO: ) light chain typical FR3 VL (SEQ ID NO:) antibodies pr( FR4 VL (SEQ ID NO: ) 1 Tiv LAG-3 * L0 EIVLTQSPGTLLSLS PGERATLSC (129) WYQQKPGQAPR LLIY (132) GIPDRFSGSGSGTDFT LTISRLEPEDFAVYYC (136) FGQGTKVEI K (139) LI EIVLTQSPGTLLSLS PGERATLSC (129) WYQQKPGQAPR LLIY (132) GIPDRFSGSGSGTSYT LTISRLEPEDFAVYYC (137) FGQGTKVEI K (139) L2 EILLTQSPGTLLSLS PGERATLSC (130) WFQQKPGQAPR LLIY (133) GIPDRFSGSGSGTSYT LTISRLEPEDFAVYYC (137) FGQGTKVEI K (139) L3 EILLTQSPGTLLSLS PGERATLSC (130) WFQQKPGQAPR LWIY (134) GVPDRFSGSGSGTSYT LTISRLEPEDFAVYYC (138) FGQGTKVEI K (139) L4 EILLTQSPGTLLSLS PGERATLTC (131) WFQQKPGQSPR LWIY (135) GVPDRFSGSGSGTSYT LTISRLEPEDFAVYYC (138) FGQGTKVEI K (139)

* Light chain framework regions described in Table. 5 are determined based on the boundaries of the Kabat numbering system for the CDR. In other words, VL CDRs are defined using Kabat, and the framework regions are the amino acid residues surrounding the CDRs in the variable region in the format FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

Table 6 __________Typical mouse antibodies against LAG-3__________ Variable region Variable region

Heavy chain antibody SEQ ID NO: light chain SEQ ID NO

Р01С09 ϊ 2

P05E01 3 4

P01A12 5 6 P13B01 7 8 P13C10 9 10 P13C08 eleven 12 P13E02 13 10 P13F02 14 10 P13B02 15 16 P13A06 17 18 P14C04 19 8 P14A04 20 21 P15F06 22 23 P13B03 24 10 P15C02 9 25 P16D04 9 26 P13F01 20 2 P13A04 27 28 P16H05 29 thirty

- 55 044026

P13F09 31 32 P13G01 33 34 Р13Н05 35 10 P13D04 36 37 P14G01 38 21 P14G03 39 40 P13G05 9 2 P13F06 9 41 Р13В11 42 43 P14F01 9 44 P14F06 45 32 P13D05 46 47 P13G04 48 10 P15E06 20 49 P15G05 33 50 Р15В06 51 52 Р14В07 53 8 P05E03 38 2 Р13С06 54 55

Table 7

Representative humanized anti-LAG-3 antibodies

Antibody Heavy Chain Variable Region (SEQ ID NO:) Light chain variable region SEQ ID NO:) Р13В02-01 BUT (56) L0 (73) Р13В02-02 BUT (56) LI (74) Р13В02-03 BUT (56) L2 (75) Р13В02-04 BUT (56) L3 (76) Р13В02-05 BUT (56) L4 (77) Р13В02-06 H1 (57) L0 (73) Р13В02-07 H1 (57) LI (74) Р13В02-08 H1 (57) L2 (75) Р13В02-09 H1 (57) L3 (76) Р13В02-10 H1 (57) L4 (77) Р13В02-11 H2 (58) L0 (73) Р13В02-12 H2 (58) LI (74) Р13В02-13 H2 (58) L2 (75) Р13В02-14 H2 (58) L3 (76) Р13В02-15 H2 (58) L4 (77) Р13В02-16 NZ (59) L0 (73) Р13В02-17 NZ (59) LI (74) Р13В02-18 NZ (59) L2 (75) Р13В02-19 NZ (59) L3 (76) Р13В02-20 NZ (59) L4 (77) Р13В02-21 H4 (60) L0 (73) Р13В02-22 H4 (60) LI (74)

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Р13В02-23 H4 (60) L2 (75) Р13В02-24 H4 (60) L3 (76) Р13В02-25 H4 (60) L4 (77) Р13В02-26 H4_R98K (61) L4 (77) Р13В02-27 H4_D100E (62) L4 (77) Р13В02-28 H1_R98K (63) L0 (73) Р13В02-29 H1_R98K_K23T (64) L0 (73) Р13В02-30 H1_R98K_L4M (65) L0 (73) Р13В02-31 H1_R98K_L4M_K23T (66) L0 (73) Р13В02-32 H1_R98K_L4M_V5K (67) L0 (73) Р13В02-33 H1_R98K_L4M_V5K_K23T (68) L0 (73) Р13В02-34 H1_R98K_V5K (69) LO (73) Р13В02-35 H1_R98K_V5K_K23T (70) LO (73) Р13В02-36 H2_R98K (71) LO (73) Р13В02-37 H3_R98K (72) LO (73)

Table 8

Human germline sequences

SEQ ID NO: Description Amino acid sequence 153 IGHV1-46*O1 QVQLVQS GAEVKKPGASVKVS CKAS GYT FT S YYMHWV RQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDT STSTVYMELSSLRSEDTAVYYCAR 154 IGHV1-69-2*01 EVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWV QQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADT STDTAYMELSSLRSEDTAVYYCAT 155 IGHV1-3*O1 QVQLVQS GAEVKKPGASVKVS CKAS GYT FT S YAMHWV RQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDT SASTAYMELSSLRSEDTAVYYCAR 156 IGHV1-24*O1 QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWV RQAPGKGLEWMGGFDPEDGETIYAQKFQGRVTMTEDT STDTAYMELSSLRSEDTAVYYCAT 157 IGHV1-2*O1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWV RQAPGQGLEWMGRINPNSGGTNYAQKFQGRVTSTRDT SISTAYMELSRLRSDDTWYYCAR 158 IGHV1-45*O1 QMQLVQSGAEVKKTGSSVKVSCKASGYTFTYRYLHWV RQAPGQALEWMGWITPFNGNTNYAQKFQDRVTITRDR SMSTAYMELSSLRSEDTAMYYCAR 159 IGHV1-18*O1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWV RQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDT

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STS TAYME LRS LRS DDTAVY YCAR 200 IGHJl*01 AEYFQHWGQGTLVTVSS 160 IGKV3-20*01 EIVLTQSPGTLLSLSPGERATLSCRASQSVSSSYLAWY QQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTL TISRLEPEDFAVYYCQQYGSSP 161 IGKV3D-15*01 or IGKV3-15*01 EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQ QKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLT ISSLQSEDFAVYYCQQYNNWP 162 IGKV3D-20*01 EIVLTQSPATLSLSPGERATLSCGASQSVSSSYLAWY QQKPGLAPRLLIYDASSRATGIPDRFSGSGSGTDFTL TISRLEPEDFAVYYCQQYGSSP 163 IGKV3D-7*01 EIVMTQSPATLSLSPGERATLSCRASQSVSSSYLSWY QQKPGQAPRLLIYGASTRATGIPARFSGSGSGTDFTL TISSLQPEDFAVYYCQQDYNLPP 164 IGKV1-9*O1 DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQ QKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLT ISSLQPEDFATYYCQQLNSYP 165 IGKV3-ll*01 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQ QKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLT ISSLEPEDFAVYYCQQRSNWP 201 IGKJl*01 WTFGQGTKVEIK

Table 9

Exemplary LAG-3 sequences

SEQ ID NO: Description Amino acid sequence 166 Immature protein Human LAG-3 (P18627-1) MWEAQFLGLLFLQPLWVAPVKPLQPGAEVPWWAQEG APAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAA APGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRS GRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYR AAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWV ILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLA ESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTV LGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTA KWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTC HIHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCE VTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLS

- 58 044026

QPWQCQLYQGERLLGAAVYFTELSSPGAQRSGRAPGA LPAGHLLLFLILGVLSLLLLVTGAFGFHLWRRQWRPR RFSALEQGIHPPQAQSKIEELEQEPEPEPEPEPEPEPEP EPEPEQL 167 Mature squirrel LAG-3 people VPWWAQEGAPAQLPCS PT IPLQDLSLLRRAGVTWQH QPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVL SVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPA RRADAGEYRAAVHLRDRALSCRLRLGQASMTASPP GSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPV RESPHHHLAESFLFLPQ VSPMDSGPWGCILTYRDGFN VSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAG VGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVS QAQAGTYTCHIHLQEQQLNATVTLAIITVTPKSFGSP GSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWL EAQEAQLLSQPWQCQLYQGERLLGAAVYFT ELSSPGA QRSGRAPGALPAGHLLLFLILGVLSLLLLVTGAFGFH LWRRQWRPRRFSALEQGIHPPQAQSKIEELEQEPEPE PEPEPEPEPEPEPEQL 210 Mature squirrel LAG-3 people LQPGAEVPWWAQEGAPAQLPCSPTIPLQDLSLLRRA GVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRP RRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFS LWLRPARRADAGEYRAAVHLRDRALSCRLRLGQAS MTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRG QGRVPVRE S PHHHLAE S FL FL PQVS PMDS GPWGCIL T YRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLP CRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTL RLEDVSQAQAGTYTCHIHLQEQQLNATVTLAIITVTP KSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRS FSGPWLEAQEAQLLSQPWQCQLYQ GERLLGAAVYFTE LSSPGAQRSGRAPGALPAGHLLLFLILGVLSLLLLVT GAFGFHLWRRQWRPRRFSALEQGIHPPQAQSKIEELE QEPEPEPEPEPEPEPEPEPEQL 217 Human LAG-3 fragment LQPGAEVPWWAQEGAPAQLPCSPTIPLQDLSLLRRA GVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRP RRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFS

- 59 044026

LWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQAS MTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRG QGRVPVRE S PHHHLAE S FL FL PQVS PMDS GPWGCIL T YRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLP CRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTL RLEDVSQAQAG TYTCHIHLQEQQLNATVTLAIITVTP KSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRS FSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTE LSSPGAQRSGRAPGALPAGHL 199 Human LAG3 30 amino acid loop GPPAAAPGHPLAPGPHPAAPSSWGPRPRRY 211 LAG-3 epitope PTIPLQD 212 LAG-3 epitope SPTIPLQD 213 LAG-3 epitope SPTIPLQDL 214 LAG-3 epitope SPTIPLQDLS 215 LAG-3 epitope SPTIPLQDLSL 216 LAG-3 epitope SPTIPLQDLSLL

In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (eg, human LAG-3), the antibody comprising a VH domain containing one, two, or all three of the VH domain CDRs listed in Table 1. 1, 2, 6 and 7 in this document. In certain embodiments, the antibody comprises CDRH1 of one of the VH domains listed in table. 1, 2, 6 and 7. In certain embodiments, the antibody comprises CDRH2 of one of the VH domains listed in table. 1, 2, 6 and 7. In certain embodiments, the antibody contains CDRH3 of one of the VH domains listed in table. 1, 2, 6 and 7.

In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (eg, human LAG-3), the antibody comprising a VL domain comprising one, two, or all three of the VL domain CDRs listed in Table 1. 1, 3, 6 and 7 in this document. In certain embodiments, the antibody comprises CDRL1 of one of the VL domains listed in table. 1, 3, 6 and 7. In certain embodiments, the antibody comprises CDRL2 of one of the VL domains listed in table. 1, 3, 6 and 7. In certain embodiments, the antibody contains CDRL3 of one of the VL domains listed in table. 1, 3, 6 and 7.

In certain embodiments, CDR antibodies can be defined according to Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest (1991), each of which is incorporated herein by reference in its entirety.

In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (eg, human LAG-3), the antibody comprising the VH Kabat CDRs of VH disclosed in Table 1. 1, 6 and 7 in this document. In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (eg, human LAG-3), the antibody comprising the VL Kabat CDRs of VL disclosed in Table 1. 1, 6 and 7 in this document. In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (eg, human LAG-3), the antibody comprising the Kabat CDR VH and Kabat CDR VL antibodies disclosed in Table 1. 1, 6 and 7 in this document.

In certain embodiments, antibody CDRs may be defined according to the Chothia numbering scheme, which refers to the location of immunoglobulin structural loops (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917; Al- Lazikani B et al., (1997) J Mol Biol 273: 927-948; Chothia C et al., (1992) J Mol Biol 227: 799-817; Tramontano A et al., (1990) J Mol Biol 215( 1): 175-82 and US Pat. No. 7,709,226, all of which are incorporated herein by reference in their entirety). Typically, using the Kabat numbering convention, the CDRH1 Chothia loop is present at heavy chain amino acids 26 to 32, 33, or 34, the CDRH2 Chothia loop is present at heavy chain amino acids 52 to 56, and the CDRH3 Chothia loop is present at heavy chain amino acids 95 to 102 , while the CDRL1 Chothia loop is present in light chain amino acids 24-34, the CDRL2 Chothia loop is present in light chain amino acids 50-56, and the CDRL3 Chothia loop is present in light chain amino acids 89-97. The end of the CDRH1 Chothia loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if there is neither 35A nor 35B,

-60044026 loop ends at 32, if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34).

In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (eg, human LAG-3), the antibody comprising the VH Chothia CDRs of VH disclosed in Table 1. 1, 6 and 7 in this document. In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (eg, human LAG-3), the antibody comprising the VL Chothia CDRs from VL disclosed in Table 1. 1, 6 and 7 in this document. In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (eg, human LAG-3), the antibody comprising the VH Chothia CDR and the Chothia CDR VL antibodies disclosed in Table 1. 1, 6 and 7 in this document. In certain embodiments, antibodies that specifically bind to LAG-3 (eg, human LAG-3) contain one or more CDRs, wherein the Chothia and Kabat CDRs have the same amino acid sequence. In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (eg, human LAG-3) and contains combinations of Kabat CDR and Chothia CDR.

In certain embodiments, antibody CDRs may be defined in accordance with the IMGT numbering system as described in Lefranc M-P, (1999) The Immunologist 7: 132-136 and Lefranc M-P et al., (1999) Nucleic Acids Res 27 : 209-212, each of which is incorporated herein by reference in its entirety. According to the IMGT numbering scheme, CDRH1 is at positions 26-35, CDRH2 is at positions 51-57, CDRH3 is at positions 93-102, CDRL1 is at positions 27-32, CDRL2 is at positions 50-52, and CDRL3 is at positions 89 -97.

In certain embodiments, the present invention relates to antibodies that specifically bind to LAG-3 (eg, human LAG-3) and contain the CDR antibodies disclosed in table. 1, 6 and 7, as defined by the IMGT numbering system, for example as described in Lefranc MP (1999) above and Lefranc MP et al. (1999) above.

In certain embodiments, antibody CDRs may be defined according to an AbM numbering scheme that refers to AbM hypervariable regions that represent a compromise between Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular AbM antibody modeling software (Oxford Molecular Group, Inc.). In a specific embodiment, this invention relates to antibodies that specifically bind to LAG-3 (eg, human LAG-3) and contain the CDR antibodies disclosed in table. 1, 6 and 7, as defined by the AbM numbering scheme. In certain embodiments, antibody CDRs may be defined in accordance with MacCallum RM et al., (1996) J Mol Biol 262: 732-745, which is incorporated herein by reference in its entirety. See also, for example, Martin A. Protein Sequence and Structure Analysis of Antibody Variable Domains, in Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001), which is incorporated herein by reference in its entirety. In a specific embodiment, this invention relates to antibodies that specifically bind to LAG-3 (eg, human LAG-3) and contain the CDR antibodies disclosed in table. 1, 6 and 7 as described in MacCallum RM et al., (1996) supra.

In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), the antibody comprising a heavy chain variable region comprising the amino acid sequences of the CDRH1, CDRH2, and CDRH3 region of the VH domain set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 14, 15, 17, 19, 20, 22, 24, 27, 29, 31, 33, 35, 36, 38, 39, 42, 45 , 46, 48, 51, 53, 54, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72, and a light chain variable region , containing the amino acid sequences of the CDRL1, CDRL2 and CDRL3 region of the VL domain specified in SEQ ID NO: 2, 4, 6, 8, 10, 12, 16, 18, 21, 23, 25, 26, 28, 30, 32, 34 , 37, 40, 41, 43, 44, 47, 49, 50, 52, 55, 73, 74, 75, 76 or 77, each CDR defined in accordance with the Kabat definition, the Chothia definition, a combination of the Kabat definition and the Chothia definition , IMGT numbering system, AbM definition or CDR MacCallum definition.

In certain embodiments, the invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), wherein the antibody comprises:

(a) CDRH1 contains the amino acid sequence DX1YX ₂ X ₃ (SEQ ID NO: 140), where

X1 represents T or N,

X ₂ represents I or M, and

X ₃ represents H, Y or D; and/or (b) CDRH2 contains the amino acid sequence X1IDPANX ₂ X ₃ X4X5X ₆ X ₇ PX ₈ X ₉ QX ₁₀ (SEQ ID NO: 142), where

X1 represents E, R, S or K,

X2 represents D or G,

X3 represents N or H,

X ₄ represents T or S,

X ₅ represents K or H,

- 61 044026

X ₆ represents Y or F,

X7 represents D or A,

X ₈ represents K or R,

X ₉ represents F or L, and

X ₁₀ represents G or D; and/or (c) CDRH3 contains the amino acid sequence YX1X2X3YX4VGGX5DY (SEQ ID NO: 144), where

X ₁ represents Y, F or S,

X ₂ represents Y or D,

X ₃ represents K or R,

X4 represents D or E, and

X ₅ represents F or C; and/or (d) CDRL1 contains the amino acid sequence SVSSX1ISSSX2LX3 (SEQ ID NO: 147), where

X ₁ represents S or G,

X ₂ represents N or T, and

X ₃ represents H or Y; and/or (e) CDRL2 contains the amino acid sequence GTSNLAS (SEQ ID NO: 104); and/or (f) CDRL3 contains the amino acid sequence QQWX1X2YPX3T (SEQ ID NO: 149), where

X ₁ represents S, N or R,

X2 represents S, T or R, and

X ₃ represents F, L, H or W.

In certain embodiments, CDRH1 comprises the amino acid sequence DX1YX2X3 (SEQ ID NO: 141), where X ₁ represents T or N; X ₂ represents I or M; and X ₃ is H or Y. In certain embodiments, CDRH2 comprises the amino acid sequence X1IDPANX2X3X4KX5X6PX7FQX8 (SEQ ID NO: 143), where X ₁ is E, R or S; X ₂ represents D or G; X ₃ represents N or H; X ₄ represents T or S; X ₅ represents Y or F; X ₆ represents D or A; X ₇ represents K or R; and X ₈ is G or D. In certain embodiments, CDRH3 comprises the amino acid sequence YX1X2X3YDVGGX4DY (SEQ ID NO: 145), where X1 is Y, F or S; X ₂ represents Y or D; X ₃ represents K or R; and X ₄ is F or C. In certain embodiments, CDRH3 comprises the amino acid sequence YYYX1YX2VGGFDY (SEQ ID NO: 146), where Xi is K or R; and X ₂ is D or E. In certain embodiments, CDRL1 contains the amino acid sequence SVSSSISSSNLX1 (SEQ ID NO: 148), where X1 is H or Y. In certain embodiments, CDRL3 contains the amino acid sequence QQWX1SYPX2T (SEQ ID NO: 150) where X1 represents S, N or R; and X ₂ represents F, L or H.

In certain embodiments, the invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), wherein the antibody comprises:

(a) CDRH1 contains the amino acid sequence DTYIH (SEQ ID NO: 79); and/or (b) CDRH2 contains the amino acid sequence EIDPANDNTKYDPKFQG (SEQ ID NO: 90); and/or (c) CDRH3 contains the amino acid sequence YYYX1YX2VGGFDY (SEQ ID NO: 146), where X1 represents K or R; and X ₂ represents D or E; and/or (d) CDRL1 contains the amino acid sequence SVSSSISSSNLH (SEQ ID NO: 100); and/or (e) CDRL2 contains the amino acid sequence GTSNLAS (SEQ ID NO: 104); and/or (f) CDRL3 contains the amino acid sequence QQWSSYPFT (SEQ ID NO: 105).

In certain embodiments, CDRH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 78-82. In certain embodiments, CDRH2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 83-93. In certain embodiments, CDRH3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 94-99. In certain embodiments, CDRL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 100-103. In certain embodiments, CDRL3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 105-112.

In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), wherein the antibody comprises a VH domain comprising the amino acid sequences CDRH1, CDRH2, and CDRH3 set forth in SEQ ID NO: 78. 83 and 94; 78, 85 and 95; 78, 86 and 96; 78, 86 and 97; 78, 91 and 94; 78, 92 and 96; 79, 84 and 95; 79, 88 and 95; 79, 89 and 95; 79, 90 and 95; 79, 90 and 98; 79, 90 and 99; 80, 85 and 96; 81, 87 and 96 or 82, 93 and 95 respectively. In certain embodiments, this invention relates to an isolated antibody

- 62 044026 lu, which specifically binds to LAG-3 (eg, human LAG-3), the antibody containing a VH domain containing the amino acid sequences CDRH1, CDRH2 and CDRH3 presented in SEQ ID NO: 79, 90 and 95, respectively. In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), wherein the antibody comprises a VH domain comprising the amino acid sequences CDRH1, CDRH2, and CDRH3 set forth in SEQ ID NO: 79. 90 and 98 respectively.

In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), wherein the antibody comprises a VL domain comprising the amino acid sequences CDRL1, CDRL2, and CDRL3 set forth in SEQ ID NO: 100. 104 and 105; 100, 104 and 106; 100, 104 and 107; 100, 104 and 109; 100, 104 and 110; 101, 104 and 108; 102, 104 and 105; 102, 104 and 112 or 103, 104 and 111 respectively. In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), wherein the antibody comprises a VL domain comprising the amino acid sequences CDRL1, CDRL2, and CDRL3 set forth in SEQ ID NO: 100. 104 and 105 respectively.

In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), the antibody comprising a heavy chain variable region comprising the CDRH1, CDRH2 and CDRH3 regions, and a light chain variable region comprising regions CDRL1, CDRL2 and CDRL3, wherein the regions CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 contain the amino acid sequences presented in

SEQ ID NO: 78, 83, 94, 100, 104 and 105; 78, 85, 95, 100, 104 and 105; 78, 86, 96, 100, 104 and 105; 78, 86, 96, 100, 104 and 109; 78, 86, 96, 100, 104 and 110; 78, 86, 96, 101, 104 and 108; 78, 86, 96, 103, 104 and 111; 78, 86, 97, 102,

104 and 112; 78, 91, 94, 100, 104 and 107; 78, 92, 96, 100, 104 and 105; 78, 92, 96, 100, 104 and 109; 79, 84, 95,100,

104 and 105; 79, 84, 95, 100, 104 and 106; 79, 84, 95, 102, 104 and 105; 79, 88, 95, 100, 104 and 105; 79, 89, 95,100,

104 and 105; 79, 90, 95, 100, 104 and 105; 79, 90, 98, 100, 104 and 105; 79, 90, 99, 100, 104 and 105; 80, 85, 96,100,

104 and 105; 81, 87, 96, 100, 104 and 105; 81, 87, 96, 100, 104 and 107 or 82, 93, 95, 100, 104 and 105 respectively. In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), the antibody comprising a heavy chain variable region comprising the CDRH1, CDRH2 and CDRH3 regions, and a light chain variable region comprising the CDRL1, CDRL2 and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 regions contain the amino acid sequences shown in SEQ ID NO: 79, 90, 95, 100, 104 and 105, respectively. In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), the antibody comprising a heavy chain variable region comprising the CDRH1, CDRH2 and CDRH3 regions, and a light chain variable region comprising the CDRL1, CDRL2 and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 regions contain the amino acid sequences shown in SEQ ID NO: 79, 90, 98, 100, 104 and 105, respectively.

In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), wherein the antibody comprises a heavy chain variable region (VH) comprising one, two, or all three of the antibody's VH CDRs table 1, 2, 6 and 7. In some embodiments, the antibody contains one, two, three, or all four VH framework regions described herein. In specific embodiments, the antibody comprises one, two, three, or all four of the VH framework regions (FRs) listed in Table. 4 (e.g., one, two, three, or four framework regions in one row in Table 4), In certain embodiments, the antibody comprises one, two, three, or all four framework regions of the heavy chain variable region sequence SEQ ID NO: 151 or 222 In certain embodiments, the antibody contains one, two, three, or all four variable heavy chain sequence framework regions of SEQ ID NO: 218 or 223. In certain embodiments, the antibody contains one, two, three, or four heavy chain variable region sequence framework regions , which are at least 75, 80, 85, 90, 95 or 100% (for example, at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) identical to one, two, three, or four heavy chain variable region sequence framework regions selected from the group consisting of SEQ ID NOs: 56-72 and 220. In certain embodiments, the antibody comprises a heavy chain variable region framework that is either is derived from an amino acid sequence encoded by a human gene, wherein the amino acid sequence is selected from the group consisting of IGHV1-46 (e.g., IGHV1-46*01, e.g., having the amino acid sequence SEQ ID NO: 153), IGHV1-69-2 (e.g., IGHV1-69-2*01, for example, having the amino acid sequence SEQ ID NO: 154), IGHV1-3 (for example, IGHV1-3*01, for example, having the amino acid sequence SEQ ID NO: 155), IGHV1-24 (for example, IGHV1-24*01, for example, having the amino acid sequence SEQ ID NO: 156), IGHV1-2 (for example, IGHV1-2*01, for example, having the amino acid sequence SEQ ID NO: 157), IGHV1-45 (for example, IGHV1- 45*01, for example, having the amino acid sequence SEQ ID NO: 158), and IGHV1-18 (for example, IGHV1-18*01,

- 63 044026 for example, having the amino acid sequence SEQ ID NO: 159). In specific embodiments, the heavy chain variable framework that is derived from said amino acid sequence consists of said amino acid sequence with up to 20 amino acid substitutions, deletions and/or insertions, preferably up to 20 amino acid substitutions. In a specific embodiment, the heavy chain variable framework that is derived from said amino acid sequence consists of said amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17, 18, 19 or 20 amino acid residues replacing an amino acid found at a similar position in the corresponding non-human heavy chain variable framework. In some embodiments, the antibody comprises a heavy chain variable framework region that is derived from the amino acid sequence of SEQ ID NO: 153), wherein at least one amino acid in the amino acid sequence of SEQ ID NO: 153 is replaced by an amino acid at a similar position in the corresponding heavy chain variable region, not received from a person. In a specific embodiment, the amino acid substitution is at an amino acid position selected from the group consisting of 4, 5, 12, 23, 27, 28, 29, 30, 48, 69, 71, 75, 76, 80, 81 and 94, wherein amino acid position is indicated according to the Kabat numbering system. In specific embodiments, the amino acid substitution is selected from the group consisting of 4M, 5K, 12V, 23T, 27F, 28N, 29I, 30K, 48I, 69I, 71A, 75S, 76N, 80L, 81Q, and 94T, wherein the position of the amino acid substitution is indicated in according to the Kabat numbering system. In another specific embodiment, the amino acid substitution is at an amino acid position selected from the group consisting of 4, 27, 28, 29, 30, 69, 71 and 94, where the amino acid position is indicated in accordance with the Kabat numbering system. In specific embodiments, the amino acid substitution is selected from the group consisting of 4M, 27F, 28N, 29I, 30K, 69I, 71A, and 94T, with the position of the amino acid substitution indicated in accordance with the Kabat numbering system.

In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), wherein the antibody comprises a light chain variable region (VL) comprising one, two, or all three of the antibody's VL CDRs table 1, 3, 6, and 7. In some embodiments, the antibody comprises one, two, three, or all four VL framework regions described herein. In specific embodiments, the antibody comprises one, two, three, or all four of the VL framework regions (FRs) listed in Table. 5 (e.g., one, two, three, or four framework regions in one row in Table 5), In certain embodiments, the antibody comprises one, two, three, or all four framework regions of the light chain variable region sequence SEQ ID NO: 152 or 224 In certain embodiments, the antibody contains one, two, three, or four light chain variable region sequence framework regions that are at least 75, 80, 85, 90, 95, or 100% (e.g., at least 86, 87, 88) , 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to one, two, three, or four light chain variable region sequence framework regions selected from the group consisting of SEQ ID NO: 73-77. In certain embodiments, the antibody comprises a light chain variable framework region that is or is derived from an amino acid sequence encoded by a human gene, wherein the amino acid sequence is selected from the group consisting of IGKV3-20 (e.g., IGKV3-20*01, for example, having the amino acid sequence SEQ ID NO: 160), IGKV3D-15 (for example, IGKV3D-15*01, for example, having the amino acid sequence SEQ ID NO: 161), IGKV3-15 (for example, IGKV3-15*01, for example, having the amino acid sequence SEQ ID NO: 161), IGKV3D-20 (for example, IGKV3D-20*01, for example, having the amino acid sequence SEQ ID NO: 162), IGKV3D-7 (for example, IGKV3D-7*01, for example, having the amino acid sequence SEQ ID NO: 163), IGKV1-9 (e.g., IGRV1-9*01, e.g., having the amino acid sequence SEQ ID NO: 164), and IGKV3-11 (e.g., IGKV3-11*01, e.g., having the amino acid sequence SEQ ID NO: 165 ). In specific embodiments, the light chain variable framework that is derived from said amino acid sequence consists of said amino acid sequence with up to 20 amino acid substitutions, deletions and/or insertions, preferably up to 20 amino acid substitutions. In a specific embodiment, the light chain variable framework that is derived from said amino acid sequence consists of said amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17, 18, 19 or 20 amino acid residues replacing an amino acid found at a similar position in the corresponding non-human light chain variable framework. In some embodiments, the antibody comprises a light chain variable framework region that is derived from the amino acid sequence of SEQ ID NO: 160, wherein at least one amino acid in the amino acid sequence of SEQ ID NO: 160 is replaced by an amino acid at a similar position in the corresponding light chain variable region derived from not from a person. In a specific embodiment, the amino acid substitution is at an amino acid position selected from the group consisting of 3, 22, 36, 43, 47, 58, 70 and 71, where the amino acid position is indicated in accordance with the Kabat numbering system. In specific embodiments, the amino acid substitution is selected from the group consisting of 3L, 22T, 36F, 43S, 47W, 58V, 70S, and 71Y, with the position of the amino acid substitution indicated in accordance with the Kabat numbering system.

In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), wherein the antibody comprises a heavy chain variable region (VH) comprising one, two, or all three of the antibody's VH CDRs table 1, 2, 6 and 7 (eg, VH CDRs in one row of Table 2) and a light chain variable region (VL) containing one, two or all three VL antibody CDRs in Table 2. 1, 3, 6 and 7 (for example, CDR VL in one row of Table 3). In some embodiments, the antibody comprises the VH framework regions and the VL framework regions described herein. In specific embodiments, the antibody comprises the VH framework regions (FRs) listed in Table 1. 4 (for example, one, two, three or four frame regions in one row in Table 4), and the frame regions (FR) VL specified in Table. 5 (for example, one, two, three or four frame regions in one row in Table 5).

In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), wherein the antibody comprises a VH domain comprising the amino acid sequences CDRH1, CDRH2, and CDRH3 set forth in SEQ ID NO: 79. 90 and 95; or 79, 90 and 98 respectively. In certain embodiments, the antibody comprises one, two, three, or all four VH framework regions derived from a human or primate VH antibody. In some embodiments, the antibody comprises the antibody VH framework regions shown in Table 1. 4.

In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), wherein the antibody comprises a VL domain comprising the amino acid sequences CDRL1, CDRL2, and CDRL3 set forth in SEQ ID NO: 100. 104 and 105 respectively. In certain embodiments, the antibody comprises one, two, three, or all four VL framework regions derived from a human or primate VL antibody. In some embodiments, the antibody comprises the antibody VL framework regions shown in Table 1. 5.

In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), the antibody comprising a heavy chain variable region comprising the CDRH1, CDRH2 and CDRH3 regions, and a light chain variable region comprising the CDRL1, CDRL2 and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 regions contain the amino acid sequences presented in SEQ ID NO: 79, 90, 95, 100, 104 and 105 or 79, 90, 98, 100, 104 and 105 respectively. In certain embodiments, the antibody comprises one, two, three, or all four VH framework regions derived from a human or primate VH antibody, and one, two, three, or all four VL framework regions derived from a human or primate VL antibody. In some embodiments, the antibody comprises the VH framework regions and the VL framework regions of the antibody shown in Table 1. 4 and 5 respectively.

In certain embodiments, this invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3) comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 151 or 222. In certain embodiments, this invention provides The invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3) comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 218 or 223. In certain embodiments, the invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3) containing a heavy chain variable region containing an amino acid sequence that is at least 75, 80, 85, 90, 95, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) identical to the amino acid sequence given in SEQ ID NO: 56, 57, 58, 59, 60, 61, 62 , 63, 64, 65, 220, 66, 67, 68, 69, 70, 71, or 72. In certain embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 220, 66, 67, 68, 69, 70, 71 or 72, optionally, wherein the amino acid residue at position 1 of the heavy chain variable region is converted to pyroglutamate. In certain embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 65. In certain embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 220. In certain embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 220. X in SEQ ID NO: 220 is Q. In certain embodiments, X in SEQ ID NO: 220 is pyroglutamate. In certain embodiments, the antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 168, 225, 169, 226, 170, 227, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180 , 181, 182, 183, 184, 185 or 186, optionally wherein the amino acid residue at position 1 of the heavy chain is converted to pyroglutamate. In certain embodiments, the antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 168. In certain embodiments, the antibody contains a heavy chain having the amino acid sequence set forth in SEQ ID NO: 225. In certain embodiments, the antibody contains a heavy chain having the amino acid sequence set forth in SEQ ID NO: 225. X in SEQ ID NO: 225 is Q. In certain embodiments, X in SEQ ID NO: 225 is pyroglutamate. In certain embodiments, the antibody contains a heavy chain having the amino acid sequence set forth in SEQ ID NO: 169. In certain embodiments, the antibody contains a heavy chain having the amino acid sequence set forth in SEQ ID NO: 226. In certain embodiments, X in SEQ ID NO: 226 is Q. In certain embodiments, X in SEQ ID NO: 226 is pyroglutamate. In certain embodiments, the antibody contains a heavy chain having the amino acid sequence set forth in SEQ ID NO: 170. In certain embodiments, the antibody contains a heavy chain having the amino acid sequence set forth in SEQ ID NO: 227. In certain embodiments, X in SEQ ID NO: 227 is Q. In certain embodiments, X in SEQ ID NO: 227 is pyroglutamate.

In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3) comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 152 or 224. In certain embodiments, the present invention refers to an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3) comprising a light chain variable region containing an amino acid sequence that is at least 75, 80, 85, 90, 95, or 100% (e.g. , at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 73, 74, 75, 76 or 77. In certain embodiments, the antibody comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 73, 74, 75, 76, or 77, optionally wherein the amino acid residue at position 1 of the light chain variable region is converted to a pyroglutamate . In certain embodiments, the antibody comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 73. In certain embodiments, the antibody comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 221. In certain embodiments, the antibody comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 221. X in SEQ ID NO: 221 is E. In certain embodiments, X in SEQ ID NO: 221 is pyroglutamate. In certain embodiments, the antibody comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 187, 188, 189, 190, or 191, optionally wherein the amino acid residue at position 1 of the light chain is converted to a pyroglutamate. In certain embodiments, the antibody comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 187. In certain embodiments, the antibody comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 228. In certain embodiments, the antibody comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 228. X in SEQ ID NO: 228 is E. In certain embodiments, X in SEQ ID NO: 228 is pyroglutamate.

In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3) comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 151 or 222, and a light chain variable region, comprising the amino acid sequence of SEQ ID NO: 152 or 224. In certain embodiments, the invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3) comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO : 218 or 223, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 152 or 224. In certain embodiments, the invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3) comprising a heavy chain variable region comprising an amino acid sequence that is at least 75, 80, 85, 90, 95, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) identical to the amino acid sequence given in SEQ ID NO: 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 220, 66, 67, 68, 69 , 70, 71, or 72, and a light chain variable region comprising an amino acid sequence that is at least 75, 80, 85, 90, 95, or 100% (e.g., at least 86, 87, 88, 89, 90 , 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 73, 221, 74, 75, 76, or 77. In certain embodiments, the antibody comprises a variable region heavy chain having the amino acid sequence set forth in SEQ ID NO: 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 220, 66, 67, 68, 69, 70, 71 or 72, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 73, 221, 74, 75, 76, or 77. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NO: 56 and 73; 56 and 74; 56 and 75; 56 and 76; 56 and 77;

- 66 044026 and 73; 57 and 74; 57 and 75; 57 and 76; 57 and 77; 58 and 73; 58 and 74; 58 and 75; 58 and 76; 58 and 77; 59 and 73; 59 and 74; 59 and 75; 59 and 76; 59 and 77; 60 and 73; 60 and 74; 60 and 75; 60 and 76; 60 and 77; 61 and 77; 62 and 77; 63 and 73; 64 and 73; 65 and 73; 220 and 73; 65 and 221; 220 and 221; 66 and 73; 67 and 73; 68 and 73; 69 and 73; 70 and 73; 71 and 73 or 72 and 73 respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 56 and 73, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 220 and 221, respectively. In certain embodiments, X in SEQ ID NO: 220 is Q. In certain embodiments, X in SEQ ID NO: 220 is pyroglutamate. In certain embodiments, X in SEQ ID NO: 221 is E. In certain embodiments, X in SEQ ID NO: 221 is pyroglutamate. In certain embodiments, X in SEQ ID NO: 220 is Q and X in SEQ ID NO: 221 is E. In certain embodiments, X in SEQ ID NO: 220 is Q and X in SEQ ID NO: 221 is a pyroglutamate. In certain embodiments, X in SEQ ID NO: 220 is pyroglutamate and X in SEQ ID NO: 221 is E. In certain embodiments, X in SEQ ID NO: 220 is pyroglutamate and X in SEQ ID NO: 221 is a pyroglutamate. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 56 and 74, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 56 and 75, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 56 and 76, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 56 and 77, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 57 and 73, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 57 and 74, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 57 and 75, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 57 and 76, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 57 and 77, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 58 and 73, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 58 and 74, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 58 and 75, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 58 and 76, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 58 and 77, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 59 and 73, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 59 and 74, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 59 and 75, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 59 and 76, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 59 and 77, respectively. In certain embodiments, the antibody comprises a variable region

- 67 044026 light chain and light chain variable region having the amino acid sequences shown in SEQ ID NO: 60 and 73, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 60 and 74, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 60 and 75, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 60 and 76, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 60 and 77, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 61 and 77, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 62 and 77, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 63 and 73, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 64 and 73, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 65 and 73, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 66 and 73, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 67 and 73, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 68 and 73, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 69 and 73, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 70 and 73, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 71 and 73, respectively. In certain embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 72 and 73, respectively. In certain embodiments, the amino acid residue at position 1 of the heavy chain variable region is converted to pyroglutamate. In certain embodiments, the amino acid residue at position 1 of the light chain variable region is converted to a pyroglutamate. In certain embodiments, the amino acid residue at position 1 of the heavy chain variable region is converted to pyroglutamate, and the amino acid residue at position 1 of the light chain variable region is converted to pyroglutamate.

In certain embodiments, the present invention provides an isolated antibody that competes for binding to LAG-3 (eg, human LAG-3) with an antibody comprising the heavy and light chain variable region amino acid sequences set forth in SEQ ID NOs: 1 and 2; 3 and 4; 5 and 6; 7 and 8; 9 and 10; 11 and 12; 13 and 10; 14 and 10; 15 and 16; 17 and 18; 19 and 8; 20 and 21; 22 and 23; 24 and 10; 9 and 25; 9 and 26; 20 and 2; 27 and 28; 29 and 30; 31 and 32; 33 and 34; 35 and 10; 36 and 37; 38 and 21; 39 and 40; 9 and 2; 9 and 41; 42 and 43; 9 and 44; 45 and 32; 46 and 47; 48 and 10; 20 and 49; 33 and 50; 51 and 52; 53 and 8; 38 and 2 or 54 and 55 respectively. In certain embodiments, the present invention provides an isolated antibody that competes for binding to LAG-3 (eg, human LAG-3) with an antibody comprising the heavy and light chain variable region amino acid sequences set forth in SEQ ID NOs: 56 and 73; 56 and 74; 56 and 75; 56 and 76; 56 and 77; 57 and 73; 57 and 74; 57 and 75; 57 and 76; 57 and 77; 58 and 73; 58 and 74; 58 and 75; 58 and 76; 58 and 77; 59 and 73; 59 and 74; 59 and 75; 59 and 76; 59 and 77; 60 and 73; 60 and 74; 60 and 75; 60 and 76; 60 and 77; 61 and 77; 62 and 77; 63 and 73; 64 and 73; 65 and 73; 220 and 73; 65 and 221; 220 and 221; 66 and 73; 67 and 73; 68 and 73; 69 and 73; 70 and 73; 71 and 73 or 72 and 73 respectively.

In certain embodiments, the present invention provides an isolated antibody that binds to the same or overlapping LAG-3 epitope (e.g., a human LAG3 epitope) as an antibody described herein, e.g., an antibody comprising heavy and light variable region amino acid sequences Circuits shown in SEQ ID NO:

- 68 044026 and 2; 3 and 4; 5 and 6; 7 and 8; 9 and 10; 11 and 12; 13 and 10; 14 and 10; 15 and 16; 17 and 18; 19 and 8; 20 and 21; 22 and 23; 24 and 10; 9 and 25; 9 and 26; 20 and 2; 27 and 28; 29 and 30; 31 and 32; 33 and 34; 35 and 10; 36 and 37; 38 and 21; 39 and 40; 9 and 2; 9 and 41; 42 and 43; 9 and 44; 45 and 32; 46 and 47; 48 and 10; 20 and 49; 33 and 50; 51 and 52; 53 and 8; 38 and 2 or 54 and 55 respectively. In certain embodiments, the present invention provides an isolated antibody that binds to the same or overlapping LAG-3 epitope (e.g., human LAG-3 epitope) as an antibody described herein, e.g., an antibody containing heavy and light amino acid sequences variable region chains set forth in SEQ ID NOs: 56 and 73; 56 and 74; 56 and 75; 56 and 76; 56 and 77; 57 and 73; 57 and 74; 57 and 75; 57 and 76; 57 and 77; 58 and 73; 58 and 74; 58 and 75; 58 and 76; 58 and 77; 59 and 73; 59 and 74; 59 and 75; 59 and 76; 59 and 77; 60 and 73; 60 and 74; 60 and 75; 60 and 76; 60 and 77; 61 and 77; 62 and 77; 63 and 73; 64 and 73; 65 and 73; 220 and 73; 65 and 221; 220 and 221; 66 and 73; 67 and 73; 68 and 73; 69 and 73; 70 and 73; 71 and 73 or 72 and 73 respectively. In some embodiments, an antibody epitope may be determined, for example, by NMR spectroscopy, surface plasmon resonance (BIAcore®), X-ray diffraction crystallography studies, ELISA assays, hydrogen/deuterium exchange coupled with mass spectrometry (e.g., mass liquid chromatography spectrometry, electrospray mass spectrometry), oligopeptide scanning matrix analysis and/or mutagenesis mapping (eg, site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be performed using any of the methods known in the art (eg, Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303, all of which are incorporated herein by reference in their entirety). Antibody:antigen crystals can be studied using well-known x-ray diffraction techniques and can be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, for example, Meth Enzymol (1985) vol. 114 & 115, eds Wyckoff HW et al.; US publication no. 2004/0014194) and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49 (Pt. 1):37-60; Bricogne G ( 1997) Meth Enzymol 276A: 361-423, ed Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10): 1316-1323), all of which are incorporated herein by reference in their entirety volume. Mutagenesis mapping studies can be performed using any method known to one skilled in the art. See, for example, Champe M et al., (1995) supra and Cunningham BC & Wells JA (1989) supra for descriptions of mutagenesis methods, including alanine scan mutagenesis methods. In a specific embodiment, the antibody epitope is determined using alanine scanning mutagenesis studies. In addition, antibodies that recognize and bind to the same or overlapping epitopes of LAG-3 (e.g., human LAG-3) can be identified using routine methods such as immunoassays, for example by demonstrating the ability of one antibody to block the binding of another antibody with the target antigen, i.e. Competitive binding assay. Competitive binding assays can also be used to determine whether two antibodies have similar epitope binding specificity. Competitive binding can be determined in an assay in which the test immunoglobulin inhibits the specific binding of a reference antibody to a common antigen such as LAG-3 (eg, human LAG-3). Numerous types of competitive binding assays are known, for example: direct or indirect radioimmunoassay (RIA), direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli C et al., (1983) Methods Enzymol 9: 242- 253); solid phase direct biotin-avidin EIA (see Kirkland TN et al., (1986) J Immunol 137: 3614-9); direct-labeling solid-phase assay, direct-labeling solid-phase assay (see Harlow E & Lane D, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press); direct-labeled solid-phase RIA using the I-125 tag (see Morel GA et al., (1988) Mol Immunol 25(1): 7-15); direct-labeled solid-phase EIA (see Cheung RC et al., (1990) Virology 176: 546-52) and direct-labeled RIA (see Moldenhauer G et al., (1990) Scand J Immunol 32: 77-82) , all of which are incorporated herein by reference in their entirety. Typically, such an assay involves the use of purified antigen (eg, LAG-3, such as human LAG-3) bound to a solid surface, or cells bearing either of them, an unlabeled test immunoglobulin, and a labeled control immunoglobulin. Competitive inhibition can be measured by determining the amount of label bound to a solid surface or cells in the presence of a test immunoglobulin. Usually the immunoglobulin test is present in excess. Typically, when a competing antibody is present in excess, it will inhibit the specific binding of the reference antibody to the common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more. The competitive binding assay can be configured in a variety of different formats using either a labeled antigen or a labeled antibody. In the conventional version of this assay, the antigen is immobilized in a 96-well plate. The ability of unlabeled antibodies to block the binding of labeled antibodies to antigen is then measured using radioactive or enzyme labels. For more information, see, for example, Wagener C et al., (1983) J Immunol 130: 2308-2315; Wagener C et al., (1984) J Immunol Methods 68: 269-274; Kuroki M

- 69 044026 et al., (1990) Cancer Res 50: 4872-4879; Kuroki M et al., (1992) Immunol Invest 21: 523-538; Kuroki M et al., (1992) Hybridoma 11: 391-407 and Antibodies: A Laboratory Manual, Ed Harlow E & Lane D editors supra, pp.

386-38 9, all of which are incorporated herein by reference in their entirety.

In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), the antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 168, 225, 169, 226, 170, 227, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185 or 186, optionally wherein the amino acid residue at position 1 of the heavy chain is converted to pyroglutamate. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 168. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 225. In certain embodiments, X in SEQ ID NO: 225 is Q. In certain embodiments, X in SEQ ID NO: 225 is pyroglutamate. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 169. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 226. In certain embodiments, X in SEQ ID NO: 226 is Q. In certain embodiments, X in SEQ ID NO: 226 is pyroglutamate. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 170. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 227. In certain embodiments, X in SEQ ID NO: 227 is Q. In certain embodiments, X in SEQ ID NO: 227 is pyroglutamate. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 171. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 172. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 173. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 174. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 175. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 176. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 177. In certain embodiments embodiment, the antibody contains a heavy chain containing the amino acid sequence set forth in SEQ ID NO: 178. In certain embodiments, the antibody contains a heavy chain containing the amino acid sequence set forth in SEQ ID NO: 179. In certain embodiments, the antibody contains a heavy chain containing the amino acid the sequence set forth in SEQ ID NO: 180. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 181. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 182. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 183. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 184. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 185. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 186. In certain embodiments, the amino acid residue at position 1 of the heavy chain is converted to a pyroglutamate .

In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), the antibody comprising a light chain comprising the amino acid sequence set forth in SEQ ID NO: 187, 228, 188, 189, 190 or 191, optionally, wherein the amino acid residue at position 1 of the light chain is converted to pyroglutamate. In certain embodiments, the antibody contains a light chain containing the amino acid sequence set forth in SEQ ID NO: 187. In certain embodiments, the antibody contains a light chain containing the amino acid sequence set forth in SEQ ID NO: 228. In certain embodiments, X in SEQ ID NO: 228 is E. In certain embodiments, X in SEQ ID NO: 228 is pyroglutamate. In certain embodiments, the antibody contains a light chain containing the amino acid sequence set forth in SEQ ID NO: 188. In certain embodiments, the antibody contains a light chain containing the amino acid sequence,

- 70 044026 set forth in SEQ ID NO: 189. In certain embodiments, the antibody contains a light chain containing the amino acid sequence set forth in SEQ ID NO: 190. In certain embodiments, the antibody contains a light chain containing the amino acid sequence set forth in SEQ ID NO: : 191. In certain embodiments, the amino acid residue at position 1 of the light chain is converted to pyroglutamate.

In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), the antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 168 and a light chain comprising the amino acid sequence SEQ ID NO: 187. In certain embodiments, the invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), wherein the antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 225, and a light chain comprising the amino acid sequence of SEQ ID NO: 228. In certain embodiments, X in SEQ ID NO: 225 is Q and X in SEQ ID NO: 228 is pyroglutamate. In certain embodiments, X in SEQ ID NO: 225 is pyroglutamate and X in SEQ ID NO: 228 is E. In certain embodiments, X in SEQ ID NO: 225 is pyroglutamate and X in SEQ ID NO: 228 is a pyroglutamate.

In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), the antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 169 and a light chain comprising the amino acid sequence SEQ ID NO: 187. In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), wherein the antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 226, and light chain comprising the amino acid sequence of SEQ ID NO: 228. In certain embodiments, X in SEQ ID NO: 226 is Q and X in SEQ ID NO: 228 is pyroglutamate. In certain embodiments, X in SEQ ID NO: 226 is pyroglutamate and X in SEQ ID NO: 228 is E. In certain embodiments, X in SEQ ID NO: 226 is pyroglutamate and X in SEQ ID NO: 228 is a pyroglutamate.

In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), the antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 170 and a light chain comprising the amino acid sequence SEQ ID NO: 187. In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), wherein the antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 227, and light chain comprising the amino acid sequence of SEQ ID NO: 228. In certain embodiments, X in SEQ ID NO: 227 is Q and X in SEQ ID NO: 228 is pyroglutamate. In certain embodiments, X in SEQ ID NO: 227 is pyroglutamate and X in SEQ ID NO: 228 is E. In certain embodiments, X in SEQ ID NO: 227 is pyroglutamate and X in SEQ ID NO: 228 is a pyroglutamate.

In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), the antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 171 and a light chain comprising the amino acid sequence SEQ ID NO: 187. In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), wherein the antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 172, and a light chain comprising the amino acid sequence of SEQ ID NO: 187. In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 173, and a light chain comprising the amino acid sequence of SEQ ID NO: 187. In certain embodiments, the invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), wherein the antibody comprises a heavy a chain comprising the amino acid sequence of SEQ ID NO: 174, and a light chain comprising the amino acid sequence of SEQ ID NO: 187. In certain embodiments, the invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3) wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 175 and a light chain comprising the amino acid sequence of SEQ ID NO: 187. In certain embodiments, the invention provides an isolated antibody that specifically binds to LAG-3 (eg , human LAG-3), the antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 176, and a light chain containing

- 71 044026 amino acid sequence SEQ ID NO: 187. In certain embodiments, this invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), wherein the antibody comprises a heavy chain containing the amino acid sequence SEQ ID NO: 177, and a light chain comprising the amino acid sequence of SEQ ID NO: 187. In certain embodiments, the invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 178, and a light chain comprising the amino acid sequence of SEQ ID NO: 187. In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 179 and a light chain comprising the amino acid sequence of SEQ ID NO: 187. In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g. human LAG-3), the antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 180 and a light chain comprising the amino acid sequence of SEQ ID NO: 187. In certain embodiments, the invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), the antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 181 and a light chain containing the amino acid sequence of SEQ ID NO: 187. In certain embodiments, the invention relates to an isolated an antibody that specifically binds to LAG-3 (e.g., human LAG-3), wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 182 and a light chain comprising the amino acid sequence of SEQ ID NO: 187. In certain embodiments The present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), the antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 183 and a light chain containing the amino acid sequence of SEQ ID NO: 183 : 187. In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), the antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 184, and a light chain, comprising the amino acid sequence SEQ ID NO: 187. In certain embodiments, this invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), wherein the antibody comprises a heavy chain comprising the amino acid sequence SEQ ID NO: 185, and a light chain comprising the amino acid sequence of SEQ ID NO: 187. In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), wherein the antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 186, and a light chain comprising the amino acid sequence of SEQ ID NO: 187. In certain embodiments, the amino acid residue at position 1 of the heavy chain is converted to pyroglutamate. In certain embodiments, the amino acid residue at position 1 of the light chain is converted to pyroglutamate. In certain embodiments, the amino acid residue at position 1 of the heavy chain is converted to pyroglutamate and the amino acid residue at position 1 of the light chain is converted to pyroglutamate.

Any Ig constant region can be used in the antibodies disclosed herein. In certain embodiments, the Ig region is a human immunoglobulin molecule IgG, IgE, IgM, IgD, IgA, or IgY, any class (e.g., IgG1, IgG ₂ , IgG ₃ , IgG4, IgA1, and IgA ₂ ), or any subclass (e.g., IgG _2a and IgG _2b ) immunoglobulin molecules.

In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), the antibody comprising a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 192, 193, 194, 195 , 196, 197, 208, or 209. In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), the antibody comprising a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 194. In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), wherein the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 195. B In certain embodiments, the invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), wherein the antibody comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 198. In certain embodiments, the invention provides refers to an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), the antibody comprising a light chain constant region containing the amino acid sequence of SEQ ID NO: 219.

In certain embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of an antibody described herein (e.g., the CH2 domain (residues 231-340 of human IgG1) and/or the CH3 domain (residues 341-447 of human IgG1) and/or a hinge region numbered according to the EU numbering system to change one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding and/or antigen- dependent cell cytotoxicity.

In certain embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) such that the number of cysteine residues in the hinge region is changed (e.g., increased or decreased), as described, for example, in US patent No. 5677425. The number of cysteine residues in the hinge region of the CH1 domain can be changed, for example, to facilitate the assembly of light and heavy chains or to change (eg, increase or decrease) the stability of the antibody.

In a specific embodiment, one, two, or more amino acid mutations (e.g., substitutions, insertions, or deletions) are introduced into an IgG constant domain or an FcRn-binding fragment thereof (preferably an Fc domain fragment or an Fc hinge) to alter (e.g., decrease or increase) half-life of the antibody in vivo. See, for example, international publications No. WO 02/060919; WO 98/23289 and WO 97/34631, and US Patent Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745, all of which are incorporated herein by reference in their entirety, for examples of mutations that alter (eg, decrease or increase) the period half-life of an antibody in vivo. In certain embodiments, one, two, or more amino acid mutations (eg, substitutions, insertions, or deletions) are introduced into an IgG constant domain or an FcRn-binding fragment thereof (preferably an Fc domain fragment or an Fc hinge) to reduce the half-life of the antibody in vivo. In other embodiments, one, two, or more amino acid mutations (eg, substitutions, insertions, or deletions) are introduced into the IgG constant domain or FcRn-binding fragment thereof (preferably an Fc domain fragment or Fc hinge) to increase the half-life of the antibody in vivo. In a specific embodiment, the antibodies may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgG1) and/or in the third constant (CH3) domain (residues 341-447 of human IgG1) , are numbered according to the EU numbering system. In a specific embodiment, the constant region of an IgG1 antibody described herein comprises a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 2, 54, and threonine. (T) by replacing glutamic acid (E) at position 256, numbered according to the EU numbering system. See US Pat. No. 7,658,921, which is incorporated herein by reference in its entirety. This type of mutant IgG, called the YTE mutant, has been shown to exhibit a fourfold increase in half-life compared to wild-type versions of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem. 281: 23514-24 , which is incorporated herein by reference in its entirety). In certain embodiments, the antibody comprises an IgG constant domain comprising one, two, three, or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the numbering system EU.

In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of an antibody described herein (e.g., CH2 domain (residues 231-340 of human IgG1) and/or CH3 domain (residues 341-340) 447 human IgG1) and/or hinge region numbered according to the EU numbering system to increase or decrease the affinity of the antibody for an Fc receptor (e.g., activated Fc receptor) on the surface of an effector cell. Mutations in the Fc region of an antibody that decrease or increase the affinity of an antibody for the Fc receptor, and methods for introducing such mutations into the Fc receptor or a fragment thereof are known to one skilled in the art.Examples of mutations in the Fc receptor of an antibody that can be made to alter the affinity of the antibody for the Fc receptor are described, for example, in Smith P et al., (2012) PNAS 109: 6181-6186, US patent No. 6737056, and international publications No. WO 02/060919; wO 98/23289 and WO 97/34631, all of which are included herein by reference in full.

In a further embodiment, one, two or more amino acid substitutions are introduced into the Fc region of the IgG constant domain to alter the effector function(s) of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322, numbered in accordance with the EU numbering system, can be replaced by another amino acid residue such that the antibody has an altered affinity for the effector ligand , but will retain the antigen-binding ability of the parent antibody. The effector ligand for which the affinity is changed may, for example, be an Fc receptor or a complement component C1. This approach is described in more detail in US Patent Nos. 5,624,821 and 5,648,260, each of which is incorporated herein by reference in its entirety. In some embodiments, deletion or inactivation (via point mutations or other means) of the constant region domain can reduce circulating antibody Fc receptor binding, thereby increasing tumor localization. See, for example, US Patent Nos. 5,585,097 and 8,591,886, each of which is incorporated herein by reference in its entirety, for descriptions of mutations that delete or inactivate the constant domain and thereby increase tumor localization. In certain embodiments, one or more amino acid substitutions may be introduced into the Fc region of an antibody described herein to remove potential glycosylation sites in the Fc region that may reduce Fc receptor binding (see, e.g., Shields RL et al., (2001) J Biol Chem 276: 6591-604, which is incorporated herein by reference in its entirety). In various embodiments, one or more of the following mutations may be made in the constant region of an antibody described herein: N297A substitution; replacement N297Q; replacement of L235A and replacement of L237A; replacement of L234A and replacement of L235A; replacement of E233P; replacement L234V; replacement L235A; removal of C236; replacing P238A; replacement D265A; A327Q replacement; or replacement P329A, numbered according to the EU numbering system. In certain embodiments, a mutation selected from the group consisting of D265A, P329A, and combinations thereof may be introduced into the constant region of an antibody described herein.

In a specific embodiment, the antibody described herein contains an IgG1 constant domain with an amino acid substitution N297Q or N297A, numbered in accordance with the EU numbering system. In one embodiment, the antibody described herein contains an IgG1 constant domain with a mutation selected from the group consisting of D265A, P329A, and combinations thereof, numbered according to the EU numbering system. In another embodiment, the antibody described herein contains an IgG1 constant domain with a mutation selected from the group consisting of L234A, L235A, and combinations thereof, numbered according to the EU numbering system. In certain embodiments, the amino acid residues in the constant region of the antibody described herein at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain, numbered according to the EU numbering index, are not L, L and D, respectively. This approach is described in detail in International Publication No. WO 14/108483, which is incorporated herein by reference in its entirety. In a specific embodiment, the amino acids corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A; or A, A and A respectively.

In certain embodiments, one or more amino acids selected from amino acid residues 329, 331, and 322 in the constant region of the antibody described herein, numbered in accordance with the EU numbering system, can be replaced with another amino acid residue such that the antibody has altered C1q binding and /or will reduce or abolish complement dependent cytotoxicity (CDC). This approach is described in more detail in US patent No. 6194551 (Idusogie et al.), which is incorporated herein by reference in its entirety. In some embodiments, one or more amino acid residues at amino acid positions 231 to 238 in the N-terminal region of the CH2 domain of an antibody described herein are altered to thereby alter the ability of the antibody to fix complement. This approach is described in detail in International Publication No. WO 94/29351, which is incorporated herein by reference in its entirety. In certain embodiments, the Fc region of an antibody described herein is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for the Fcy receptor by mutating one or more amino acids (e.g., introducing amino acid substitutions) to the following provisions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289,

290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 328, 329, 330,

331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437,

438 or 439, numbered according to the EU numbering system. This approach is described in detail in International Publication No. WO 00/42072, which is incorporated herein by reference in its entirety.

In certain embodiments, the antibody described herein comprises an IgG ₄ antibody constant region, and the serine at amino acid residue 228 of the heavy chain, numbered according to the EU numbering system, is replaced by proline. In certain embodiments, this invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), wherein the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 196. In certain embodiments, this The invention relates to an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), the antibody comprising a heavy chain constant region containing the amino acid sequence of SEQ ID NO: 197.

In certain embodiments, any mutations or modifications to the constant region described herein can be introduced into one or both heavy chain constant regions of an antibody described herein having two heavy chain constant regions.

In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (eg, human LAG-3) and functions as an antagonist.

In certain embodiments, the present invention provides an isolated antibody,

- 74 044026 which specifically binds to LAG-3 (for example, human LAG-3) and reduces the activity of LAG-3 (for example, human LaG-3) by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98 or 99%, as assessed by methods described herein and/or known to one skilled in the art, relative to LAG- activity 3 (eg, human LAG-3) without any antibody or with an unrelated antibody (eg, an antibody that does not specifically bind to LAG-3 (eg, human LAG-3)).

In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (eg, human LAG-3) and reduces the activity of LAG-3 (eg, human LAG-3) by at least about 1.2, 1 ,3, 1.4, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 , 60, 70, 80, 90, or 100-fold, as assessed by methods described herein and/or known to one skilled in the art, relative to LAG-3 (e.g., human LAG-3) activity without or with any antibody an unrelated antibody (eg, an antibody that does not specifically bind to LAG-3 (eg, human LAG-3)). Non-limiting examples of LAG-3 (e.g., human LAG-3) activity may include LAG-3 (e.g., human LAG-3) signaling, LAG-3 (e.g., human LAG-3) binding to LAG-3 ligand (e.g., LAG -3 humans) (eg, MHC class II), and inhibition of cytokine production (eg, IL-2 and/or TNF-α). In certain embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (eg, human LAG-3) and inactivates, reduces, or inhibits the activity of LAG-3 (eg, human LAG-3). In specific embodiments, the reduction in LAG-3 (eg, human LAG-3) activity is assessed as described in the examples below.

In specific embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (eg, human LAG-3) and reduces the binding of LAG3 (eg, human LAG-3) to its ligand (eg, MHC class II) by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98 or 99%, as estimated by methods described herein (see examples below) or known to one skilled in the art, LAG-3 (eg, human LAG-3) binds to its ligand (eg, MHC class II) without any antibody or with an unrelated antibody (eg, an antibody that does not specifically bind to LAG-3 (eg, human LAG-3)). In specific embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3) and reduces the binding of LAG-3 (e.g., human LAG-3) to its ligand (e.g., MHC class II) at least about 1.2 times, 1.3 times. 1.4 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times, as assessed by methods described herein (see examples below) or known one skilled in the art regarding the binding of LAG-3 (eg, human LAG-3) to its ligand (eg, MHC class II) without any antibody or to an unrelated antibody (eg, an antibody that does not specifically bind LAG-3). 3 (e.g. human LAG-3)).

In specific embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3) and increases cytokine production (e.g., IL-2 and/or TNF-α) by at least about 5. 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98 or 99%, as assessed using the methods described herein document (see examples below) or known to one skilled in the art regarding the production of cytokines without any antibody or with an unrelated antibody (eg, an antibody that does not specifically bind to LAG-3 (eg, human LAG-3)). In specific embodiments, the present invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3) and increases the production of cytokines (e.g., IL-2 and/or TNF-α) by at least about 1. 2 times, 1.3 times. 1.4 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times, as assessed by methods described herein (see examples below) or known one skilled in the art regarding the production of cytokines without any antibody or with an unrelated antibody (eg, an antibody that does not specifically bind to LAG-3 (eg, human LAG-3)).

In specific embodiments, the invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3) and, either alone or in combination with an anti-PD-1 antibody (e.g., pembrolizumab or nivolumab), an anti-PD antibody -L1 (eg, avelumab, durvalumab, or atezolizumab) or anti-CTLA-4 antibody (eg, ipilimumab) increases IL-2 production in human peripheral blood mononuclear cells (PBMCs) in response to stimulation with Staphylococcus enterotoxin A (SEA) by at least about 1.2, 1.3. 1.4, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100-fold, as assessed by methods described herein (see examples below) or known to one skilled in the art for IL-2 production without any antibody or with an unrelated antibody

- 75 044026 (for example, an antibody that does not specifically bind to LAG-3 (for example, human LAG-3)).

In certain embodiments, human peripheral blood mononuclear cells (PBMCs) stimulated with staphylococcal enterotoxin A (SEA) in the presence of an antibody described herein that specifically binds to LAG-3 (e.g., human LAG-3) have increased levels of IL production -2 to at least about 1.2, 1.3. 1.4, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70-, 80-, 90-, or 100-fold, as assessed by methods described herein (see examples below) or known to one skilled in the art, relative to PBMCs stimulated with SEA alone without any antibody or with an unrelated antibody (e.g. an antibody that does not specifically bind to LAG-3 (eg, human LAG-3)) as assessed by methods described herein (see examples below) or known to one skilled in the art.

In specific embodiments, the invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3) and, either alone or in combination with an anti-PD-1 antibody (e.g., pembrolizumab or nivolumab), increases TNFa production in tumor infiltrating lymphocytes (TILs) in response to stimulation by anti-CD3 antibodies and anti-CD28 antibodies in at least about 1.2, 1.3. 1.4, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100-fold, as assessed by methods described herein (see examples below) or known to one skilled in the art for TNFa production without any antibody that specifically binds to LAG-3 ( e.g. human LAG-3). In one embodiment, the TILs are a renal cell carcinoma tumor. In another embodiment, the TILs are a colorectal cancer tumor.

In some embodiments, tumor infiltrating lymphocytes (TILs) stimulated with anti-CD3 and anti-CD28 antibodies in the presence of an antibody described herein that specifically binds to LAG-3 (e.g., human LAG-3) have increased TNFa production at at least about 1.2, 1.3. 1.4, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70-, 80-, 90-, or 100-fold relative to TILs stimulated with anti-CD3 antibodies alone and anti-CD28 antibodies without an antibody that specifically binds to LAG-3 (eg, human LAG-3), as assessed using the methods described herein (see examples below) or known to one skilled in the art. In one embodiment, the TILs are a renal cell carcinoma tumor. In another embodiment, the TILs are a colorectal cancer tumor.

6.3. Pharmaceutical compositions.

Provided herein are compositions (e.g., pharmaceutical compositions) containing an anti-LAG-3 antibody (e.g., human LAG-3) described herein having the desired degree of purity in a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1990) ) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations used and include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

In certain embodiments, the composition comprises one or more antibodies against LAG-3 (e.g., human LAG-3), as described herein, wherein in the antibody portion, the N-terminal amino acid residue(s) of the heavy chain and/or light chain have been converted to pyroglutamate (for example, as a result of post-translational cyclization of the free amino group of the N-terminal residue E or Q). In certain embodiments, the N-terminal amino acid residue of at least 50% (e.g., at least 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99%) of the heavy chains in the composition has been converted to pyroglutamate. In certain embodiments, the N-terminal amino acid residue of no more than 50% (e.g., no more than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, or 40%) of the light chains in the composition was converted to pyroglutamate. In certain embodiments, the N-terminal amino acid residue of at least 50% (e.g., at least 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99%) of the heavy chains in the composition has been converted to pyroglutamate , and the N-terminal amino acid residue is not more than 50% (for example, not more than 1, 2, 3, 4, 5, 10, 15, 20, 25,

- 76 044026 or 40%) of the light chains in the composition were converted to pyroglutamate.

In a specific embodiment, the pharmaceutical compositions comprise an anti-LAG-3 antibody (eg, human LAG-3) described herein and, optionally, one or more additional prophylactic or therapeutic agents in a pharmaceutically acceptable carrier. In a specific embodiment, the pharmaceutical compositions contain an effective amount of an antibody described herein and, optionally, one or more additional prophylactic or therapeutic agents in a pharmaceutically acceptable carrier. In some embodiments, the antibody is the only active ingredient included in the pharmaceutical composition. The pharmaceutical compositions described herein may be useful for inhibiting the activity of LAG-3 (eg, human LAG-3) and treating a condition such as cancer or an infectious disease.

Pharmaceutically acceptable carriers used in parenteral formulations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharmaceutically acceptable agents. Examples of aqueous vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water for injection, dextrose and lactated Ringer's injection. Non-aqueous parenteral vehicles include vegetable fatty oils, cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobial agents at bacteriostatic or fungistatic concentrations may be added to parenteral products packaged in multi-dose containers that include phenols or cresols, mercuric compounds, benzyl alcohol, chlorobutanol esters, methyl and propyl phydroxybenzoic acid, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropylmethylcellulose and polyvinylpyrrolidone. Emulsifying agents include polysorbate 80 (TWEEN® 80). The metal ion sequestering or chelating agent includes EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible carriers; and sodium hydroxide, hydrochloric acid, citric acid or lactic acid to adjust the pH.

The pharmaceutical composition can be formulated for any route of administration to a subject. Specific examples of routes of administration include intranasal, oral, pulmonary, transdermal, intradermal and parenteral.

Parenteral administration, characterized by either subcutaneous, intramuscular or intravenous injection, is also discussed herein. Injectable preparations may be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid before injection, or as emulsions. Injectables, solutions and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. In addition, if desired, the pharmaceutical compositions for administration may also contain minor amounts of non-toxic excipients such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers and other such agents as, for example, sodium, acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.

Preparations for parenteral administration of the antibody include sterile solutions ready for injection, sterile dry soluble products such as lyophilized powders, ready to be combined with a diluent immediately before use, including tablets for subcutaneous injection, sterile suspensions ready for injection, sterile dry insoluble products prepared for use with the carrier immediately before use and sterile emulsions. Solutions may be aqueous or non-aqueous.

For intravenous administration, suitable carriers include physiological saline or phosphate buffered saline (PBS) and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol and polypropylene glycol, and mixtures thereof.

Topical mixtures containing the antibody are prepared as described for topical and systemic administration. The resulting mixture may be a solution, suspension, emulsion or the like and may be formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, irrigants, sprays, suppositories, dressings, skin patches or any other formulations suitable for topical use.

The anti-LAG-3 antibody (e.g., human LAG-3) described herein can be formulated as a topical aerosol, such as by inhalation (see, e.g., US Pat. Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for the delivery of a steroid useful in the treatment of inflammatory diseases, in particular asthma, and incorporated by reference in

- 77 044026 in full). These respiratory compositions may be in the form of an aerosol or nebulizer solution or as a fine powder for insufflation, alone or in combination with an inert carrier such as lactose. In such a case, the drug particles will in one embodiment have diameters less than 50 microns, in one embodiment less than 10 microns.

The anti-LAG-3 (e.g., human LAG-3) antibody described herein can be formulated for topical or topical use, for example, for topical application to the skin and mucous membranes, such as the eyes, in the form of gels, creams, and lotions, and for application to the eye, or for intracisternal or intraspinal use. Topical administration is intended for transdermal delivery, as well as for ocular or mucosal administration or for inhalation therapy. Nasal antibody solutions may also be administered alone or in combination with other pharmaceutically acceptable excipients.

Transdermal patches, including iontophoretic and electrophoretic devices, are well known to those skilled in the art and can be used to administer the antibody. For example, such patches are disclosed in US Pat. Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010,715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957, all of which are incorporated herein by reference in their entirety.

In certain embodiments, the pharmaceutical composition containing the antibody described herein is a lyophilized powder that can be reconstituted for administration as solutions, emulsions, and other mixtures. It can also be reconstituted and formulated as solids or gels. The lyophilized powder is prepared by dissolving the antibody described herein, or a pharmaceutically acceptable derivative thereof, in a suitable solvent. In some embodiments, the lyophilized powder is sterile. The solvent may contain an excipient that improves stability or other pharmacological component of the powder or reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent. The solvent may also contain a buffer, such as citrate, sodium or potassium phosphate, or other such buffer known to those skilled in the art, in one embodiment, at approximately neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those skilled in the art yields the desired composition. In one embodiment, the resulting solution will be distributed into vials for lyophilization. Each vial will contain one dose or multiple doses of the compound. The lyophilized powder can be stored under suitable conditions, for example at about 4°C to room temperature. Reconstitution of this lyophilized powder with water for injection provides a formulation for parenteral use. For reconstitution, the lyophilized powder is added to sterile water or other suitable carrier. The exact amount depends on the selected compound. This amount can be determined experimentally.

The anti-LAG-3 (e.g., human LAG-3) antibodies described herein and other compositions provided herein may also be formulated to target a specific tissue, receptor, or other area of the body of the subject being treated . Many such targeting methods are well known to those skilled in the art. All such targeting methods are contemplated herein for use in instant formulations. For non-limiting examples of targeting methods, see, for example, US Patent Nos. 6316652, 6274552, 6271359, 6253872, 6139865, 6131570, 6120751, 6071495, 6060082, 6048736, 6039975, 6 004534, 5985307, 5972366, 5900252, 5840674, 5759542 and 5709874, all of which are incorporated herein by reference in their entirety. In a specific embodiment, an antibody described herein targets a tumor.

Compositions used for in vivo administration may be sterile. This is easily achieved by filtration through, for example, sterile filter membranes.

6.4. Methods of use and application.

In another aspect, the present invention provides a method of treating a subject using anti-LAG-3 (eg, human LAG-3) antibodies disclosed herein. Any disease or disorder in a subject that would benefit from inhibition of LAG-3 (eg, human LAG-3) function can be treated using anti-LAG-3 (eg, human LAG-3) antibodies described herein. Antibodies against LAG-3 (eg, human LAG-3) disclosed herein are particularly useful for inhibiting immune system resistance to tumors and accordingly can be used as immunotherapy for subjects with cancer. For example, in certain embodiments, the present invention provides a method of increasing T cell activation in response to an antigen in a subject, the method comprising administering to the subject an effective amount of an anti-LAG-3 antibody (e.g., human LAG-3) or a pharmaceutical composition thereof, as described in this document. In certain embodiments, this invention provides a method of treating cancer in a subject, comprising administering to the subject an effective amount of an antibody or pharmaceutical composition as described herein.

Cancers that can be treated with anti-LAG-3 antibodies (e.g., human LAG-3) or pharmaceutical compositions disclosed herein include, but are not limited to, solid tumor, hematologic cancer, leukemia, lymphoma, osteosarcoma, rhabdomyosarcoma, neuroblastoma, kidney cancer, renal transitional cell cancer, bladder cancer, Wilm's cancer, ovarian cancer, pancreatic cancer, breast cancer (eg, characterized by a mutation in BRCA1 and/or BRCA2), prostate cancer, bone cancer, lung cancer (eg, non-small cell cancer lung or small cell lung cancer), stomach cancer, colorectal cancer, cervical cancer, synovial sarcoma, head and neck cancer, squamous cell carcinoma, multiple myeloma, renal cell carcinoma, retinoblastoma, hepatoblastoma, hepatocellular carcinoma, melanoma, renal rhabdoid tumor, sarcoma Ewing's, chondrosarcoma, brain cancer, glioblastoma, meningioma, pituitary adenoma, vestibular schwannoma, primitive neuroectodermal tumor, medulloblastoma, astrocytoma, anaplastic astrocytoma, oligodendroglioma, ependymoma, choroidal papillomas, polycythemia vera, thrombocythemia, idiopathic myelfibrosis, soft tissue sarcoma, thyroid cancer glands, endometrial cancer, carcinoid cancer, liver cancer, epithelial cancer and peritoneal cancer. In certain embodiments, the cancer is a metastatic cancer, such as the varieties described above.

In certain embodiments, the cancer is a solid tumor, hematologic cancer (eg, leukemia, lymphoma, myeloma), and metastatic lesions thereof. In one embodiment, the cancer is a solid tumor. Examples of solid tumors include malignancies such as sarcomas and carcinomas (eg, adenocarcinomas) of various organ systems, such as those affecting the lung, breast, lymphoid, gastrointestinal or colorectal, genital or genitourinary tract (eg, kidney, urothelial, bladder cells) ), pharynx, central nervous system (eg, brain, nerve or glial cells), skin (eg, melanoma), head and neck (eg, head and neck squamous cell carcinoma (HNCC)), and pancreas. For example, melanoma, colon cancer, gastric cancer, rectal cancer, renal cell cancer, breast cancer (for example, breast cancer that does not express one, two or all estrogen receptors, progesterone receptor or Her2/neu, e.g. triple negative breast cancer), liver cancer, lung cancer (eg, non-small cell lung cancer (NSCLC) (eg, NSCLC with squamous cell and/or squamous cell histology) or small cell lung cancer), prostate cancer, head or neck cancer (eg, HPV+ squamous cell carcinoma), small bowel cancer and esophageal cancer.

In one embodiment, the cancer is a hematologic cancer, such as leukemia, lymphoma, or myeloma. In one embodiment, the cancer is a leukemia, for example, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloid leukemia ( CML), chronic myelomonocytic leukemia (CMML), chronic lymphocytic leukemia (CLL) or hairy cell leukemia. In one embodiment, the cancer is a lymphoma, e.g., B cell lymphoma, diffuse large B cell lymphoma (DLBCL), activated B cell (ABC), diffuse large B cell lymphoma, germinal center cell lymphoma (GCL), diffuse large B -cell lymphoma, mantle cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, recurrent non-Hodgkin's lymphoma, refractory non-Hodgkin's lymphoma, recurrent follicular non-Hodgkin's lymphoma, lymphoma

Burkitt's lymphoma, small lymphatic lymphoma, follicular lymphoma, lymphoplastic lymphoma, or extranodal marginal zone lymphoma. In one embodiment, the cancer is myeloma, such as multiple myeloma.

In certain embodiments, the present invention provides a method of preventing or treating an infectious disease in a subject, comprising administering to the subject an effective amount of an anti-LAG-3 antibody (eg, human LAG-3) or a pharmaceutical composition thereof, as disclosed herein. In one embodiment, provided herein are methods of preventing and/or treating an infection (eg, a viral infection, a bacterial infection, a fungal infection, a protozoan infection, or a parasitic infection). The infection prevented and/or treated in accordance with the methods may be caused by an infectious agent identified herein. In a specific embodiment, the anti-LAG-3 antibody (eg, human LAG-3) described herein, or a composition thereof, is the only active agent administered to the subject. In some embodiments, an anti-LAG-3 antibody (eg, human LAG-3) described herein, or a composition thereof, is used in combination with anti-infective interventions (eg, antiviral, antibacterial, antifungal, or anthelminthic agents) to treat infectious diseases.

Infectious diseases that can be treated and/or prevented by antibodies against LAG-3 (e.g., human LAG-3) or pharmaceutical compositions described herein are caused by infectious agents, including, but not limited to, bacteria , parasites, fungi, protozoa and viruses. In a specific embodiment, the infectious disease treated or prevented by an anti-LAG-3 antibody (eg, human LAG-3) or pharmaceutical composition disclosed herein is caused by a virus. Viral diseases or viral infections that can be prevented and/or treated in accordance with the methods described herein include, but are not limited to, diseases caused by hepatitis type A, hepatitis type B, hepatitis type C, influenza (eg, influenza A or influenza B), chickenpox, adenovirus, herpes simplex virus type I (HSV-I), herpes simplex virus type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papillomavirus, papova virus, cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackie virus, mumps virus, measles virus, rubella virus, polio virus, smallpox, Epstein-Barr virus, human immunodeficiency virus type I (HIV-I), human immunodeficiency virus type II (HIV-II) and agents of viral diseases such as viral meningitis, encephalitis, dengue fever or smallpox.

Bacterial infections that can be prevented and/or treated include those caused by Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Proteus vulgaris, Staphylococcus viridans and Pseudomonas aeruginosa. Bacterial diseases caused by bacteria (eg, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Proteus vulgaris, Staphylococcus viridans and Pseudomonas aeruginosa) that can be prevented and/or treated according to the methods described herein include, but are not limited to : Mycobacteria rickettsia, Mycoplasma, Neisseria, S. pneumonia, Borrelia burgdorferi (Lyme disease), Bacillus anthracis (anthrax), tetanus, Streptococcus, Staphylococcus, mycobacteria, whooping cough, cholera, plague, diphtheria, chlamydia, S. aureus and legionella.

Protozoal diseases or protozoal infections caused by protozoa that can be prevented and/or treated in accordance with the methods described herein include, but are not limited to, leishmania, coccidiosis, trypanosome, schistosome or malaria. Parasitic diseases or parasitic infections caused by parasites that can be prevented and/or treated in accordance with the methods described herein include, but are not limited to, chlamydia and rickettsia.

Fungal diseases or fungal infections that can be prevented and/or treated in accordance with the methods described herein include, but are not limited to, Candida infections, zygomycosis, candidal mastitis, progressive disseminated trichosporonosis with latent trichosporonemia, disseminated candidiasis, pulmonary diseases, paracoccidioidomycosis, pulmonary aspergillosis, Pneumocystis pneumonia, cryptococcal meningitis, coccidioidal meningoencephalitis and spinal vasculitis, infections caused by Aspergillus niger, Fusarium keratitis, paranasal sinus mycoses, endocarditis caused by Aspergillus fumigatus, tibial dyschondrosis plasia, vaginitis caused by Candida glabrata, oropharyngeal candidiasis, X-associated chronic granulomatosis, tinea pedis, cutaneous candidiasis, mycotic placentitis, disseminated trichosporonosis, allergic bronchopulmonary aspergillosis, mycotic keratitis, infections caused by Cryptococcus neoformans, fungal peritonitis, infections caused by Curvularia geniculata, staphylococcal endophthalosis mit, sporotrichosis and dermatophytosis.

In certain embodiments, the present invention provides a method of preventing or treating a disease or disorder of the nervous system in a subject, the method comprising administering to the subject an effective amount of an anti-LAG-3 antibody (eg, human LAG-3) or a pharmaceutical composition thereof, as described herein. In some embodiments, the disease or disorder of the nervous system is a synucleinopathy. In some embodiments, the nervous system disease or disorder is Parkinson's disease.

In some embodiments, these methods further comprise administering an additional therapeutic agent to the subject. In certain embodiments, the additional therapeutic agent is a chemotherapeutic agent, a radiotherapeutic agent, or a checkpoint-targeting agent. In certain embodiments, the chemotherapeutic agent is a hypomethylating agent (eg, azacitidine). In certain embodiments, the checkpoint-targeting agent is selected from the group consisting of an anti-CTLA-4 antagonist antibody, an anti-PD-L1 antagonist antibody, an anti-PD-L2 antagonist antibody, an anti-PD-1 antagonist antibody, an anti-TIM-antagonist antibody, 3, anti-LAG-3 antagonist antibody, anti-CEACAM1 antagonist antibody, anti-GITR agonist antibody, anti-OX40 agonist antibody, anti-TIGIT antagonist antibody, anti-CD137 agonist antibody, anti-VISTA antagonist antibody, anti-CD73 antagonist antibody, and anti-CD96 antagonist antibody.

In one embodiment, the present invention provides an antibody and/or pharmaceutical composition of the present invention for use in a method of the present invention, wherein

- 80 044026 the method further includes administering an additional therapeutic agent to the subject. In one embodiment, the present invention relates to (a) an antibody and/or pharmaceutical composition of the present invention and (b) an additional therapeutic agent for use as a drug. In one embodiment, the present invention provides (a) an antibody and/or pharmaceutical composition of the present invention and (b) an additional therapeutic agent for use in a method of treating cancer. In another embodiment, the present invention relates to a pharmaceutical composition, kit or set of parts containing (a) an antibody and/or pharmaceutical composition of the present invention and (b) an additional therapeutic agent. In one embodiment, the additional therapeutic agent is a chemotherapeutic agent, a radiotherapeutic agent, or a checkpoint-targeting agent.

In certain embodiments, an anti-PD-1 antibody is used in the methods disclosed herein. In some embodiments, the anti-PD-1 antibody is nivolumab, also known as BMS-936558 or MDX1106, developed by Bristol-Myers Squibb. In certain embodiments, the anti-PD-1 antibody is pembrolizumab, also known as lambrolizumab or MK-3475, developed by Merck & Co. In certain embodiments, the anti-PD-1 antibody is pidilizumab, also known as CT-011, developed by CureTech. In certain embodiments, the anti-PD-1 antibody is MEDI0680, also known as AMP-514, developed by Medimmune. In certain embodiments, the anti-PD-1 antibody is PDR001 developed by Novartis Pharmaceuticals. In certain embodiments, the anti-PD-1 antibody is REGN2810, developed by Regeneron Pharmaceuticals. In certain embodiments, the anti-PD-1 antibody is PF-06801591 developed by Pfizer. In certain embodiments, the anti-PD-1 antibody is BGB-A317, developed by BeiGene. In certain embodiments, the anti-PD-1 antibody is TSR-042, developed by AnaptysBio and Tesaro. In certain embodiments, the anti-PD-1 antibody is SHR-1210 developed by Hengrui.

Additional non-limiting examples of anti-PD-1 antibodies that can be used in the treatments disclosed herein are disclosed in the following patents and patent applications, all of which are incorporated herein by reference in their entirety: US Patent No. 6,808,710; US Patent No. 7332582; US Patent No. 7488802; US Patent No. 8008449; US Patent No.

8114845; US Patent No. 8168757; US Patent No. 8354509; US Patent No. 8686119; US Patent No.

8735553; US Patent No. 8747847; US Patent No. 8779105; US Patent No. 8927697; US Patent No.

8993731; US Patent No. 9102727; US Patent No. 9205148; US publication No. US 2013/0202623 A1;

US publication No. US 2013/0291136 A1; US publication No. US 2014/0044738 A1; US publication No. US 2014/0356363 A1; US Publication No. US 2016/0075783 A1 and PCT Publication No. WO 2013/033091 A1; PCT publication No. WO 2015/036394 A1; PCT publication No. WO 2014/179664 A2; PCT publication No. WO 2014/209804 A1; PCT publication No. WO 2014/206107 A1; PCT publication No. WO 2015/058573 A1; PCT publication No. WO 2015/085847 A1; PCT publication No. WO 2015/200119 A1; PCT publication No. WO 2016/015685 A1 and PCT publication No. WO 2016/020856 A1.

In certain embodiments, an anti-PD-L1 antibody is used in the methods disclosed herein. In certain embodiments, the anti-PD-L1 antibody is atezolizumab developed by Genentech. In certain embodiments, the anti-PD-L1 antibody is durvalumab developed by AstraZeneca, Celgene and Medimmune. In certain embodiments, the anti-PD-L1 antibody is avelumab, also known as MSB0010718C, developed by Merck Serono and Pfizer. In certain embodiments, the anti-PD-L1 antibody is MDX-1105 developed by Bristol-Myers Squibb. In certain embodiments, the anti-PD-L1 antibody is AMP-224, developed by Amplimmune and GSK.

Non-limiting examples of anti-PD-L1 antibodies that can be used in the treatments disclosed herein are disclosed in the following patents and patent applications, all of which are incorporated herein by reference in their entirety: US Patent No. 7,943,743; US Patent No. 8168179; US Patent No. 8217149; US Patent No. 8552154; US Patent No. 8779108; US Patent No. 8981063; US Patent No. 9175082; US Publication No. US 2010/0203056 A1; US Publication No. US 2003/0232323 A1; US Publication No. US 2013/0323249 A1; US Publication No. US 2014/0341917 A1; US publication No. US 2014/0044738 A1; US Publication No. US 2015/0203580 A1; US Publication No. US 2015/0225483 A1; US Publication No. US 2015/0346208 A1; US Publication No. US 2015/0355184 Al and PCT Publication No. WO 2014/100079 A1; PCT publication No. WO 2014/022758 A1; PCT publication No. WO 2014/055897 A2; PCT publication No. WO 2015/061668 A1; PCT publication No. WO 2015/109124 A1; PCT publication No. WO 2015/195163 A1; PCT publication No. WO 2016/000619 A1 and PCT publication No. WO 2016/030350 A1.

In certain embodiments, an anti-CTLA-4 antibody is used in the methods disclosed herein. In certain embodiments, the anti-CTLA-4 antibody is ipilimumab developed by Bristol-Myers Squibb. In certain embodiments, the anti-CTLA-4 antibody is ipilimumab developed by Bristol-Myers Squibb. In certain embodiments, the anti-CTLA-4 antibody is tremelimumab developed by Pfizer and Medimmune.

Non-limiting examples of anti-CTLA-4 antibodies that can be used in the treatments disclosed herein are disclosed in the following patents and patent applications, all of which are incorporated herein by reference in their entirety: US Patent No. 6,984,720; US Patent No. 7411057; US Patent No. 7034121; US Patent No. 8697845; US publication No. US 2009/0123477 A1; US publication No. US 2014/0105914 A1; US publication No. US 2013/0267688 A1; US publication No. US 2016/0145355 A1; PCT publication No. WO 2014/207064 A1 and PCT publication No. WO 2016/015675 A1.

In certain embodiments, an anti-LAG-3 antibody (e.g., human LAG-3) disclosed herein is administered to a subject in combination with a compound that targets immunomodulatory enzyme(s), such as IDO (indoleamine-(2,3) -dioxygenase) and/or TDO (tryptophan 2,3-dioxygenase). In certain embodiments, such a compound is selected from the group consisting of epacadostat (Incyte Corp; see, for example, WO 2010/005958 which is incorporated herein by reference in its entirety), BMS-986205 (Flexus Biosciences/Bristol-Myers Squibb), indoximod (NewLink Genetics) and NLG919 (NewLink Genetics). In one embodiment, the compound is epacadostat. In another embodiment, the compound is BMS-986205. In another embodiment, the compound is indoximod. In another embodiment, the compound is NLG919. In a specific embodiment, an anti-LAG-3 antibody (eg, human LAG-3) disclosed herein is administered to a subject in combination with an IDO inhibitor for the treatment of cancer. An IDO inhibitor, as described herein for use in the treatment of cancer, is present in a solid dosage form of a pharmaceutical composition, such as a tablet, pill or capsule, the pharmaceutical composition comprising an IDO inhibitor and a pharmaceutically acceptable excipient. As such, the antibody, as described herein, and the IDO inhibitor, as described herein, can be administered separately, sequentially, or simultaneously in separate dosage forms. In one embodiment, the antibody is administered parenterally and the IDO inhibitor is administered orally. In specific embodiments, the inhibitor is selected from the group consisting of epacadostat (Incyte Corporation), BMS-986205 (Flexus Biosciences/Bristol-Myers Squibb), indoximod (NewLink Genetics) and NLG919 (NewLink Genetics). Epacadostat is described in PCT publication No. WO 2010/005958, which is incorporated herein by reference in its entirety. In one embodiment, the inhibitor is epacadostat. In another embodiment, the inhibitor is BMS-986205. In another embodiment, the inhibitor is indoximod. In another embodiment, the inhibitor is NLG919.

In certain embodiments, the invention provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3) in combination with an anti-PD-1 antibody (e.g., pembrolizumab or nivolumab) and an indoleamine 2,3-dioxygenase inhibitor (IDO). In certain embodiments, the anti-PD-1 antibody is pembrolizumab. In certain embodiments, the anti-PD-1 antibody is nivolumab. In certain embodiments, the IDO inhibitor is selected from the group consisting of epacadostat, BMS-986205, indoximod, and NLG919. In certain embodiments, the IDO inhibitor is epacadostat. In certain embodiments, the IDO inhibitor is BMS-986205. In certain embodiments, the IDO inhibitor is indoximod.

In certain embodiments, an anti-LAG-3 antibody (eg, human LAG-3) disclosed herein is administered to a subject in combination with another anticancer agent. Typical anticancer agents include antibodies such as trastuzumab (Herceptin), antibodies to costimulatory or coinhibitory molecules such as CTLA-4, CD137 and PD-1, and antibodies to cytokines such as IL-10 and TGF-β.

In certain embodiments, the additional therapeutic agent is a JAK, PI3Kdelta, BRD, PI3Kgamma, or Axl/Mer inhibitor. In certain embodiments, the additional therapeutic agent is a JAK inhibitor, including JAK1 and/or JAK2. In certain embodiments, the additional therapeutic agent is a PI3Kdelta inhibitor. In certain embodiments, the additional therapeutic agent is a BRD inhibitor. In certain embodiments, the additional therapeutic agent is a PI3Kgamma inhibitor. In certain embodiments, the additional therapeutic agent is an Axl/Mer inhibitor.

Additional examples of anticancer agents include those that block the migration of immune cells, such as antagonists of chemokine receptors, including CCR2 and CCR4, and those that enhance the immune system, such as adjuvants or adoptive transfers of T cells.

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One or more additional immune checkpoint modulators can be used in combination with an anti-LAG-3 antibody (e.g., human LAG-3) disclosed herein to treat any of the diseases, disorders, or conditions described herein, e.g. or pathological conditions associated with TAM. Typical immune checkpoint modulators include modulators against immune checkpoint molecules such as CD27, CD28, CD40, CD122, CD96, CD73, CD47, CD96, CD137, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase , CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG-3, TIM-3, TIGIT, VISTA, PD-1, PD-L1 and PD -L2. In some embodiments, the immune checkpoint molecule is a co-stimulatory checkpoint molecule selected from CD27, CD28, CD40, iCoS, OX40, GITR, and CD137. In some embodiments, the immune checkpoint molecule is a coinhibitory checkpoint molecule selected from A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, PD-1, TIM-3, and VISTA. In some embodiments, an anti-LAG-3 antibody (e.g., human LAG-3) disclosed herein may be used in combination with one or more agents selected from KIR inhibitors, TIGIT inhibitors, LAIR1 inhibitors, CD160 inhibitors, 2B4 inhibitors, and TGFR beta inhibitors.

In some embodiments, the immune checkpoint molecule modulator is an anti-PD1 antagonist antibody, an anti-PD-L1 antagonist antibody, or an anti-CTLA-4 antagonist antibody.

In some embodiments, the immune checkpoint molecule modulator is a GITR agonist, such as an anti-GITR agonist antibody. In some embodiments, the anti-GITR agonist antibody is TRX518 or MK-4166.

In some embodiments, the immune checkpoint molecule modulator is an OX40 agonist, such as an anti-OX40 agonist antibody or an OX40L fusion protein. In some embodiments, the anti-OX40 agonistic antibody is MEDI0562. In some embodiments, the OX40L fusion protein is MEDI6383.

An anti-LAG-3 (eg, human LAG-3) antibody described herein can be used in combination with one or more agents to treat diseases such as cancer. In some embodiments, the agent is an alkylating agent, a proteasome inhibitor, a corticosteroid, or an immunomodulatory agent. Examples of the alkylating agent include cyclophosphamide (CY), melphalan (MEL) and bendamustine. In some embodiments, the proteasome inhibitor is carfilzomib. In some embodiments, the corticosteroid is dexamethasone (DEX). In some embodiments, the immunomodulatory agent is lenalidomide (LEN) or pomalidomide (POM).

In certain embodiments, an anti-LAG-3 antibody (eg, human LAG-3) disclosed herein is administered to a subject in combination with a vaccine. The vaccine may be, for example, a peptide vaccine, a DNA vaccine or an RNA vaccine. In certain embodiments, the vaccine is a heat shock protein tumor vaccine or a heat shock protein pathogen vaccine. In a specific embodiment, an anti-LAG-3 antibody (eg, human LAG-3) disclosed herein is administered to a subject in combination with a heat shock protein-based tumor vaccine. Heat shock proteins (HSPs) are a family of highly conserved proteins found ubiquitously across all species. Their expression can be strongly induced to much higher levels by heat shock or other forms of stress, including exposure to toxins, oxidative stress, or glucose deprivation. Five families have been classified by molecular weight: HSP-110, -90, -70, -60 and -28. HSPs deliver immunogenic peptides through cross-presentation pathway to antigen presenting cells (APCs) such as macrophages and dendritic cells (DCs), leading to T cell activation. HSPs function as chaperone carriers of tumor-associated antigenic peptides, forming complexes capable of inducing tumor-specific immunity. Once released from dying tumor cells, HSP-antigen complexes are taken up by antigen presenting cells (APCs), in which the antigens are converted into peptides that bind MHC class I and class II molecules, resulting in activation of antitumor CD8+ and CD4+ T cells. Immunity induced by tumor-derived HSP complexes is specifically directed against the unique antigenic peptide repertoire expressed by each subject's cancer. Therefore, in one embodiment, the present invention relates to (a) an antibody and/or pharmaceutical composition of the present invention and (b) a vaccine for use as a medicine, for example, for use in a method of treating cancer. In one embodiment, the present invention relates to a pharmaceutical composition, kit or set of parts containing (a) an antibody and/or pharmaceutical composition of the present invention and (b) a vaccine. In one embodiment, the vaccine is a heat shock protein-based antitumor vaccine. In one embodiment, the vaccine is an anti-pathogen vaccine

- 83 044026 based on heat shock protein.

Heat shock protein peptide complex (HSPPC) is a protein peptide complex consisting of heat shock protein non-covalently complexed with antigenic peptides. HSPPCs trigger both innate and adaptive immune responses. In a specific embodiment, the antigenic peptide(s) exhibit antigenicity to the cancer being treated. HSPPCs are efficiently captured by APCs through membrane receptors (mainly CD91) or by binding to Toll-like receptors. Internalization of HSPPC leads to the functional maturation of APCs with the production of chemokines and cytokines, leading to the activation of natural killer (NK) cells, monocytes, and Th1 and Th-2-mediated immune responses. In certain embodiments, the HSPPCs used in the methods disclosed herein comprise one or more heat shock proteins from the hsp60, hsp70, or hsp90 family of stress proteins complexed with antigenic peptides. In certain embodiments, HSPPCs include hsc70, hsp70, hsp90, hsp110, grp170, gp96, calreticulin, or combinations of two or more of these.

In a specific embodiment, the heat shock protein peptide complex (HSPPC) comprises recombinant heat shock proteins (eg, hsp70 or hsc70) or a peptide binding domain thereof complexed with recombinant antigenic peptides. Recombinant heat shock proteins can be produced using recombinant DNA technology, for example, using the sequence of human hsc70, as described in Dworniczak and Mirault, Nucleic Acids Res. 15:5181-5197 (1987) and GenBank accession number P11142 and/or Y00371, each of which is incorporated herein by reference in its entirety. In certain embodiments, the Hsp70 sequences are as described in Hunt and Morimoto Proc. Natl. Acad. Sci. U.S.A. 82 (19), 6455-6459 (1985) and GenBank accession number P0DMV8 and/or M11717, each of which is incorporated herein by reference in its entirety. Antigenic peptides can also be produced by recombinant DNA methods known in the art.

In certain embodiments, the antigenic peptides contain a modified amino acid. In certain embodiments, the modified amino acid comprises a post-translational modification. In certain embodiments, the modified amino acid comprises a post-translational modification mimetic. In certain embodiments, the modified amino acid is a Tyr, Ser, Thr, Arg, Lys, or His that has been phosphorylated on a hydroxyl or amine side chain. In certain embodiments, the modified amino acid is a mimetic of a Tyr, Ser, Thr, Arg, Lys, or His amino acid that has been phosphorylated on a hydroxyl or amine side chain.

In a specific embodiment, an anti-LAG-3 antibody (e.g., human LAG-3) disclosed herein is administered to a subject in combination with a heat shock protein peptide complex (HSPPC), e.g., heat shock protein peptide complex-96 (HSPPC-96 ), for the treatment of cancer. HSPPC-96 contains a 96 kDa heat shock protein (Hsp), dr9b, which forms a complex with antigenic peptides. HSPPC-96 is a cancer immunotherapy made from a subject's tumor and contains the cancer's antigenic fingerprint. In some embodiments, the fingerprint contains unique antigens that are present only on specific cancer cells of that particular subject, and the vaccine injection is designed to stimulate the subject's immune system to recognize and attack any cells with the specific cancer fingerprint. Therefore, in one embodiment, the present invention provides an antibody and/or pharmaceutical composition of the present invention in combination with a heat shock protein peptide complex (HSPPC) for use as a drug and/or for use in a method of treating cancer.

In certain embodiments, HSPPC, for example, HSPPC-96, is obtained from tumor tissue of a subject. In a specific embodiment, HSPPC (eg, HSPPC-96) is obtained from the tumor type of cancer or metastasis thereof being treated. In another specific embodiment, the HSPPC (eg, HSPPC-96) is autologous to the subject being treated. In certain embodiments, the tumor tissue is necrotic tumor tissue. In certain embodiments, at least 1 g (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 grams) of necrotic tumor tissue is used to prepare the vaccination regimen. In certain embodiments, following surgical resection, tissue other than necrotic tumor is frozen prior to use in the vaccine formulation. In some embodiments, HSPPC, such as HSPPC96, is isolated from tumor tissue by purification methods, filtered, and prepared for an injectable vaccine. In certain embodiments, the subject is administered 6-12 doses of HSPPC, such as HSPCC-96. In such embodiments, doses of HSPPC, for example, HSPPC-96, can be administered weekly for the first 4 doses, and then biweekly for 2-8 additional doses.

Additional examples of HSPPC that may be used in accordance with the methods described herein are disclosed in the following patents and patent applications, all of which are incorporated herein by reference in their entirety: US Patent Nos. 6,391,306, 6,383,492,

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6403095, 6410026, 6436404, 6447780, 6447781 and 6610659, all of which are incorporated herein by reference in their entirety.

In certain embodiments, an anti-LAG-3 antibody disclosed herein is administered to a subject in combination with an adjuvant. Various adjuvants may be used depending on the treatment context.

Non-limiting examples of suitable adjuvant include, but are not limited to, Complete Freund's Adjuvant (CFA), Incomplete Freund's Adjuvant (IFA), Montanide ISA (Incomplete Adjuvant Septic), Ribi Adjuvant System (RAS), Titer Max, Muramyl Peptides, Syntex Adjuvant Formulation (SAF), alum (aluminum hydroxide and/or aluminum phosphate), aluminum salt adjuvants, Gerbu® adjuvants, nitrocellulose absorbed antigen, encapsulated or captured antigen, 3 De-O-acylated monophosphoryl lipid A (3 D-MPL) immunostimulatory oligonucleotides , toll-like receptor (TLR) ligands, mannan-binding lectin (MBL) ligands, STING agonists, immunostimulatory complexes such as saponins, Quil A, QS-21, QS-7, ISCOMATRIX and others. Other adjuvants include CpG oligonucleotides and double-stranded RNA molecules such as poly(A) and poly(U). Combinations of the above adjuvants can also be used. See, for example, US Pat. No. 6,645,495; 7029678 and 7858589, all of which are incorporated herein by reference in their entirety. In one embodiment, the adjuvant used herein is QS-21 STIMULON.

In certain embodiments, an anti-LAG-3 antibody disclosed herein is administered to a subject in combination with an additional therapeutic agent comprising a TCR. In certain embodiments, the additional therapeutic agent is a soluble TCR. In certain embodiments, the additional therapeutic agent is a cell expressing a TCR. Therefore, in one embodiment, the present invention provides an antibody and/or pharmaceutical composition of the present invention in combination with an additional therapeutic agent, including a TCR, for use as a drug and/or for use in a method of treating cancer.

In certain embodiments, an anti-LAG-3 antibody disclosed herein is administered to a subject in combination with a cell expressing a chimeric antigen receptor (CAR). In certain embodiments, the cell is a T cell.

In certain embodiments, an anti-LAG-3 antibody disclosed herein is administered to a subject in combination with a TCR-mimicking antibody. In certain embodiments, the TCR-mimicking antibody is an antibody that specifically binds to the peptide-MHC complex. For non-limiting examples of TCR-mimicking antibodies, see, for example, US Pat. No. 9,074,000 and US Publication No. US 2009/0304679 A1 and US 2014/0134191 A1, all of which are incorporated herein by reference in their entirety.

Anti-LAG-3 antibody (eg, human LAG-3) and an additional therapeutic agent (eg, chemotherapy, radiotherapy, checkpoint-targeting agent, IDO inhibitor, vaccine, adjuvant, soluble TCR, TCR-expressing cell, chimeric-expressing cell antigen receptor and/or TCR-mimicking antibody) may be administered separately, sequentially, or simultaneously in separate dosage forms. In one embodiment, an anti-LAG-3 antibody (eg, human LAG-3) is administered parenterally and the IDO inhibitor is administered orally.

The antibody or pharmaceutical composition described herein can be delivered to a subject in a variety of ways. These include, but are not limited to, parenteral, intranasal, intratracheal, oral, intradermal, topical, intramuscular, intraperitoneal, transdermal, intravenous, intratumoral, conjunctival, intraarterial, and subcutaneous routes. Pulmonary administration can also be used, for example, using an inhaler or nebulizer and a composition with an aerosol agent for use as a spray. In certain embodiments, the antibody or pharmaceutical composition described herein is delivered subcutaneously or intravenously. In certain embodiments, an antibody or pharmaceutical composition described herein is delivered intratumorally. In certain embodiments, an antibody or pharmaceutical composition described herein is delivered intra-arterially. In certain embodiments, an antibody or pharmaceutical composition described herein is delivered to a lymph node draining a tumor. In certain embodiments, an antibody or pharmaceutical composition described herein is delivered intranasally.

The amount of antibody or composition that will be effective in treating and/or preventing the condition will depend on the nature of the disease and can be determined by standard clinical techniques.

The exact dosage to be used in the composition will also depend on the route of administration and the severity of the infection or disease it causes, and should be selected in accordance with the judgment of the practitioner and the circumstances of each subject. For example, effective doses may also vary depending on the route of administration, the target site, the physiological condition of the patient (including age, body weight and health status), whether the patient is human or animal, other drugs administered, or whether whether the treatment is preventive or therapeutic. Typically the patient is a human, but non-human mammals, including transgenic mammals, may also be treated. Treatment doses are optimally adjusted to optimize safety and effectiveness.

The anti-LAG-3 (e.g., human LAG-3) antibody described herein can also be used to analyze LAG-3 (e.g., human LAG-3) protein levels in a biological sample using classical immunohistological techniques known to those skilled in the art. , including immunoassays such as enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, or Western blotting. Suitable labels for antibody assays are known in the art and include enzyme labels such as glucose oxidase; radioisotopes such as iodine ( ¹²⁵ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹²¹ In) and technetium ( ⁹⁹ Tc); luminescent labels such as luminol and fluorescent labels such as fluorescein and rhodamine, and biotin. Such labels can be used to label the antibodies described herein. Alternatively, a second antibody that recognizes an anti-LAG-3 antibody (e.g., human LAG-3) described herein can be labeled and used in combination with an anti-LAG-3 antibody (e.g., human LAG-3) antibody to detect LAG-3 protein levels (eg human LAG-3). Therefore, in one embodiment, the present invention relates to the use of an antibody of the present invention for the in vitro detection of LAG-3 protein (eg, human LAG-3 protein) in a biological sample. In a further embodiment, the present invention relates to the use of an anti-LAG-3 antibody of the invention to analyze and/or detect levels of LAG-3 protein (e.g., human LAG3) in a biological sample in vitro, optionally when the anti-LAG-3 antibody is conjugated with a radionuclide or a detectable label, and/or carries a label described herein, and/or where an immunohistological method is used.

Analysis of the expression level of a LAG-3 protein (e.g., human LAG-3) is intended to involve qualitatively or quantitatively measuring or assessing the level of a LAG-3 protein (e.g., human LAG-3) in a first biological body, either directly (e.g., by determining or assessing absolute protein level), or relative (for example, by comparison with the disease-associated protein level in a second biological sample). The expression level of a LAG-3 polypeptide (e.g., human LAG-3) in a first biological sample may be measured or assessed and compared to a standard level of LAG-3 protein (e.g., human LAG-3), the standard being taken from a second biological sample obtained from individual without the disorder, or determined by averaging levels from a population of individuals without the disorder. As will be understood in the art, when a standard level of LAG-3 polypeptide (eg, human LAG-3) is known, it can be repeatedly used as a reference standard. Therefore, in a further embodiment, the present invention relates to an in vitro method for analyzing and/or detecting levels of LAG-3 protein, for example levels of human LAG-3 protein, in a biological sample, comprising qualitatively or quantitatively measuring or estimating the level of LAG-3 protein, for example, human LAG-3 protein, in a biological sample by immunohistological method.

As used herein, the term biological sample refers to any biological sample obtained from a subject, cell line, tissue, or other source of cells potentially expressing LAG-3 (eg, human LAG-3). Methods for obtaining biopsies of tissues and biological fluids from animals (eg, humans) are well known in the art. Biological samples include peripheral blood mononuclear cells.

The anti-LAG-3 antibody described herein (e.g., human LAG-3) can be used for prognostic, diagnostic, monitoring and screening applications, including in vitro and in vivo applications well known and standard to those skilled in the art. techniques, and based on the description of this invention. Prognostic, diagnostic, monitoring and screening assays and kits for in vitro assessment and evaluation of the immune system and/or immune response can be used for prognosis, diagnosis and monitoring to evaluate patient samples, including those known to have or are suspected of having for the presence of immune system dysfunction or in relation to an expected or desired immune system response, response to an antigen or response to a vaccine. Assessing and determining the status of the immune system and/or immune response is also useful in determining a patient's suitability for a clinical drug trial or for administration of a particular chemotherapeutic agent, radiotherapeutic agent or antibody, including combinations thereof, as opposed to another agent or antibody. This type of prognostic and diagnostic monitoring and evaluation is already in practice using antibodies against the HER2 protein in breast cancer (HercepTest™, Dako), where the assay is also used to evaluate patients for antibody therapy using Herceptin®. In vivo applications include targeted cell therapy and modulation of the immune system, as well as

- 86 044026 visualization of immune responses. Thus, in one embodiment, the present invention provides an anti-LAG-3 antibody and/or a pharmaceutical composition of the present invention for use as a diagnostic agent. In one embodiment, the present invention relates to an anti-LAG-3 antibody and/or a pharmaceutical composition of the present invention for use in a method of predicting, diagnosing and/or monitoring a subject having or suspected of having immune system dysfunction and/or expected or desired immune system response, antigen response, or vaccine response. In another embodiment, the invention relates to the use of an anti-LAG-3 antibody of the invention for predicting, diagnosing and/or monitoring a subject having or suspected of having immune system dysfunction and/or an expected or desired immune system response, response to an antigen or response to a vaccine by analyzing and/or detecting LAG-3 protein levels in a biological sample of the subject in vitro.

In one embodiment, an anti-LAG-3 antibody (eg, human LAG-3) can be used in immunohistochemistry of biopsy samples. In another embodiment, an anti-LAG-3 (e.g., human LAG-3) antibody can be used to determine levels of LAG-3 (e.g., human LAG-3) or levels of cells that contain LAG-3 (e.g., human LAG-3 ) on their membrane surface, levels of which can then be associated with specific disease symptoms. Anti-LAG-3 (eg, human LAG-3) antibodies described herein may be detectably or functionally labeled. When fluorescent labels are used, currently available microscopy and fluorescence-activated cell sorting (FACS) analysis, or a combination of both methods known in the art, can be used to identify and quantify specific binding elements. Anti-LAG-3 (eg, human LAG-3) antibodies described herein may be fluorescently labeled. Typical fluorescent labels include, for example, reactive and conjugated probes such as aminocoumarin, fluorescein and Texas Red, Alexa Fluor dyes, Cy dyes and DyLight dyes. An anti-LAG-3 antibody (eg, human LAG-3) may carry a radiolabel such as the isotopes ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁶⁷ Cu , ⁹⁰ Y, ⁹⁹ Tc, ¹ⁿ In, ¹¹⁷ Lu, ¹²¹ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ¹⁹⁸ Au, ²¹¹ At, ²¹³ Bi, ²²⁵ Ac and ¹⁸⁶ Re. When radioactive labels are used, currently available counting procedures known in the art can be used to identify and quantify the specific binding of an anti-LAG-3 antibody (e.g., human LAG-3) to LAG-3 (e.g., LAG-3 person). In the case where the label is an enzyme, detection can be performed by any of the currently used colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric methods known in the art. This can be achieved by contacting the sample or control with an anti-LAG-3 antibody (eg, human LAG-3) under conditions that allow complex formation between the antibody and LAG-3 (eg, human LAG-3). Any complexes formed between the antibody and LAG-3 (eg, human LAG-3) are detected and compared in the sample and control. In light of the specific binding of the antibodies described herein for LAG-3 (eg, human LAG-3), the antibodies can be used to specifically detect the expression of LAG-3 (eg, human LAG-3) on the surface of cells. The antibodies described herein can also be used to purify LAG-3 (eg, human LAG-3) using immunoaffinity purification. Also included herein is an assay system that can be formulated as a test kit to quantify the presence of, e.g., LAG-3 (e.g., human LAG-3) or LAG-3 ligand complexes (e.g., human LAG-3) /LAG-3 (for example, human LAG-3). The system or test kit, kit, or set of parts may contain a labeled component, such as a labeled antibody, and one or more additional immunochemical reagents.

6.5. Polynucleotides, vectors and methods for producing antibodies against LAG-3.

In another aspect, provided herein are polynucleotides comprising a nucleotide sequence encoding an antibody described herein, or a fragment thereof (e.g., a light chain variable region and/or a heavy chain variable region) that specifically binds to LAG-3 antigen and vectors (eg, human LAG-3), for example, vectors containing such polynucleotides for recombinant expression in host cells (eg, E. coli and mammalian cells). Provided herein are polynucleotides containing nucleotide sequences encoding the heavy and/or light chain of any of the antibodies provided herein, as well as vectors containing such polynucleotide sequences, such as expression vectors for efficient expression thereof in host cells, e.g. in mammalian cells.

As used herein, the term isolated polynucleotide or nucleic acid molecule is a molecule that is separated from other nucleic acid molecules that are present in a natural source (eg, mouse or human) of the nucleic acid molecule. In addition, an isolated nucleic acid molecule, such as a cDNA molecule, may

- 87 044026 be substantially free of other cellular material or culture medium when produced by recombinant methods, or substantially free of chemical precursors or other chemicals when chemically synthesized. For example, the expression essentially free includes preparations of polynucleotide or nucleic acid molecules having less than about 15, 10, 5, 2, 1, 0.5 or 0.1% (especially less than about 10%) of other material, e.g. cellular material, culture medium, other nucleic acid molecules, chemical precursors and/or other chemicals. In a specific embodiment, the nucleic acid molecule(s) encoding an antibody described herein is isolated or purified.

In specific aspects, provided herein are polynucleotides comprising nucleotide sequences encoding antibodies that specifically bind to a LAG-3 polypeptide (e.g., human LAG-3) and contain an amino acid sequence as described herein, as well as antibodies that compete with such antibodies to bind to a LAG-3 (eg, human LAG-3) polypeptide (eg, dose dependent) or that binds to the same epitope as such antibodies.

In certain aspects, provided herein are polynucleotides comprising a nucleotide sequence encoding the light chain or heavy chain of an antibody described herein. The polynucleotides may contain nucleotide sequences encoding a light chain containing the FR VL and CDR of the antibodies described herein (see, for example, Tables 1, 3, 5, 6 and 7), or nucleotide sequences encoding a heavy chain containing the FR VH and CDR of the antibodies described herein (see, for example, Tables 1, 2, 4, 6 and 7).

Also provided herein are polynucleotides encoding an anti-LAG-3 antibody (eg, human LAG-3) that are optimized, for example, by codon/RNA optimization, replacement with heterologous signal sequences, and removal of mRNA instability elements. Methods for producing optimized nucleic acids encoding an anti-LAG3 antibody (e.g., human LAG-3) or a fragment thereof (e.g., light chain, heavy chain, VH domain, or VL domain) for recombinant expression by introducing codon changes and/or eliminating inhibitory regions in mRNA can be implemented by adapting optimization methods described, for example, in US patent No. 5965726; 6174666; 6291664; 6414132; and 6,794,498, respectively, all of which are incorporated herein by reference in their entirety. For example, potential splice sites and instability elements (eg, A/T-rich or A/U-rich elements) in RNA can be mutated without changing the amino acids encoded by the nucleic acid sequences to increase the stability of the RNA for recombinant expression. The changes exploit degeneracy in the genetic code, such as the use of an alternative codon for an identical amino acid. In some embodiments, it may be desirable to change one or more codons to encode a conservative mutation, for example, a similar amino acid with similar chemical structure and properties and/or function as the parent amino acid. Such methods can increase the expression of an anti-LAG-3 antibody (e.g., human LAG-3) or fragment thereof by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times or more relative to anti-LAG-3 antibody expression (e.g., human LAG-3) , encoded by polynucleotides that have not been optimized.

In certain embodiments, an optimized polynucleotide sequence encoding an anti-LAG-3 antibody (e.g., human LAG-3) described herein, or a fragment thereof (e.g., a VL domain and/or a VH domain) may hybridize to an antisense polynucleotide (e.g., complementary) to a non-optimized polynucleotide sequence encoding an anti-LAG-3 antibody (eg, human LAG-3) described herein, or a fragment thereof (eg, VL domain and/or VH domain). In specific embodiments, an optimized nucleotide sequence encoding an anti-LAG-3 antibody (e.g., human LAG-3) described herein or fragment hybridizes under high stringency conditions to an antisense polynucleotide of a non-optimized polynucleotide sequence encoding an anti-LAG-3 antibody (e.g. , human LAG-3) described herein, or a fragment thereof. In a specific embodiment, an optimized nucleotide sequence encoding an anti-LAG-3 antibody (e.g., human LAG-3) described herein, or a fragment thereof, is hybridized under high stringency, intermediate, or lower stringency hybridization conditions to an antisense polynucleotide of the non-optimized nucleotide sequence , encoding an anti-LAG-3 antibody (eg, human LAG-3) described herein, or a fragment thereof. Information regarding hybridization conditions has been described, see, for example, US Patent Application Publication No. US 2005/0048549 (eg, claims 72-73), which is incorporated herein by reference in its entirety.

Polynucleotides can be prepared and the nucleotide sequence of the polynucleotides can be determined by any method known in the art. Nucleotide sequences encoding the antibodies described herein, for example, the antibodies described in table. 1, 6 and 7, and modified versions of these antibodies can be determined using methods well known in the art, that is, nucleotide codons that are known to encode specific amino acids are assembled in such a way to generate nucleic acid , which codes for an antibody.

Such an antibody-encoding polynucleotide can be assembled from chemically synthesized oligonucleotides (for example, as described in Kutmeier G et al., (1994) BioTechniques 17:242-6, which is incorporated herein by reference in its entirety), which is summarized , involves the synthesis of overlapping oligonucleotides containing sections of the antibody coding sequence, annealing and ligation of these oligonucleotides, and then amplification of the ligated oligonucleotides using PCR.

Alternatively, a polynucleotide encoding an antibody described herein can be obtained from nucleic acid from a suitable source (eg, a hybridoma) using methods well known in the art (eg, PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers that hybridize to the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells producing the antibody of interest. Such PCR amplification methods can be used to produce nucleic acids containing the sequence encoding the light chain and/or heavy chain of an antibody. Such PCR amplification methods can be used to produce nucleic acids containing a sequence encoding a light chain variable region and/or a heavy chain variable region of an antibody.

The amplified nucleic acids can be cloned into vectors for expression in host cells and for further cloning, for example, to produce chimeric and humanized antibodies.

If a clone containing the nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, the nucleic acid encoding the immunoglobulin can be chemically synthesized or obtained from a suitable source (for example, an antibody cDNA library or a cDNA library generated from the nucleic acid or, preferably , polyA+RNA isolated from any tissue or cells expressing an antibody, such as hybridoma cells selected to express the antibody described herein), by PCR amplification using synthetic primers hybridized at the 3' and 5' ends sequence or by cloning using an oligonucleotide probe specific for a particular gene sequence to identify, for example, a cDNA clone from a cDNA library that encodes an antibody. The amplified nucleic acids obtained by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

DNA encoding the anti-LAG-3 antibodies (e.g., human LAG-3) described herein can be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that are capable of specifically binding to genes encoding severe and anti-LAG-3 antibody light chains (e.g. human LAG-3).Hybridoma cells can serve as a source of such DNA.Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells, such as E. coli cells, cells Monkey COS, Chinese hamster ovary (CHO) cells (eg, CHO cells from the GS™ CHO system (Lonza)) or myeloma cells that do not otherwise produce immunoglobulin protein to produce antibodies against LAG-3 (eg, LAG-3 human) in recombinant host cells.

To generate whole antibodies, PCR primers comprising VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to amplify VH or VL sequences in scFv clones. Using cloning techniques known to those skilled in the art, PCR-amplified VH domains can be cloned into vectors expressing a heavy chain constant region, such as the human gamma-4 constant region, and PCR-amplified VL domains can be cloned into vectors expressing a heavy chain constant region, such as the human gamma-4 constant region. expressing a light chain constant region, such as human kappa or lambda constant regions. In certain embodiments, the VH or VL domain expression vectors contain an EF-1a promoter, a secretion signal, a cloning site for the variable region, constant domains, and a selection marker such as neomycin. The VH and VL domains can also be cloned into a single vector expressing the necessary constant regions. The heavy chain conversion vectors and the light chain conversion vectors are then co-transfected into cell lines to generate stable or transient cell lines that express full-length antibodies, such as IgG, using methods known to those skilled in the art.

The DNA may also be modified, for example, by replacing the coding sequence for the human heavy and light chain constant domains instead of the murine sequences, or by covalently attaching all or

- 89 044026 part of the coding sequence for the non-coding sequence of an immunoglobulin polypeptide.

Also provided are polynucleotides that hybridize under high stringency, medium or low stringency hybridization conditions with polynucleotides that encode an antibody described herein. In specific embodiments, the polynucleotides described herein hybridize under high stringency, intermediate, or lower stringency hybridization conditions with polynucleotides encoding the VH domain and/or VL domain provided herein.

Hybridization conditions have been described in the art and are known to one skilled in the art. For example, hybridization under stringent conditions may involve hybridization with filter-bound DNA in 6x sodium chloride/sodium citrate (SSC) at about 45°C followed by one or more washes in 0.2xSSC/0.l% SDS at about 50-65°C; hybridization under very stringent conditions may include hybridization with the filter-bound nucleic acid in 6xSSC at about 45°C followed by one or more washes in 0.1xSSC/0.2% SDS at about 68°C. Hybridization under other stringent hybridization conditions is known to those who have been described, see, for example, Ausubel FM et al., eds., (1989) Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York on pages 6.3.1 through 6.3.6 and 2.10.3, which are incorporated herein by reference in their entirety.

In certain aspects, provided herein are cells (e.g., host cells) expressing (e.g., recombinant) antibodies described herein that specifically bind to LAG-3 (e.g., human LAG-3) and related polynucleotides and expression vectors . Provided herein are vectors (eg, expression vectors) containing polynucleotides containing nucleotide sequences encoding antibodies against LAG-3 (eg, human LAG-3) or fragment for recombinant expression in host cells, preferably mammalian cells. Also provided herein are host cells containing such vectors for the recombinant expression of anti-LAG-3 antibodies (eg, human LAG-3) described herein (eg, human or humanized antibodies). In a specific aspect, this document provides methods for producing an antibody described herein, comprising expressing such antibody from a host cell.

Recombinant expression of an antibody described herein (e.g., a full-length antibody, a heavy and/or light chain antibody, or a single chain antibody described herein) that specifically binds to LAG-3 (e.g., human LAG-3) involves constructing an expression vector containing a polynucleotide encoding an antibody. Once a polynucleotide encoding an antibody molecule, an antibody heavy and/or light chain, or a fragment thereof (e.g., heavy and/or light chain variable regions) described herein has been obtained, a vector for producing the antibody molecule can be produced using recombinant technology DNA using methods well known in the art. Thus, this document describes methods for producing a protein by expressing a polynucleotide containing an antibody or antibody fragment (eg, light chain or heavy chain) encoding a nucleotide sequence. Methods that are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding an antibody or antibody fragment (eg, light chain or heavy chain) and corresponding transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA methods, in vivo synthesis methods, and genetic recombination. Also provided are replicable vectors containing a nucleotide sequence encoding an antibody molecule described herein, an antibody heavy or light chain, a heavy or light chain variable region of an antibody or fragment thereof, or a heavy or light chain CDR operably linked to a promoter. Such vectors may, for example, include a nucleotide sequence encoding the constant region of an antibody molecule (see, for example, International Publications Nos. WO 86/05807 and WO 89/01036; and US Pat. No. 5,122,464, which are incorporated herein by reference in their entirety ) and antibody variable regions can be cloned into such a vector to express the entire heavy chain, the entire light chain, or both the entire heavy and light chain.

The expression vector can be transferred into a cell (eg, a host cell) by conventional methods, and the resulting cells can then be cultured by conventional methods to produce the antibody described herein, or a fragment thereof. Thus, provided herein are host cells containing a polynucleotide encoding an antibody described herein, or fragments thereof, or a heavy or light chain, or fragment thereof, or a single chain antibody described herein, operably linked to a promoter to express such sequences in the host cell. In certain embodiments, for the expression of double chain antibodies, vectors encoding both the heavy and light chains separately can be co-expressed in a host cell to express the entire immunoglobulin molecule, as described in detail below. In certain embodiments, the host cell contains a vector containing a polynucleotide encoding both a heavy chain and a light chain of an antibody described herein, or a fragment thereof. In specific embodiments, the host cell contains two different vectors, a first vector containing a polynucleotide encoding the heavy chain or heavy chain variable region of an antibody described herein or a fragment thereof, and a second vector containing a polynucleotide encoding the light chain or variable region the light chain of an antibody described herein, or a fragment thereof. In other embodiments, the first host cell contains a first vector containing a polynucleotide encoding the heavy chain or heavy chain variable region of an antibody described herein or a fragment thereof, and the second host cell contains a second vector containing a polynucleotide encoding the light chain or the light chain variable region of an antibody described herein. In specific embodiments, the heavy chain/heavy chain variable region is expressed by a first cell associated with the light chain/light chain variable region of a second cell to form an anti-LAG-3 antibody (eg, human LAG-3) described herein. In certain embodiments, a population of host cells is provided herein, including such a first host cell and such a second host cell.

In a specific embodiment, provided herein is a population of vectors comprising a first vector comprising a polynucleotide encoding the light chain/light chain variable region of an anti-LAG-3 antibody (e.g., human LAG-3) described herein and a second vector comprising a polynucleotide encoding the heavy chain/heavy chain variable region of an anti-LAG-3 antibody (eg, human LAG-3) described herein.

A variety of expression host vector systems can be used to express the antibody molecules described herein (see, for example, US Pat. No. 5,807,715, which is incorporated herein by reference in its entirety). Such expression host systems are vehicles through which coding sequences of interest can be produced and subsequently purified, but are also cells that can, when transformed or transfected with the appropriate nucleotide coding sequences, express the antibody molecule described herein in situ. These include, but are not limited to, microorganisms such as bacteria (eg, E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences; yeast (eg, Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (eg, baculovirus) containing antibody coding sequences; plant cell systems (eg, green algae such as Chlamydomonas reinhardtii) infected with recombinant virus expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (eg, Ti plasmids) containing antibody coding sequences; or mammalian cell systems (eg, COS (eg, COS1 or COS), CHO, BHK, MDCK, HEK 293, NSO, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa and NIH 3T3, HEK-293T, HepG2, SP210 cells R1.1, BW, LM, BSC1, BSC40, YB/20 and BMT10), carrying recombinant expression constructs containing promoters derived from the genome of mammalian cells (for example, the metallothionein promoter) or from mammalian viruses (for example, the adenovirus late promoter) promoter cowpox virus 7.5K). In a specific embodiment, the antibody expression cells described herein are CHO cells, such as CHO cells from the GS™ CHO System (Lonza). In certain embodiments, the heavy chain and/or light chain of an antibody produced by a CHO cell may have an N-terminal glutamine or glutamate residue replaced with pyroglutamate. In a specific embodiment, the antibody expression cells described herein are human cells, such as human cell lines. In a specific embodiment, the mammalian expression vector is pOptiVEC™ or pcDNA3.3. In a specific embodiment, bacterial cells, such as Escherichia coli, or eukaryotic cells (eg, mammalian cells), especially for expressing a whole recombinant antibody molecule, are used to express the recombinant antibody molecule. For example, mammalian cells, such as Chinese hamster ovary (CHO) cells, in combination with a vector, such as the core intermediate early gene promoter element from human cytomegalovirus, are an effective expression system for antibodies (Foecking MK & Hofstetter H (1986) Gene 45: 101-5 and Cockett MI et al., (1990) Biotechnology 8(7): 662-7, each of which is incorporated herein by reference in its entirety). In certain embodiments, the antibodies described herein are produced by CHO cells or NS0 cells. In a specific embodiment, the expression of nucleotide sequences described herein encoding antibodies that specifically bind LAG-3 (e.g., human LAG-3) is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

- 91 044026 promoter.

In bacterial systems, a number of expression vectors may advantageously be selected depending on the intended use of the antibody molecule being expressed. For example, when large quantities of such an antibody are needed to create pharmaceutical compositions of the antibody molecule, vectors that direct expression of high levels of fusion protein products that are easily purified may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983) EMBO J 2: 1791-1794), in which the antibody coding sequence can be ligated individually into the vector in frame with the coding sequence the lac Z region so that a fusion protein is formed; pIN vectors (Inouye S & Inouye M (1985) Nuc Acids Res 13: 3101-3109; Van Heeke G & Schuster SM (1989) J Biol Chem 24: 5503-5509) and the like, all of which are incorporated herein by links in full. For example, pGEX vectors can also be used to express foreign polypeptides as glutathione 5-transferase (GST) fusion proteins. Typically, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to glutathione-agarose matrix beads followed by elution in the presence of free glutathione. pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST fragment.

In an insect system, for example, Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector for the expression of foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence can be individually cloned into non-essential regions (eg, the polyhedrin gene) of the virus and placed under the control of the AcNPV promoter (eg, the polyhedrin promoter).

A number of viral expression systems can be used in mammalian host cells. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest can be ligated to the adenovirus transcription/translation control complex, such as a late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination.

Insertion into a non-essential region of the viral genome (eg the E1 or E3 region) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (eg see Logan J & Shenk T (1984). PNAS 81 (12) : 3655-9, which is incorporated herein by reference in its entirety). Specific initiation signals may also be required for efficient translation of integrated antibody coding sequences. These signals include the ATG start codon and adjacent sequences. In addition, the start codon must be in phase with the reading frame of the desired coding sequence to allow translation of the entire insert. These exogenous translational control signals and start codons can have various origins, both natural and synthetic. Expression efficiency can be increased by including appropriate transcription enhancer elements, transcription terminators, etc. (See, for example, Bitter G et al., (1987) Methods Enzymol. 153: 516-544, which is incorporated herein by reference in its entirety.)

In addition, a host cell strain may be selected that modulates the expression of the inserted sequences or modifies and processes the gene product in a particular desired manner. Such modifications (eg, glycosylation) and processing (eg, cleavage) of protein products may be important for protein function. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Suitable cell lines or host systems can be selected to ensure proper modification and processing of the expressed foreign protein. For this purpose, eukaryotic host cells that possess the cellular machinery to properly process the primary transcript, glycosylate, and phosphorylate the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a murine myeloma cell line that is not endogenously produces no immunoglobulin chains), cells CRL7O3O, COS (for example, COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB/20, BMT10 and HsS78Bst. In certain embodiments, anti-LAG-3 (eg, human LAG-3) antibodies described herein are produced in mammalian cells, such as CHO cells.

In a specific embodiment, the antibodies described herein have reduced or no fucose content. Such antibodies can be prepared using techniques known to one skilled in the art. For example, antibodies may be expressed in cells deficient in or lacking the ability to fucosylate. In a specific example, cell lines with knockout of both a1,6-fucosyltransferase alleles can be used to produce antibodies with a reduced fucose content. The Potelligent® system (Lonza) is an example of such a system that can be used to produce antibodies with reduced fucose content.

For long-term production of recombinant proteins in high yield, stable expression cells can be obtained. For example, cell lines can be constructed that stably express an anti-LAG-3 antibody (eg, human LAG-3) described herein. In specific embodiments, a cell provided herein stably expresses a light chain/light chain variable region and a heavy chain/heavy chain variable region that bind to form an antibody described herein.

In certain aspects, instead of using expression vectors that contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (eg, promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. After the introduction of foreign DNA/polynucleotide, the constructed cells can grow for 1-2 days in an enriched medium, followed by transplantation into a selective medium. A selectable marker in a recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci, which in turn can be cloned and expanded into cell lines. This method can advantageously be used to construct cell lines that express an anti-LAG-3 antibody (eg, human LAG-3) described herein, or a fragment thereof. Such engineered cell lines may be particularly useful in screening and evaluating compositions that directly or indirectly interact with an antibody molecule.

A number of selection systems can be used, including, but not limited to, herpes simplex virus thymidine kinase (Wigler M et al., (1977) Cell 11 (1): 223-32), hypoxanthine guanine phosphoribosyltransferase (Szybalska EH & Szybalski W (1962) PNAS 48 (12): 2026-2034) and adenine phosphoribosyltransferase genes (Lowy I et al., (1980) Cell 22 (3): 817-23) in tk-, hgprt- or aprt-cells, respectively, all of which are included in this document by reference in its entirety. In addition, antimetabolite resistance can be used as a basis for selection of the following genes: dhfr, which confers resistance to methotrexate, (Wigler M et al., (1980) PNAS 77(6): 3567-70; O'Hare K et al., (1981) PNAS 78: 1527-31); gpt, which confers resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4): 2072-6); neo, which confers resistance to aminoglycoside G-418 (Wu GY & Wu CH (1991) Biotherapy 3: 87-95; Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32: 573-596; Mulligan RC (1993) Science 260: 926 -932 and Morgan RA & Anderson WF (1993) Ann Rev Biochem 62: 191-217; Nabel GJ & Feigner PL (1993) Trends Biotechnol 11(5): 211-5); and hygro, which confers resistance to hygromycin (Santerre RF et al., (1984) Gene 30(1-3): 147-56), all of which are incorporated herein by reference in their entirety. Methods well known in the art of recombinant DNA technology can typically be used to select the desired recombinant clone, and such methods are described, for example, in Ausubel F. M. et al., (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY ( 1993); Kriegler M, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990) and in chapters 12 and 13, Dracopoli NC et al., (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY ( 1994); ColbereGarapin F et al., (1981) J Mol Biol 150: 1-14, which are incorporated herein by reference in their entirety.

Expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington CR & Hentschel CCG, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987), which is incorporated herein by reference in its entirety). When a marker in a vector system expressing an antibody is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is associated with the antibody gene, antibody production will also be increased (Crouse GF et al., (1983) Mol Cell Biol 3: 257-66, which is incorporated herein by reference in its entirety).

A host cell can be co-transfected with two or more expression vectors described herein, a first vector encoding a heavy chain-derived polypeptide and a second vector encoding a light chain-derived polypeptide. The two vectors may contain identical selectable markers that provide equal expression of the heavy and light chain polypeptides. Host cells can be co-transfected with varying amounts of two or more expression vectors. For example, host cells can be transfected with any of the following ratios of the first expression vector and the second expression vector: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1: 8, 1:9, 1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45 or 1:50.

Alternatively, a single vector may be used that encodes and is capable of expressing both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid excess toxin-free heavy chain (Proudfoot NJ (1986) Na-93 044026 ture 322: 562-565 and Kohler G (1980) PNAS 77: 2197-2199, each which are incorporated herein by reference in their entirety). The coding sequences for the heavy and light chains may include cDNA or genomic DNA. The expression vector can be monocistronic or multicistronic. A multicistronic nucleic acid construct may encode 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, or in the range of 2-5, 5-10 or 10-20 genes/nucleotide sequences. For example, a bicistronic nucleic acid construct may comprise, in the order of a promoter, a first gene (eg, the heavy chain of an antibody described herein) and a second gene (eg, a light chain of an antibody described herein). In such an expression vector, the transcription of both genes can be driven by a promoter, while the translation of the mRNA from the first gene can be carried out by a cap-dependent scanning mechanism, and the translation of the mRNA from the second gene can be carried out by a cap-independent scanning mechanism, for example, IRES.

Once an antibody molecule described herein has been produced by recombinant expression, it can be purified by any method known in the art to purify an immunoglobulin molecule, for example, chromatography (e.g., ion exchange, affinity, particularly with affinity for a specific antigen after protein A and calibration chromatography), centrifugation, differential solubility or any other standard protein purification method. In addition, the antibodies described herein can be fused to heterologous polypeptide sequences described herein or other methods known in the art to facilitate purification.

In certain embodiments, an antibody described herein is isolated or purified. Typically, an isolated antibody is an antibody that is substantially free of other antibodies with antigenic specificity other than the isolated antibody. For example, in a particular embodiment, the antibody preparation described herein is substantially free of cellular material and/or chemical precursors. The expression essentially free of cellular material includes antibody preparations in which the antibody is separated from the cellular components of the cells from which it is isolated or recombinantly produced. Thus, an antibody that is substantially free of cellular material includes antibody preparations having less than about 30, 20, 10, 5, 2, 1, 0.5, or 0.1% (by dry weight) heterologous protein (also referred to herein as a contaminant protein) and/or variants of the antibody, such as various post-translationally modified forms of the antibody or other different variants of the antibody (eg, antibody fragments). When an antibody is produced recombinantly, it is also typically substantially free of culture medium, that is, the culture medium constitutes less than about 20, 10, 2, 1, 0.5, or 0.1% by volume of the protein preparation. When an antibody is produced by chemical synthesis, it is typically substantially free of chemical precursors or other chemicals, that is, it is separated from chemical precursors or other chemicals that are involved in protein synthesis. Accordingly, such antibody preparations contain less than about 30, 20, 10, or 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest. In a specific embodiment, the antibodies described herein are isolated or purified.

Antibodies or fragments thereof that specifically bind to LAG-3 (eg, human LAG-3) can be prepared by any antibody synthesis method known in the art, such as chemical synthesis or recombinant expression methods. The methods described herein use, unless otherwise noted, generally accepted methods in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the field. technology. These techniques are described, for example, in the references cited herein and are fully explained in the literature. See, for example, Maniatis T et al., (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook J et al., (1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel F. M. et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates) Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren B et al., (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press, all of which are incorporated herein by reference in their entirety.

In a specific embodiment, an antibody described herein is an antibody (eg, a recombinant antibody) produced, expressed, created, or isolated by any method that includes the creation, for example, by synthesis, of genetically engineering DNA sequences. In certain embodiments, such an antibody contains sequences (eg, DNA sequences or amino acid sequences) that do not naturally exist in the germline repertoire of an animal or mammalian (eg, human) antibody in vivo.

- 94 044026

In one aspect, provided herein is a method of producing an antibody that specifically binds to LAG-3 (eg, human LAG-3), comprising culturing a cell or host cell described herein. In one embodiment, the method is performed in vitro. In a certain aspect, provided herein is a method of producing an antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising expressing (e.g., recombinant expression) the antibody using a cell or host cell described herein (e.g., , cell or host cell containing polynucleotides encoding the antibody described herein). In a specific embodiment, the cell is an isolated cell. In a specific embodiment, exogenous polynucleotides have been introduced into the cell. In a specific embodiment, the method further includes the step of purifying an antibody obtained from a cell or host cell.

Methods for producing polyclonal antibodies are known in the art (see, for example, Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., eds., John Wiley and Sons, New York, which incorporated herein by reference in its entirety).

Monoclonal antibodies can be produced using a wide range of techniques known in the art, including the use of hybridoma, recombinant and phage display technologies, or combinations thereof. For example, monoclonal antibodies can be produced using hybridoma techniques, including those known in the art and described, for example, in Harlow E & Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988 ); Hammerling GJ et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, N.Y., 1981), each of which is incorporated herein by reference in its entirety. As used herein, the term monoclonal antibody is not limited to antibodies produced by hybridoma technology. For example, monoclonal antibodies can be produced recombinantly from host cells exogenously expressing an antibody described herein, or a fragment thereof, such as the light chain and/or heavy chain of such an antibody.

In specific embodiments, a monoclonal antibody, as used herein, is an antibody produced by a single cell (e.g., a hybridoma or a host cell producing a recombinant antibody), wherein the antibody specifically binds to LAG-3 (e.g., human LAG-3) as determined, for example, by ELISA or other antigen binding or competitive binding assay known in the art or in the examples provided herein. In specific embodiments, the monoclonal antibody may be a chimeric antibody or a humanized antibody. In certain embodiments, the monoclonal antibody is a monovalent antibody or a multivalent (eg, bivalent) antibody. In specific embodiments, the monoclonal antibody is a monospecific or multispecific antibody (eg, a bispecific antibody). The monoclonal antibodies described herein may be produced, for example, by the hybridoma method as described in Kohler G & Milstein C (1975) Nature 256: 495, which is incorporated herein by reference in its entirety, or may be, for example , isolated from phage libraries, for example, using the techniques described herein. Other methods for generating clonal cell lines and the monoclonal antibodies they express are well known in the art (see, for example, Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., supra).

Methods for producing and screening specific antibodies using hybridoma technology are standard and well known in the art. For example, in the hybridoma method, a mouse or other suitable host animal, such as a sheep, goat, rabbit, rat, hamster, or macaque, is immunized to detect lymphocytes that produce or are capable of producing antibodies that specifically bind to a protein (eg, LAG-3 (eg human LAG-3)) used for immunization. Alternatively, lymphocytes can be immunized in vitro. Lymphocytes are then fused with myeloma cells using a suitable fusion agent such as polyethylene glycol to form a hybridoma cell (Goding JW (Ed), Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986), which is included in this document as a reference in its entirety). Additionally, the RIMMS (repeated immunization multiple site) technique can be used to immunize an animal (Kilpatrick KE et al., (1997) Hybridoma 16:381-9, which is incorporated herein by reference in its entirety).

In some embodiments, mice (or other animals such as rats, monkeys, donkeys, pigs, sheep, hamsters, or dogs) can be immunized with an antigen (e.g., LAG-3 (e.g., human LAG-3)) and exhibit a single immune response For example, antigen-specific antibodies are detected in mouse serum, mouse spleens are collected, and splenocytes are isolated. The splenocytes are then fused by well-known methods with any suitable myeloma cells, for example cells from the SP20 cell line available from the American Type Culture Collection (ATCC®) (Manassas, VA), to form hybridomas. Hybridomas are selected and cloned by limited breeding. In certain embodiments, lymph nodes are immune

- 95 044026 nized mice are collected and fused with NS0 myeloma cells.

The hybridoma cells thus obtained are seeded and grown in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), hybridoma culture medium typically includes hypoxanthine, aminopterin, and thymidine (HAT medium), which inhibits the growth of HGPRT-deficient cells.

In particular embodiments, myeloma cells are used that efficiently fuse, support stable high-level antibody production by selected antibody-producing cells, and are sensitive to a medium, such as a HAT medium. Among these myeloma cell lines are mouse myeloma lines, such as the NS0 cell line or mouse tumor-derived lines MOPC-21 and MPC-11, which are available from the Salk Institute Cell Distribution Center, San Diego, California, USA, and SP cells -2 or X63-Ag8.653, available from the American Type Culture Collection, Rockville, Maryland, USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies. (Kozbor D (1984) J Immunol 133: 3001-5; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 5163 (Marcel Dekker, Inc., New York, 1987), each of which is included herein in as a reference in full).

The culture medium in which hybridoma cells grow is assayed for the production of monoclonal antibodies directed against LAG-3 (eg, human LAG-3).

The binding specificity of monoclonal antibodies produced by hybridoma cells is determined by methods known in the art, for example, immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

Once hybridoma cells that produce antibodies of the desired specificity, affinity, and/or activity have been identified, clones can be subcloned by limiting dilution procedures and grown using standard methods (Goding JW (Ed), Monoclonal Antibodies: Principles and Practice, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI 1640. In addition, hybridoma cells can be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid or serum using conventional immunoglobulin purification procedures, such as, for example, protein A-Sepharose chromatography, hydroxyapatite, gel electrophoresis, dialysis or affinity chromatography.

The antibodies described herein include antibody fragments that recognize specific LAG-3 (eg, human LAG-3) and can be prepared by any method known to those skilled in the art. For example, the Fab and F(ab') ₂ fragments described herein can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') ₂ fragments) . The Fab fragment corresponds to one of two identical arms of the antibody molecule and contains the complete light chain paired with the VH and CH1 domains of the heavy chain. The F(ab') ₂ fragment contains two antigen-binding arms of the antibody molecule linked by disulfide bonds at the hinge region.

In addition, the antibodies described herein can also be produced using various phage display methods known in the art. In phage display methods, the functional domains of antibodies are displayed on the surface of phage particles that carry the polynucleotide sequences encoding them. In particular, DNA sequences encoding the VH and VL domains are amplified from animal cDNA libraries (eg, human or mouse diseased tissue cDNA libraries). The DNA encoding the VH and VL domains is recombined along with the scFv linker by PCR and cloned into the phagemid vector. The vector is electroporated into E. coli, and E. coli is infected with a helper phage. The phage used in these methods is usually a filamentous phage, including fd and M13, and the VH and VL domains are usually recombinantly fused to a phage III gene or VIII gene. A phage expressing an antigen-binding domain that binds to a particular antigen can be selected or identified with the antigen, for example, using a labeled antigen or an antigen bound or captured on a solid surface or bead. Examples of phage display methods that can be used to produce the antibodies described herein include those disclosed in Brinkman U et al., (1995) J Immunol Methods 182: 4150; Ames RS et al., (1995) J Immunol Methods 184: 177-186; Kettleborough CA et al., (1994) Eur J Immunol 24: 952-958; Persic L et al., (1997) Gene 187: 9-18; Burton DR & Barbas CF (1994) Advan Immunol 57: 191280; PCT Publication No. pCt/GB91/001134; International Publication No. WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, WO 95/20401 and WO 97/13844; and US Patent No. 5698426, 5223409, 5403484, 5580717, 5427908, 5750753, 5821047, 5571698, 5427908, 5516637, 5780225,

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5658727, 5733743 and 5969108, all of which are incorporated herein by reference in their entirety.

As described in the above references, after phage selection, antibody coding regions from the phage can be isolated and used to create whole antibodies, including human antibodies or any other desired antigen binding moiety, and expressed in any desired host, including mammalian cells, insect cells, plant cells , yeast and bacteria, for example as described below. Techniques for recombinant production of antibody fragments such as Fab, Fab' and F(ab') ₂ fragments can also be used using methods known in the art, such as those disclosed in PCT Publication No. WO 92/22324; Mullinax RL et al., (1992) BioTechniques 12(6): 864-9; Sawai H et al., (1995) Am J Reprod Immunol 34: 26-34 and Better M et al., (1988) Science 240: 1041-1043, all of which are incorporated herein by reference in their entirety.

In certain embodiments, PCR primers comprising VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to generate whole antibodies to amplify VH or VL sequences from a template, such as scFv clones. Using cloning techniques known to those skilled in the art, PCR amplified VH domains can be cloned into vectors expressing the VH constant region, and PCR amplified VL domains can be cloned into vectors expressing a VL constant region, e.g. human kappa and lambda. The VH and VL domains can also be cloned into a single vector expressing the necessary constant regions. The heavy chain conversion vectors and the light chain conversion vectors are then co-transfected into cell lines to generate stable or transient cell lines that express full-length antibodies, such as IgG, using methods known to those skilled in the art.

A chimeric antibody is a molecule in which different parts of the antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody may comprise the variable region of a mouse or rat monoclonal antibody fused to the constant region of a human antibody. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison SL (1985) Science 229: 1202-7; Oi VT & Morrison SL (1986) BioTechniques 4: 214-221; Gillies SD et al., (1989) J Immunol Methods 125: 191-202; and US Patent Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415, all of which are incorporated herein by reference in their entirety.

The humanized antibody is capable of binding to a predetermined antigen and contains a framework region having essentially the amino acid sequence of a human immunoglobulin, and CDRs having essentially the amino acid sequence of a non-human immunoglobulin (eg, mouse immunoglobulin). In specific embodiments, the humanized antibody also contains at least a portion of an immunoglobulin constant region (Fc), typically a portion of a human immunoglobulin. The antibody may also include the CH1 region, the hinge region, the CH2, CH3 and CH4 regions of the heavy chain. The humanized antibody may be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG1, _IgG2 , _IgG3 and IgG4. Humanized antibodies can be produced using various techniques known in the art, including, but not limited to, CDR transplantation (European Patent No. EP 239400; International Publication No. WO 91/09967 and US Patent Nos. 5225539, 5530101 and 5585089), venation or modification of the surface (European patents no. EP 592106 and EP 519596; Padlan EA (1991) Mol Immunol 28(4/5): 489-498; Studnicka GM et al., (1994) Prot Engineering 7(6): 805- 814 and Roguska MA et al., (1994) PNAS 91: 969-973), chain shuffling (US Patent No. 5565332), and techniques disclosed in, for example, US Patent No. 6407213, US Patent No. 5766886, International Publication No. WO 93/17105; Tan P et al., (2002) J Immunol 169: 1119-25; Caldas C et al., (2000) Protein Eng. 13(5): 353-60; Morea V et al., (2000) Methods 20(3): 267-79; Baca M et al., (1997) J Biol Chem 272(16): 10678-84; Roguska MA et al., (1996) Protein Eng 9(10): 895 904; Couto JR et al., (1995) Cancer Res. 55 (23 Supp): 5973s-5977s; Couto JR et al., (1995) Cancer Res 55(8): 1717-22; Sandhu JS (1994) Gene 150(2): 409-10 and Pedersen JT et al., (1994) J Mol Biol 235(3): 959-73, all of which are incorporated herein by reference in their entirety. See also US Application Publication No. US 2005/0042664 A1 (Feb. 24, 2005), which is incorporated herein by reference in its entirety.

Methods for producing multispecific (eg, bispecific antibodies) have been described, see, for example, US patent No. 7951917; 7183076; 8227577; 5837242; 5989830; 5869620; 6132992 and 8586713, all of which are incorporated herein by reference in their entirety.

Single domain antibodies, for example, antibodies lacking light chains, can be produced by methods well known in the art. See Riechmann L & Muyldermans S (1999) J Immunol 231: 25-38; Nuttall SD et al., (2000) Curr Pharm Biotechnol 1(3): 253-263; Muyldermans S, (2001) J Biotechnol 74(4): 277-302, US Patent No. 6005079, and International Publications No. WO 94/04678; WO 94/25591 and WO 01/44301, all of which are incorporated herein by reference in their entirety.

In addition, antibodies that specifically bind to LAG-3 antigen (e.g., LAG-3

- 97 044026 human) can in turn be used to create anti-idiotypic antibodies that mimic the antigen using methods well known to those skilled in the art. See, for example, Greenspan NS & Bona CA (1989) FASEB J 7(5): 437-444 and Nissinoff A (1991) J Immunol 147(8):

2429-2438, each of which is incorporated herein by reference in its entirety.

In specific embodiments, an antibody described herein that binds to the same epitope of LAG-3 (e.g., human LAG-3) as an anti-LAG-3 antibody (e.g., human LAG-3) described herein is a human antibody. In specific embodiments, an antibody described herein that competitively blocks (e.g., in a dose-dependent manner) any one of the antibodies described herein from binding to LAG-3 (e.g., human LAG-3) is an antibody person. Human antibodies can be obtained using any method known in the art. For example, it is possible to use transgenic mice that are unable to express functional endogenous immunoglobulins, but which can express human immunoglobulin genes. In particular, gene complexes of human immunoglobulin heavy and light chains can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable region, constant region and variant region can be introduced into mouse embryonic stem cells in addition to human heavy and light chain genes. The mouse immunoglobulin heavy and light chain genes can become nonfunctional individually or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents the production of endogenous antibodies. Modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. Transgenic mice are immunized in the usual manner with a selected antigen, eg all or part of an antigen (eg LAG-3 (eg human LAG-3)). Monoclonal antibodies directed against an antigen can be generated from immunized transgenic mice using conventional hybridoma technology. Human immunoglobulin transgenes carried by transgenic mice are rearranged during B cell differentiation and subsequently undergo class switching and somatic mutations. Thus, using this technique, therapeutically useful IgG, IgA, IgM and IgE antibodies can be obtained. For a review of this technology for the production of human antibodies, see Lonberg N & Huszar D (1995) Int Rev Immunol 13:65-93, incorporated herein by reference in its entirety. For a detailed discussion of this technology for the production of human antibodies and human monoclonal antibodies and protocols for the production of such antibodies, see, for example, international publications No. WO 98/24893, WO 96/34096 and WO 96/33735 and US patent No. 5413923, 5625126, 5633425, 5569825 , 5661016, 5545806, 5814318 and 5939598, all of which are incorporated herein by reference in their entirety. Examples of mice capable of producing human antibodies include Xenomouse™ (Abgenix, Inc.; US Patent Nos. 6075181 and 6150184), HuAb-Mouse™ (Mederex, Inc./Gen Pharm; US Patent Nos. 5545806 and 5569825), Trans Chromo Mouse™ (Kirin) and KM Mouse™ (Medarex/Kirin), all of which are incorporated herein by reference in their entirety.

Human antibodies that specifically bind to LAG-3 (eg, human LAG-3) can be produced by various methods known in the art, including the phage display methods described above, using antibody libraries derived from human immunoglobulin sequences. See also US Patent Nos. 4,444,887, 4,716,111, and 5,885,793; and International Publications No. WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741, all of which are incorporated herein by reference in their entirety volume.

In some embodiments, human antibodies can be produced using mouse-human hybridomas. For example, human peripheral blood lymphocytes transformed with Epstein-Barr virus (EBV) can be fused with mouse myeloma cells to produce mouse-human hybridomas that secrete human monoclonal antibodies, and these mouse-human hybridomas can be screened to identify those that secrete human monoclonal antibodies that specifically bind to a target antigen (eg, LAG-3 (eg, human LAG-3)). Such methods are known and described in the art, see, for example, Shinmoto H et al., (2004) Cytotechnology 46: 19-23; Naganawa Y et al., (2005) Human Antibodies 14: 27-31, each of which is incorporated herein by reference in its entirety.

6.6. Sets.

Also provided are kits containing one or more of the antibodies described herein or a pharmaceutical composition or conjugates thereof. In a specific embodiment, provided herein is a pharmaceutical kit containing one or more containers filled with one or more ingredients of the pharmaceutical compositions described herein, such as one or more antibodies provided herein. In some embodiments, the kits contain a pharmaceutical composition described herein and any prophylactic or therapeutic agent such as those described herein. In some embodiments, the kits may contain a T cell mitogen, such as, for example, phytohemagglutinin (PHA) and/or phorbol myristate acetate (PMA), or a TCR complex stimulating antibody, such as an anti-CD3 antibody and an anti-CD28 antibody. If desired, such container(s) may be accompanied by a notice in a form prescribed by the government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which notice reflects the agency's approval of the manufacture, use, or sale for human administration.

Kits are also provided that can be used in the methods described above. In one embodiment, the kit contains an antibody described herein, preferably a purified antibody, in one or more containers. In a specific embodiment, the kits described herein contain substantially isolated LAG-3 antigen (eg, human LAG-3) as a control. In another specific embodiment, the kits described herein further contain a control antibody that does not react with a LAG-3 antigen (eg, human LAG-3). In another specific embodiment, the kits described herein contain one or more elements for detecting the binding of an antibody to a LAG-3 (e.g., human LAG-3) antigen (e.g., the antibody may be conjugated to a detectable substrate, such as a fluorescent compound, an enzymatic substrate , a radioactive compound or a luminescent compound or a second antibody that recognizes the first antibody may be conjugated to a detectable substrate). In specific embodiments, a kit provided herein may comprise a recombinantly produced or chemically synthesized LAG-3 antigen (eg, human LAG-3). The LAG-3 antigen (eg, human LAG-3) provided in the kit can also be attached to a solid support. In a more specific embodiment, the detection means of the kit described above includes a solid support to which a LAG-3 antigen (eg, human LAG-3) is attached. Such a kit may also include an unattached reporter-labeled anti-human antibody or anti-mouse/rat antibody. In this embodiment, binding of an antibody to a LAG-3 antigen (eg, human LAG-3) can be detected by binding of said reporter-labeled antibody. In one embodiment, the present invention relates to the use of a kit according to the present invention for the analysis and/or in vitro detection of LAG-3 antigen (eg, human LAG-3) in a biological sample.

7. Examples

The examples in this section (i.e., Section 6) are offered as illustrations and not as limitations.

7.1. Example 1. Generation and characterization of new antibodies against human LAG-3.

This example describes the generation and characterization of antibodies that bind to human lymphocyte activation gene 3 (LAG-3), also known as CD223. In particular, this example describes the generation of mouse antibodies that specifically bind to human LAG-3 and inhibit the function of human LAG-3.

7.1.1. Generation of antibodies against LAG-3.

Antibodies against LAG-3 were identified by constructing and selecting an immunized Fab phage display library. First, total RNA was purified from a single cell suspension of splenocytes from three separate mice preimmunized with recombinant human LAG-3-Fc protein (R&D Systems, Cat No. 2319-L3-050) and recombinant cynomolgus LAG-3-Fc protein. (Evitria, Custom order). For the mouse, a Fab library was generated using randomly primed cDNA using total RNA as a template to amplify variable regions from mouse antibody genes. Heavy and kappa chain amplicons were combined and cloned into phagemid vectors. Three rounds of selection were performed against recombinant LAG-3 proteins (human LAG-3Fc, human LAG-3-6His, and/or human LAG-3 30-amino acid peptide) and/or cells expressing cynomolgus LAG-3 to identify LAG-3 -specific clones of phage Fab. The 30 amino acid human LAG-3 peptide is a biotinylated peptide containing the amino acid sequence GPPAAAPGHPLAPGPHPAAPSSWGPRPRRY (SEQ ID NO: 199). Periplasmic extracts of selected Fab clones were then screened by ELISA or flow cytometry. Antibody sequencing and dissociation rate analysis on anti-LAG-3 Fab were then performed.

A set of mouse antibodies that bind to human LAG-3 were identified and designated P01A12, P01C09, P05E01, P05E03, P13A04, P13A06, P13B01, P13B02, P13B03, P13B11, P13C06, P13C08, P13C10, P13D04, P13 D05, P13E02, P13F01 . 15F06, P15G05, P16D04 and P16H05. Information on the sequence of the variable regions of these antibodies is given in table. 6.

7.1.2. Binding of anti-LAG-3 Fab to LAG-3-expressing cells.

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Anti-LAG-3 Fab from periplasmic extracts was tested for binding to LAG-3-expressing cells using flow cytometry. Briefly, Jurkat cells expressing wild-type and human LAG-3 were seeded at 2x105 cells/well in sample buffer. (PBS (Gibco, cat. no. 10010-015) + 0.5% FBS (Gibco, cat. no. 10270-106)) in 96-well U-plates (Sarstedt). 22 μl of periplasmic anti-LAG-3 Fab extract and 83 μl of diluted anti-c-myc antibody (Gentaur, clone no. 9E10, cat. no. 04-cmyc-9E10) were incubated for 30 min at room temperature. 100 μl of the periplasmic extract/anti-c-myc antibody mixture was added to the cells and then incubated for one hour on ice. Cells were washed three times and then incubated with goat anti-mouse APC (BD Biosciences, cat. no. 550826) for 30 min on ice. Cells were washed three times and then analyzed on a FACS apparatus (BD Accuri6).

As shown in FIG. 1A-1C, all anti-LAG-3 Fabs tested exhibited binding to Jurkat cells expressing human LAG-3. The apparent low level of P05E01 binding may have been due to the low concentration of Fab in the periplasmic extracts. P05E01 binding was confirmed in later experiments (data not shown).

7.1.3. Ligand blocking activity in Fab or anti-LAG-3 antibodies.

Anti-LAG-3 Fabs were tested for their ability to block the binding of recombinant human LAG-3 to cells expressing MHC class II. LAG-3-6His (Acro Biosystems, cat. no. LA3H5222) was preincubated at a concentration of 10 μg/ml with anti-His biotin (Genscript, clone no. A00186, cat. no. A00613) at 6 μg/ml for 10 min, at 4°C. This mixture was then incubated with 10,000 ng/ml (Fig. 2A) or a serial dilution (21,170, 7056.6, 2352.2, 784.0, 261.3, 87.1, 29.0, or 9.7 ng/ml ) (Fig. 2B) Fab against LAG-3 or negative control with Fab nonspecific for LAG-3 for 60 min at 4°C and then incubated with 50,000 Raji cells for an additional 60 min at 4°C. Cultures were washed twice with sample buffer (PBS+2% FBS+0.09% sodium azide) and then incubated with streptavidin-PE (Biolegend, cat. no. 405204) in sample buffer for 30 min at 4°C. Cells were washed twice and then analyzed using a FACS Fortessa cytometer (Becton Dickinson).

As shown in FIG. 2A and 2B, all anti-LAG-3 Fabs tested blocked binding of cross-linked recombinant LAG-3-6His to MHC class II positive Raji cells.

The variable regions of the selected Fabs were cloned into the human heavy chain (Ch1, Ch2 and Ch3) and light chain (CL) constant regions and expressed as full-length chimeric IgG1 antibodies and tested in a similar ligand blocking assay as described above at various concentrations (96360 , 48180, 24090, 12045, 6022, 3011, 1505, 753, 376, 188 or 94 ng/ml).

All full-length anti-LAG-3 chimeric antibodies tested blocked the interaction between recombinant LAG-3 and MHC class II-expressing Raji cells (Fig. 3).

7.1.4. Effect of anti-LAG-3 antibody on human PBMCs stimulated with staphylococcal enterotoxin A (SEA).

The functional activity of the chimeric anti-LAG-3 antibody P13B02 was tested using primary human peripheral blood mononuclear cells (PBMCs) stimulated with Staphylococcal enterotoxin A (SEA). Briefly, cryopreserved human PBMCs (Research Blood Components) were seeded at ¹⁰⁵ cells/well in RPMI1640 supplemented with Normocin™ (Invivogen, cat. no. ant-nr-1) and 10% heat-inactivated PBS (Thermo Fisher Scientific, cat. no. ant-nr-1). No. 26140079) into 96-well NUNCLON (NUNC™) delta surface plates. Cells were cultured with 100 ng/ml SEA (Toxin Technologies, cat. no. at101red) and 10 μg/ml P13B02 or isotype control antibody for 5 days at 37°C, 5% CO ₂ and 97% humidity. The clarified supernatant was collected and stored at −80°C until analysis. IL-2 levels were determined using AlphaLISA (Perkin Elmer, cat. no. AL221C).

As shown in FIG. 4, anti-LAG-3 antibody P13B02 increased IL-2 production in human PBMCs stimulated with superantigen SEA.

7.2. Example 2: Generation and characterization of humanized antibodies against human LAG-3.

This example describes the humanization of the murine antibody P13B02 and the characterization of the humanized antibodies.

7.2.1. Humanization of mouse antibody P13B02.

Homology matching was used to select human acceptor framework regions for transplantation of the murine antibody P13B02 CDR. Databases, for example, the database of germline variable genes from human and mouse immunoglobulin loci (IMGT (International Information System ImMunoGeneTics)® database; Lefranc MP et al., (1999) Nucleic Acids Res 27(1): 209-12; Ruiz M et al., (2000) Nucleic Acids Res 28(1): 219-21; Lefranc MP (2001) Nucleic Acids Res 29(1): 207-9; Lefranc MP (2003) Nucleic Acids Res 31(1) : 307-10; Lefranc MP et al., (2005) Dev Compo Immunol 29(3): 185-203; Kaas Q et al., (2007) Briefings in Functional Genomics & Proteomics 6(4): 253-64) or VBASE2 (Retter I et al., (2005) Nucleic Acids Res 33, Database issue D671-D674) or Kabat database (Johnson G et al., (2000) Nucleic Acids Res 28: 214-218)) or publications ( for example, Kabat E. A. et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services,

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NIH Publication No. 91-3242), all of which are incorporated by reference in their entirety, can be used to identify the human subfamilies to which the mouse heavy and light chain variable regions belong, and to determine the most suitable human germline structure for use as the acceptor molecule. The selection of heavy and light chain variable region sequences within these subfamilies for use as an acceptor may be based on sequence homology and/or structural overlap between the CDR1 and CDR2 regions to help maintain the appropriate relative representation of the six CDRs after transplantation.

Search the IMGT database downloaded from IMGT.org using the BioEdit sequence alignment editor (Hall, T.A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids. Symp Ser.41:95-98, incorporated herein by reference in its entirety) indicates good homology between the P13B02 heavy chain variable region framework and members of the human IGHV1 heavy chain variable region subgroup. The highest homologies and identities of both CDR and framework sequences were observed for the germline sequences: IGHV1-46*01 (SEQ ID NO: 153) (62% identity; 61 amino acid residues out of 98); IGHV1-69-2*01 (SEQ ID NO: 154) (63% identity; 62/98); IGHV1-3*01 (SEQ ID NO: 155) (64% identity; 63/98); IGHV1-24*01 (SEQ ID NO: 156) (61% identity; 60/98); IGHV1-2*01 (SEQ ID NO: 157) (60% identity; 59/98); IGHV1-45*01 (SEQ ID NO: 158) (59% identity; 58/98) and IGHV1-18*01 (SEQ ID NO: 159) (60% identity; 59/98).

Using the same approach, the light chain variable region sequence of P13B02 showed good homology to the human light chain kappa subgroup members IGKV3 and IGKV1. The highest homologies and identities of both CDR and framework sequences were observed for the germline sequences: IGKV3-20*01 (SEQ ID NO: 160) (59% identity; 57 amino acid residues out of 96); IGKV3D-15*01 and IGKV3-15*01 (SEQ ID NO: 161) (58% identity; 56/96); IGKV3D-20*01 (SEQ ID NO: 162) (59% identity; 57/96); IGKV3D-7*01 (SEQ ID NO: 163) (58% identity; 56/96); IGKV1-9*01 (SeQ ID NO: 164) (63% identity; 61/96) and IGKV3-11*01 (SEQ ID NO: 165) (60% identity; 58/96).

As a starting point for the humanization process, a VH version of the P13B02 CDR-grafted mouse was generated using framework regions 1, 2 and 3 of human IGHV1-46*01 (SEQ ID NO: 153) and framework region 4 of human IGHJl*01 (SEQ ID NO: 153). 200) as a human acceptor scaffold. A number of back mutations have been made at positions that may affect CDR conformation or packing between variable regions and therefore may be structurally important to maintain full antibody activity (H0-H4; SEQ ID NOs: 56-60 and 220, respectively) (FIG. 5A). Similarly, a CDR-grafted version of mouse P13B02 VL was generated using human IGKV3-20*01 frameworks 1, 2 and 3 (SEQ ID NO: 160) and human IGKJ1*01 framework 4 (SEQ ID NO: 201) as human scaffold acceptor (L0; SEQ ID NO: 73) (Fig. 5B). Back mutations were created at various positions (L1-L4; SEQ ID NO: 74-77, respectively) (Fig. 5B). The sequences of humanized VH H0-H4 and humanized VL L0-L4 are presented in table. 1. In table. 10 shows positions that differ between mouse and human antibody frameworks and are reverse mutated in at least one of the humanized VH or VL described above. In table 11 shows positions that differ in the framework regions of mouse and human antibodies and are reverse mutated to H1_R98K_L4M.

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Review of backbone mutations in at least one of the humanized VH or VL

Table 10

Variable region severe chains Position by VH Р13В02 IGHV1-46*O1 (SEQ ID NO: 153) Kabat H4 M L H5 TO V H12 V TO H23 T TO H27 F Y H2 8 N Τ H2 9 I Ε ISO TO Τ H48 I M H69 I M H71 A R H75 S Τ H76 N S H80 L M H81 Q Ε H94 T R Variable region easy chains Position according to VL Р13В02 IGKV3-20*01 (SEQ ID NO: 160) Kabat L3 L V L22 T S L36 F Y L43 S A L47 W L L58 V I L70 S D L71 Y F Table 11 Overview of framed inverses mutations in H1_R98K_L4M Position by VH Р13В02 IGHV1-46*O1 (SEQ ID NO: 153) Kabat H4 M L H27 F Y H2 8 N T H2 9 I F ISO TO T H69 I M H71 A R H94 T R

In addition, HCDR3 from P13B02 contains an RYD motif, which has previously been demonstrated to mimic the RGD integrin binding motif. To test whether the RYD motif could be removed without affecting LAG-3 binding, an amino acid substitution R98K or D100E, numbered as defined by Kabat, was introduced into the heavy chain.

A panel of 37 humanized antibodies, designated P13B02-01 to P13B02-37, were designed based on the above description and created as full-length IgG1 antibodies.

- 102 044026

P13B02-30 were produced in three variants: a full-length antibody containing the IgG1 G1m17 allotype, designated P13B02-30 (IgG1); a full-length antibody containing the IgG1 G1m17 allotype with the N297A mutation, designated P13B02-30 (IgG1 G1m17 N297A); and a full-length antibody containing the IgG1 G1m3 allotype with the N297A mutation, designated P13B02-30 (IgG1 G1m3 N297A). Information on the sequence of variable regions from P13B02-01 to P13B02-37 is given in table. 7. Two batches of P13B02-30 (IgG1 G1m3 N297A) were examined for post-translational processing in producer cells. All of these 37 humanized antibodies retained binding to recombinant human LAG-3 in a surface plasmon resonance assay (data not shown).

7.2.2. Binding of humanized anti-LAG-3 antibodies to human LAG-3.

Anti-LAG-3 antibodies were tested for binding to primary human T cells using flow cytometry. Cryopreserved human PBMCs (Research Blood Components) were seeded at 10 ⁶ cells/ml in RPMI1640 supplemented with NormocinTM (Invivogen, cat. no. ant-nr-1) and 10% heat-inactivated PBS (Thermo Fisher Scientific, cat. no. 26140079) in T-75 flask (Corning) in the presence of 100 ng/ml SEA (Toxin Technologies, cat. no. at101red) for 5 days at 37°C, 5% CO ₂ and 97% humidity. Cultured PBMCs were then seeded at 105 cells/well in 96-well U-bottom plates (Nunc). Cells were incubated with 10 μg/ml anti-LAG-3 antibody for 30 min on ice. Cells were washed three times and then incubated with anti-CD4-PE/Cy7 antibody (Biolegend, clone no. OKT4, cat. no. 317414), with anti-CD8-FITC antibody (Biolegend, clone no. RPA-T8, cat. no. 301060), with antibody Anti-CD3-APC (BD Biosciences, Clone No. SP34-2, Cat. No. 557597), LIVE/DEAD®, Near-IR Dead Cell Fixative Dye (Life Technologies, Cat. No. L10119), Fc Blocker (BD Biosciences , cat. no. 422302) and goat anti-human IgGPE (ThermoFisher, cat. no. PA1-74408). Cells were incubated for 30 min on ice, washed, and analyzed on a FACS apparatus (BD Canto). As shown in FIG. 6A, chimeric antibody P13B02 and all humanized antibodies tested showed binding to primary CD4+ human T cells stimulated with superantigen SEA.

Human PBMCs were then activated using SEA superantigen as described above and incubated with serially diluted (50,000, 15,000, 4500, 1350, 407, 122, 37, or 11 ng/ml) P13B02-16 or isotype control antibody. Binding was analyzed using a FACS Fortessa cytometer (Becton Dickinson). The humanized antibody P13B02-16 bound to activated primary human CD4+ T cells in a dose-dependent manner (Fig. 6B).

7.2.3. Ligand blocking activity of humanized anti-LAG-3 antibodies.

The ability of humanized anti-LAG-3 antibodies to block the interaction between cross-linked recombinant LAG-3-6His and MHC class II expressing Raji cells was then examined as described above.

Humanized antibodies P13B02-06, P13B02-07, P13B02-16, P13B02-26 and P13B02-27, all of which include the human IgG1 constant region, were tested at 57820, 28910, 14455, 7228, 3613, 1807, 903, 452, 226 , 113 and 56 ng/ml (Fig. 7A). Humanized antibody P13B02-30 (IgG1 G1m17 N297A) was tested at 96360, 48180, 2409θ, 12ο45, 6022, 3011, 1505, 753, 376, 188 and 94 ng/ml (Fig. 7B).

As shown in FIG. 7A and 7B, all humanized antibodies tested effectively blocked LAG-3 binding to MHC class II positive Raji cells.

7.2.4. Effect of anti-LAG-3 antibody on human PBMCs stimulated with staphylococcal enterotoxin A (SEA).

The functional activity of the humanized antibody P13B02-30 (IgG1) was assessed using primary human PBMCs stimulated with Staphylococcal enterotoxin A (SEA). Cryopreserved human PBMCs (Research Blood Components) were seeded at 105 cells/well in RPMI1640 supplemented with Normocin™ (Invivogen, cat. no. ant-nr-1) and 10% heat-inactivated PBS (Thermo Fisher Scientific, cat. no. 26140079) in NUNCLON (NUNC™) 96-well delta surface plates. Cells were cultured with 100 ng/ml SEA (Toxin Technologies, cat. no. at101red) and 10 μg/ml P13B02-30 (IgG1) or isotype control antibody for 5 days at 37°C, 5% CO ₂ and 97% humidity. The clarified supernatant was collected and stored at −80°C until analysis. IL-2 levels were determined using AlphaLISA (Perkin Elmer, cat. no. AL221C).

As shown in FIG. 8A, anti-LAG-3 antibody P13B02-30 (IgG1) enhanced IL-2 production in human PBMCs stimulated with superantigen SEA.

In a similar experiment, cryopreserved human PBMCs were cultured with 100 ng ml SEA (Toxin Technologies, cat. no. at101red) and 10 μg/ml P13B02-16 (IgG1) or isotype control antibody in the presence or absence of 5 μg/ml anti-PD-1 antibody pembrolizumab (Pembro) (Myoderm), anti-PD-1 antibody nivolumab (Nivo) (Myoderm), three different anti-PD-L1 antibodies, or anti-CTLA-4 antibody ipilimumab (Ipi) (Myoderm) for 5 days at 37°C, 5% CO ₂ and 97% humidity. The clarified supernatant was collected and stored at −80°C until analysis. IL-2 levels were determined using AlphaLISA (Perkin Elmer, cat. no. AL221C). Anti-PD-L1 antibody No. 1 was derived from the variable region sequences of antibody A09-246-2 submitted to the public

- 103 044026 tion of US Patent Application No. US2014/0341917 (incorporated herein by reference in its entirety). Anti-PD-L1 antibody No. 2 was derived from the variable region sequences of the 2.14H9ORT antibody provided in US Pat. No. 8,779,108 (incorporated herein by reference in its entirety). Anti-PD-L1 antibody No. 3 was generated based on the sequences presented in US Pat. No. 8,217,149 (incorporated herein by reference in its entirety). The sequences of these three anti-PD-L1 antibodies are shown in Table. 12.

Table 12

Anti-PD-L1 antibody sequences

SEQ ID Description Amino acid sequence NO: EVQLLESGGGLVQPGGSLRLSCAASGFFSSYIMMWVRQAPGK GLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSL RAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSSASTKGPSVF heavy PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT chain FPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVD 202 antibodies KRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR vs PD- TPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS L1 No. 1 T YRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG 203 anti-PDL1 antibody light chain #1 QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPG KAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEA DYYCSSYTSSSTRVFGTGTKVTVLQPKANPTVTLFPPSSEELQ ANKATLVCLIS D FYPGAVTVAWKADGS PVKAGVE TTKPSKQSN NKYAASSYLSLTPEQWKSHR SYSCQVTHEGSTVEKTVAPTECS 204 heavy chain EVQLVESGGGLVQPGGSLRLSCAASGTFFSRYWMSWVRQAPGK GLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSL

- 104 044026

antibodies vs PD- L1 No. 2 RAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKV DKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVWDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPG 205 anti-PDL1 antibody light chain #2 EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQ APRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAV YYCQQYGSLPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSG TASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC 206 heavy chain antibodies against PD- L1 No. 3 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGK GLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSL RAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSWTVPSSSLGTQTYIC NVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPG 207 anti-PDL1 antibody light chain #3 DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKA PKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGT ASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC

As shown in FIG. 8B, P13B02-16 (IgG^, alone or in combination with anti-PD-1 antibody pembrolizumab or nivolumab, anti-PD-L1 antibody #1, #2 or #3, or anti-CTLA-4 antibody ipilimumab, increased IL-2 production in Human PBMCs in the presence of superantigen SEA.

7.2.5. Effect of humanized anti-LAG-3 antibody on cytokine production by tumor infiltrating lymphocytes.

Anti-LAG-3 antibody P13B02-30 (IgGt Glm3 N297A) was further evaluated for its ability to stimulate cytokine production by activated primary tumor infiltrating lymphocytes (TILs), alone or in combination with anti-PD-1 antibody. Single cell suspensions from fresh renal cell carcinoma (RCC) (stage I) or colorectal cancer (CRC) (stage II) tumors (UMass Medical School, Worcester, MA) were isolated by mechanical microdissection. In some cases, depending on the level of fibrosis, enzymatic digestion was necessary (Liberase and DNAsel, Roche). Cells were left alone at a concentration of 5x10 ⁴ cells/well in RPMI164 0 supplemented with Normocin™ (Invivogen, cat. no. ant-nr-1), recombinant human IL-2 (20 U/ml, R&D Systems, cat. no. 202- IL-010) and 10% heat-inactivated PBS (Thermo Fisher Scientific, cat. no. 26140079) in NUNCLON 96-well delta surface plates (NUNC™, cat. no. 143761) for 1 day. The next day, the samples were centrifuged and fresh culture medium was obtained containing the antibodies of interest, P13B02-30 (IgGt Glm3 N297A) at a concentration of 20 μg/ml and the anti-PD-1 antibody pembrolizumab (Pembro) (Myoderm) at 5 μg/ml and microbeads anti-CD3/CD28 (bead ratio 1:1 cells) was added in a final volume of 100 μl and left for 3 days incubation at 37°C and 5% CO ₂ . The cell-free supernatant was collected and stored at −80°C until analysis. TNFot levels were determined using AlphaLISA (Perkin Elmer, cat. no. AL208C).

As shown in FIG. 9A and 9B, anti-LAG-3 antibody P13B02-30 (IgGi Glm3 N297A) enhances

-105044026 TNFa production by activated primary TILs from RCC or CRC tumors.

7.2.6. Anti-LAG-3 antibody enhances T cell activation in a LAG-3-mediated cell suppression assay.

In this example, an NFAT-luciferase reporter line was used to evaluate the inhibitory effect of the anti-LAG-3 antibody P13B02-30 (IgG1 G1m3 N297A) against LAG-3 in a cell inhibition assay. Two experiments were performed as described below.

In each of the two experiments, Jurkat-NFAT-LAG-3 cells were suspended to 2.5x the working concentration of 1x106 cells/ml in assay medium (RPMI+10% heat-inactivated PBS+1% Pen/Strep). Raji cells were suspended to 3.33x working concentration of 1x106 cells/ml in assay medium. Staphylococcal enterotoxin E (Toxin Technology) was prepared at 10x working concentration of 0.04 ng/mL in assay media. Serial dilutions of anti-LAG-3 antibody or isotype antibody were prepared in assay media. In the first experiment, antibody concentrations ranged from 0.2-50 μg/ml. In the second experiment, antibody concentrations ranged from 0.1-100 μg/ml. After preparing serial antibody dilutions, 40 μl of Jurkat-NFAT-LAG-3 cells and 20 μl of antibody solution were preincubated for 30 min at 37°C and 5% CO ₂ in 96-well U-bottom plates. 30 μl of Raji cells and 10 μl of Staphylococcus enterotoxin E were added to 96-well plates and incubated for 5-6 hours. Then 100 μl of BioGlo luciferase (Promega) was added to each well and luminescence was recorded using an EnVision plate reader (Perkin Elmer) through 10-15 min.

As shown in FIG. 10A and 10B, the anti-LAG-3 antibody significantly increased the NFAT-luciferase reporter signal relative to the isotype control antibody in a dose-dependent manner.

7.3. Example 3 Epitope mapping of anti-LAG-3 antibody.

In this example, the epitope of the anti-LAG-3 antibody P13B02-30 was characterized as described below.

.3.1. Epitope mapping of anti-LAG-3 antibody using hydrogen-deuterium exchange (HDX) mass spectrometry.

F(ab') ₂ against LAG-3 was obtained from P13B02-30 (IgG ₄ S228P) using the FragIT kit (Genovis, cat no. A2-FR2-100). The interaction of F(ab') ₂ anti-LAG-3 with human LAG-3 was studied using hydrogen-deuterium exchange (HDX) mass spectrometry.

For pepsin/protease XIII digestion, 7.9 μg of recombinant human-tagged LAG-3 (Sino Biological, cat. no. 16498-H08H) was denatured in 125 μl of control buffer (50 mM phosphate, 100 mM sodium chloride, pH 7.4). , adding 125 μl of 4 M guanidine hydrochloride, 0.85 M TCEP buffer (final pH 2.5) and incubating the mixture for 5 min at 20°C. The mixture was then subjected to pepsin/protease XIII column digestion using a self-packed pepsin/protease XIII column (w/w, 1:1) and the resulting peptides were analyzed using an UHPLC-MS system consisting of a Waters UPLC Acquity coupled to a hybrid quadrupole orbitrap mass -Q-Exactive™ spectrometer (Thermo). Peptides were separated on a 50x1 mm C8 column with a 19-minute gradient from 2-30% solvent B (0.2% formic acid in acetonitrile). Peptide identification was performed by searching MS/MS data against the human LAG-3 sequence with Mascot. The mass tolerance for the procursor and product was 10 ppm and 0.05 Da, respectively.

µl of human LAG-3 (7.9 µg) or 20 µl of human LAG-3/F(ab')2 mixture (7.9 µg: 15.8 µg) was incubated with 105 µl of deuterium oxide labeling buffer (50 mM phosphate sodium, 100 mM sodium chloride, pD 7.4) for 0, 6, 300, 1800, 7200 and 14400 s at 20°C. Hydrogen/deuterium exchange was quenched by adding 125 μl of 4 M guanidine hydrochloride, 0.85 M TCEP buffer (final pH 2.5). Quenched samples were then subjected to pepsin/protease XIII column digestion and LC-MS analysis as described above. Mass spectra were recorded only in MS mode. Raw MS data were processed using HDX Workbench, software for analyzing H/D MS data (J. Am. Soc. Mass Spectrom. 2012, 23(9), 1512-1521, incorporated herein by reference in its entirety ). Deuterium levels were calculated using the average mass difference between the deuterated peptide and its native form ( _t0 ).

The sequence coverage achieved for human LAG-3 was 96.6%. While most human LAG-3 peptides showed identical or similar levels of deuterium with and without F(ab') ₂ versus LAG-3, several peptide segments were found to significantly reduce deuterium incorporation upon F(ab')2 binding. A strong reduction in deuterium uptake was observed in the region consisting of the amino acid sequence SEQ ID NO: 215 (SPTIPLQDLSL). The residues are numbered according to SEQ ID NO: 166.

The invention should not be limited in scope to the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

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Claims (17)

All references (e.g., publications or patents or patent applications) cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) has been specifically and individually stated to be incorporated by reference in its entirety for all purposes. Other embodiments are within the scope of the following claims. CLAIM 1. An isolated antibody that specifically binds to human LAG-3, comprising a heavy chain variable region comprising the complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising the complementarity determining regions CDRL1, CDRL2 and CDRL3, in which CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 contain the amino acid sequences shown in SEQ ID NO: 78, 83, 94, 100, 104 and 105; 78, 85, 95, 100, 104 and 105; 78, 86, 96, 100, 104 and 105; 78, 86, 96, 100, 104 and 109; 78, 86, 96, 100, 104 and 110; 78, 86, 96, 101, 104 and 108; 78, 86, 96, 103, 104 and 111; 78, 86, 97, 102, 104 and 112; 78, 91, 94, 100, 104 and 107; 78, 92, 96, 100, 104 and 105; 78, 92, 96, 100, 104 and 109; 79, 84, 95, 100, 104 and 105; 79, 84, 95, 100, 104 and 106; 79, 84, 95, 102, 104 and 105; 79, 88, 95, 100, 104 and 105; 79, 89, 95, 100, 104 and 105; 79, 90, 95, 100, 104 and 105; 79, 90, 98, 100, 104 and 105; 79, 90, 99, 100, 104 and 105; 80, 85, 96, 100, 104 and 105; 81, 87, 96, 100, 104 and 105; 81, 87, 96, 100, 104 and 107; or 82, 93, 95, 100, 104 and 105 respectively. 2. Isolated antibody according to claim 1, where: (a) said heavy chain variable region comprises framework regions of the heavy chain variable region sequence SEQ ID NO: 151, 222, 218 or 223; (b) said heavy chain variable region contains the amino acid sequence of SEQ ID NO: 151, 222, 218 or 223; (c) said heavy chain variable region contains an amino acid sequence that is at least 75, 80, 85, 90, 95 or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 56-72 and 220; (d) said heavy chain variable region contains framework regions derived from humans; (e) said heavy chain variable framework region is or is derived from an amino acid sequence encoded by a human gene, wherein said amino acid sequence is selected from the group consisting of IGHV1-46*01 (SEQ ID NO: 153), IGHV1-69-2*01 (SEQ ID NO: 154), IGHV1-3*01 (SEQ ID NO: 155), IGHV1-24*01 (SEQ ID NO: 156), IGHV1-2*01 (SEQ ID NO: 157), IGHV1-45 *01 (SEQ ID NO: 158) and IGHV1-18*01 (SEQ ID NO: 159), optionally wherein the heavy chain variable framework region is derived from the amino acid sequence of IGHV1-46*01 (SEQ ID NO: 153), and at least at least one amino acid in the amino acid sequence of IGHV1-46*01 (SEQ ID NO: 153) is replaced by an amino acid at a similar position in the corresponding non-human heavy chain variable framework, optionally where: (i) the amino acid substitution is at an amino acid position selected from the group consisting of 4, 5, 12, 23, 27, 28, 29, 30, 48, 69, 71, 75, 76, 80, 81 and 94, wherein the amino acid the position is indicated according to the Kabat numbering system; (ii) the amino acid substitution is selected from the group consisting of 4M, 5K, 12V, 23T, 27F, 28N, 29I, 30K, 48I, 69I, 71A, 75S, 76N, 80L, 81Q and 94T, the position of the amino acid substitution being indicated according to with Kabat numbering system; and/or (iii) the amino acid substitution is at an amino acid position selected from the group consisting of 4, 27, 28, 29, 30, 69, 71 and 94, the amino acid position being indicated in accordance with the Kabat numbering system; (f) the N-terminal amino acid residue of said heavy chain variable region is pyroglutamate; (g) said antibody contains a heavy chain containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 168-186 and 225-227; and/or (h) said antibody contains a heavy chain that is aglycosylated. 3. Isolated antibody according to claim 1 or 2, where: (a) said light chain variable region comprises framework regions of the light chain variable region sequence SEQ ID NO: 152 or 224; (b) said light chain variable region contains the amino acid sequence of SEQ ID NO: 152 or 224; (c) said light chain variable region contains an amino acid sequence that is at least 75, 80, 85, 90, 95 or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 73-77 and 221; (d) said light chain variable region comprises framework regions derived from human - 107 044026 centuries; (e) said light chain variable framework region which is or is derived from an amino acid sequence encoded by a human gene, wherein said amino acid sequence is selected from the group consisting of IGKV3-20*01 (SEQ ID NO: 160), IGKV3D-15*01 (SEQ ID NO: 161), IGKV3-15*01 (SEQ ID NO: 161), IGKV3D-20*01 (SEQ ID NO: 162), IGKV3D-7*01 (SEQ ID NO: 163), IGKV1-9 *01 (SEQ ID NO: 164) and IGKV3-11*01 (SEQ ID NO: 165), optionally wherein the light chain variable framework region is derived from the amino acid sequence of IGKV320*01 (SEQ ID NO: 160), wherein at least one an amino acid in the amino acid sequence of IGKV3-20*01 (SEQ ID NO: 160) is replaced by an amino acid at a similar position in the corresponding non-human light chain variable framework, optionally where: (i) the amino acid substitution is at an amino acid position selected from the group consisting of 3, 22, 36, 43, 47, 58, 70 and 71, wherein the amino acid position is indicated in accordance with the Kabat numbering system, and optionally where (ii) the amino acid the substitution is selected from the group consisting of 3L, 22T, 36F, 43S, 47W, 58V, 70S and 71Y, the position of the amino acid substitution being indicated in accordance with the Kabat numbering system; (f) the N-terminal amino acid residue of said light chain variable region is pyroglutamate; and/or (g) said antibody contains a light chain containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 187-191 and 228. 4. An isolated antibody that specifically binds to human LAG-3, said antibody comprising: (a) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 56-72 and 220, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 73- 77 and 221; (b) a heavy chain containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 168-186 and 225-227, and a light chain containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 187-191 and 228; (c) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region, respectively, comprise the amino acid sequences set forth in SEQ ID NOs: 56 and 73; 56 and 74; 56 and 75; 56 and 76; 56 and 77; 57 and 73; 57 and 74; 57 and 75; 57 and 76; 57 and 77; 58 and 73; 58 and 74; 58 and 75; 58 and 76; 58 and 77; 59 and 73; 59 and 74; 59 and 75; 59 and 76; 59 and 77; 60 and 73; 60 and 74; 60 and 75; 60 and 76; 60 and 77; 61 and 77; 62 and 77; 63 and 73; 64 and 73; 65 and 73; 220 and 73; 65 and 221; 220 and 221; 66 and 73; 67 and 73; 68 and 73; 69 and 73; 70 and 73; 71 and 73 or 72 and 73; or (d) a heavy chain and a light chain, wherein said heavy chain and said light chain, respectively, comprise the amino acid sequences set forth in SEQ ID NOs: 169 and 187. 5. An isolated antibody according to any of the preceding claims, characterized in that it contains (a) a heavy chain constant region selected from the group consisting of human IgG1, _IgG2 , _IgG3 , IgG4, IgA1 and _IgA2 , optionally wherein said constant region heavy chain is: (i) IgG1 containing the N297A mutation, numbered in accordance with the EU numbering system, optionally containing the amino acid sequence SEQ ID NO: 194; (ii) IgG1 containing the N297Q mutation, numbered according to the EU numbering system; (iii) non-fucosylated IgG1; and/or (iv) IgG4 containing the S228P mutation, numbered in accordance with the EU numbering system, optionally containing a heavy chain constant region containing the amino acid sequence of SEQ ID NO: 196; and/or (b) a human k or λ light chain constant region, optionally wherein said light chain constant region comprises the amino acid sequence of SEQ ID NO: 198 or 219. 6. An isolated antibody according to any of the preceding paragraphs, characterized in that said antibody: (a) binds to an epitope located in the human LAG-3 region consisting of the amino acid sequence SEQ ID NO: 216; (b) binds to an epitope located in the human LAG-3 region consisting of the amino acid sequence SEQ ID NO: 215; (c) binds to an epitope located in the human LAG-3 region consisting of the amino acid sequence SEQ ID NO: 214; (d) binds to an epitope located in the human LAG-3 region consisting of the amino acid sequence SEQ ID NO: 213; (e) binds to an epitope located in the human LAG-3 region consisting of the amino acid sequence SEQ ID NO: 212; and/or - 108 044026 (f) binds to an epitope located in the human LAG-3 region consisting of the amino acid sequence SEQ ID NO: 211. 7. An isolated antibody according to any of the preceding paragraphs, characterized in that said antibody: (a) is a humanized antibody, a murine antibody, or a chimeric antibody; (b) is antagonistic to human LAG-3; (c) inactivates, reduces or inhibits human LAG-3 activity; (d) inhibits the binding of human LAG-3 to MHC class II; (e) induces the production of IL-2 by peripheral blood mononuclear cells (PBMCs) stimulated with Staphylococcal enterotoxin A (SEA); and/or (f) induces the production of TNFa by tumor infiltrating lymphocytes (TILs) stimulated with anti-CD3 and anti-CD28 antibodies. 8. A conjugate containing an isolated antibody according to any one of claims 1 to 7 and a cytotoxic agent, cytostatic agent, toxin or radionuclide. 9. A conjugate containing an isolated antibody according to any one of claims 1-7 and a detectable label. 10. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier or excipient. 11. An isolated polynucleotide encoding the heavy chain variable region or the heavy chain of an antibody according to any one of claims 1 to 7. 12. An isolated polynucleotide encoding a light chain variable region or a light chain of an antibody according to any one of claims 1 to 7. 13. A vector containing a polynucleotide according to claim 11 or 12. 14. Recombinant host cell containing: (a) a polynucleotide according to claim 11 or 12; (b) a vector according to claim 13; (c) a polynucleotide encoding a heavy chain variable region and a light chain variable region, or a heavy chain and a light chain, antibodies according to any one of claims 1 to 7; (d) a first polynucleotide encoding a heavy chain variable region or a heavy chain of an antibody according to any one of claims 1 to 7, and a second polynucleotide encoding a light chain variable region or a light chain of an antibody according to any one of claims 1 to 7; (e) a vector containing a polynucleotide encoding a heavy chain variable region and a light chain variable region, or a heavy chain and a light chain, antibodies according to any one of claims 1 to 7; or (f) a first vector containing a polynucleotide encoding a heavy chain variable region or an antibody heavy chain according to any one of claims 1 to 7, and a second vector containing a polynucleotide encoding a light chain variable region or an antibody light chain according to any one of claims 1 -7. 15. A method for producing an antibody that binds to human LAG-3, comprising culturing a recombinant host cell containing: (a) a polynucleotide encoding a heavy chain variable region and a light chain variable region, or a heavy chain and a light chain, antibodies according to any one of claims 1 to 7; (b) a first polynucleotide encoding a heavy chain variable region or a heavy chain of an antibody according to any one of claims 1 to 7, and a second polynucleotide encoding a light chain variable region or a light chain of an antibody according to any one of claims 1 to 7; (c) a vector containing a polynucleotide encoding a heavy chain variable region and a light chain variable region, or a heavy chain and a light chain, antibodies according to any one of claims 1 to 7; or (d) a first vector containing a polynucleotide encoding a heavy chain variable region or an antibody heavy chain according to any one of claims 1 to 7, and a second vector containing a polynucleotide encoding a light chain variable region or an antibody light chain according to any one of claims 1 -7, so that the antibody is produced. 16. A method of increasing T cell activation in response to an antigen in a subject, comprising administering to the subject an effective amount of an antibody according to any one of claims 1 to 7 or a pharmaceutical composition according to claim 10. 17. A method of treating cancer in a subject, comprising administering to the subject an effective amount of an antibody according to any one of claims 1 to 7 or a pharmaceutical composition according to claim 10. - 109 -