Anti-LAG-3 antibodies and methods of use thereof

Abstract

To provide therapeutic agents designed to antagonize LAG-3 signaling, which are promising for the treatment of diseases involving LAG-3-mediated immunosuppression, given the LAG-3's role in modulating immune responses.SOLUTION: The instant disclosure provides antibodies that specifically bind to LAG-3 (e.g., human LAG-3) and antagonize LAG-3 function. Also provided are pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for making these antibodies, and methods of treating a subject using these antibodies.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Application No. 62 / 406,766, filed October 11, 2016, and U.S. Provisional Application No. 62 / 420,280, filed November 10, 2016, each of which is incorporated by reference in its entirety.

[0002] The present disclosure relates to antibodies that specifically bind to LAG-3 (eg, human LAG-3) and methods of use thereof. [Background technology]

[0003] Lymphocyte activation gene 3 (LAG-3), also known as CD223, is a type I membrane protein of the immunoglobulin (Ig) superfamily consisting of four extracellular Ig domains and a cytoplasmic domain containing conserved repeated EP motifs and a single 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 persistent antigen exposure, such as chronic pathogenic infections or within the tumor microenvironment (TME), LAG-3 expression is maintained on type 1 regulatory T cells (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).

[0004] LAG-3 functions to negatively regulate 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 cytokine secretion of 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 that were also genetically deficient for PD-1 (Okazaki et al., (2011) J Exp Med, 208:395-407). Furthermore, in vivo blockade with a monoclonal antibody against mouse LAG-3 synergized in combination with PD-1 blockade to enhance antitumor immunity in a syngeneic mouse tumor model (Woo et al., (2012) Cancer Res, 72:917-27). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 7,220,417 (B2) Summary of the Invention [Problem to be solved by the invention]

[0006] Given the role of LAG-3 in regulating immune responses, therapeutic agents designed to antagonize LAG-3 signaling hold considerable promise for the treatment of diseases involving LAG-3-mediated immunosuppression. [Means for solving the problem]

[0007] The present disclosure provides antibodies that specifically bind to LAG-3 (e.g., human LAG-3) and antagonize LAG-3 function, e.g., LAG-3-mediated immunosuppression. Pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells making these antibodies, and methods of treating a subject with these antibodies are also provided. The antibodies disclosed herein are particularly useful for treating cancer in a subject, or treating or preventing an infectious disease in a subject, because they increase T cell activation in response to an antigen (e.g., a tumor antigen, or an infectious disease antigen) and / or decrease Treg-mediated immunosuppression.

[0008] Thus, in one aspect, the disclosure provides an antibody or isolated antibody comprising a heavy chain variable region comprising complementarity determining regions CDRH1, CDRH2, and CDRH3, and a light chain variable region comprising complementarity determining regions CDRL1, CDRL2, and CDRL3, (a) CDRH1 comprises the amino acid sequence of DX1YX2X3 (SEQ ID NO:140), X1 is T or N; X2 is I or M, and X3 is H, Y, or D; (b) CDRH2 is X1IDPANX2X3X4X5X6X7PX8X9QX 10 (SEQ ID NO: 142), X1 is E, R, S, or K; X2 is D or G; X3 is N or H; X4 is T or S; X5 is K or H; X6 is Y or F; X7 is D or A; X8 is K or R; X9 is F or L, and X 10 is G or D, (c) CDRH3 comprises an amino acid sequence of YX1X2X3YX4VGGX5DY (SEQ ID NO: 144), X1 is Y, F, or S; X2 is Y or D; X3 is K or R; X4 is D or E, and X5 is F or C; (d) CDRL1 comprises the amino acid sequence of SVSSX1ISSSX2LX3 (SEQ ID NO:147), X1 is S or G; X2 is N or T, and X3 is H or Y; (e) CDRL2 comprises the amino acid sequence of GTSNLAS (SEQ ID NO: 104), and (f) CDRL3 comprises the amino acid sequence of QQWX1X2YPX3T (SEQ ID NO: 149), X1 is S, N, or R; X2 is S, T, or R; and X3 is F, L, H, or W,

[0009] In another aspect, the disclosure provides an antibody or isolated antibody that specifically binds to human LAG-3, comprising a heavy chain variable region comprising complementarity determining regions CDRH1, CDRH2, and CDRH3, and a light chain variable region comprising complementarity determining regions CDRL1, CDRL2, and CDRL3; (a) CDRH1 comprises the amino acid sequence of DX1YX2X3 (SEQ ID NO:140), X1 is T or N; X2 is I or M, and X3 is H, Y, or D; (b) CDRH2 is X1IDPANX2X3X4X5X6X7PX8X9QX 10 (SEQ ID NO: 142), X1 is E, R, S, or K; X2 is D or G; X3 is N or H; X4 is T or S; X5 is K or H; X6 is Y or F; X7 is D or A; X8 is K or R; X9 is F or L, and X 10 is G or D, (c) CDRH3 comprises an amino acid sequence of YX1X2X3YX4VGGX5DY (SEQ ID NO: 144), X1 is Y, F, or S; X2 is Y or D; X3 is K or R; X4 is D or E, and X5 is F or C; (d) CDRL1 comprises the amino acid sequence of SVSSX1ISSSX2LX3 (SEQ ID NO:147), X1 is S or G; X2 is N or T, and X3 is H or Y; (e) CDRL2 comprises the amino acid sequence of GTSNLAS (SEQ ID NO: 104), and (f) CDRL3 comprises the amino acid sequence of QQWX1X2YPX3T (SEQ ID NO: 149), X1 is S, N, or R; X2 is S, T, or R; and X3 is F, L, H, or W,

[0010] In certain embodiments, CDRH1 comprises the amino acid sequence of DX1YX2X3 (SEQ ID NO: 141), where: X1 is T or N, X2 is I or M, and X3 is H or Y. In certain embodiments, CDRH2 comprises the amino acid sequence of X1IDPANX2X3X4KX5X6PX7FQX8 (SEQ ID NO: 143), where: X1 is E, R, or S, X2 is D or G, X3 is N or H, X4 is T or S, X5 is Y or F, X6 is D or A, X7 is K or R, and X8 is G or D. In certain embodiments, CDRH3 comprises the amino acid sequence of YX1X2X3YDVGGX4DY (SEQ ID NO: 145), where X1 is Y, F, or S, X2 is Y or D, X3 is K or R, and X4 is F or C. In certain embodiments, CDRH3 comprises the amino acid sequence of YYYX1YX2VGGFDY (SEQ ID NO: 146), where X1 is K or R, and X2 is D or E. In certain embodiments, CDRL1 comprises the amino acid sequence of SVSSSISSSNLX1 (SEQ ID NO: 148), where X1 is H or Y. In certain embodiments, CDRL3 comprises the amino acid sequence of QQWX1SYPX2T (SEQ ID NO: 150), where X1 is S, N, or R, and X2 is F, L, or H.

[0011] In certain embodiments, (a) CDRH1 comprises the amino acid sequence of DTYIH (SEQ ID NO:79); (b) CDRH2 comprises the amino acid sequence of EIDPANDNTKYDPKFQG (SEQ ID NO: 90); (c) CDRH3 comprises an amino acid sequence of YYYX1YX2VGGFDY (SEQ ID NO:146), where: Xi is K or R, and X2 is D or E; (d) CDRL1 comprises the amino acid sequence of SVSSSISSSNLH (SEQ ID NO: 100); (e) CDRL2 comprises the amino acid sequence of GTSNLAS (SEQ ID NO: 104), and (f) CDRL3 contains the amino acid sequence of QQWSSYPFT (SEQ ID NO: 105).

[0012] In certain embodiments, CDRH1, CDRH2, and CDRH3 comprise the amino acid sequences set forth in SEQ ID NOs: 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%, or 99% identical to the amino acid sequence of SEQ ID NO: 65 or 220. In certain embodiments, the heavy chain variable region 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 the 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 comprising the amino acid sequence of SEQ ID NO: 169.

[0013] In certain embodiments, CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences set forth in SEQ ID NOs: 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%, or 99% identical to the amino acid sequence of SEQ ID NO: 73 or 221. In certain embodiments, the light chain variable region 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 the amino acid sequence of SEQ ID NO: 73. 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.

[0014] In certain embodiments, CDRH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 78 to 82. In certain embodiments, CDRH2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 83 to 93. In certain embodiments, CDRH3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 94 to 99. In certain embodiments, CDRL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 100 to 103. In certain embodiments, CDRL3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 105 to 112.

[0015] In certain embodiments, CDRH1, CDRH2, and CDRH3 comprise the CDRH1, CDRH2, and CDRH3 amino acid sequences 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, respectively.

[0016] In certain embodiments, CDRL1, CDRL2, and CDRL3 comprise the CDRL1, CDRL2, and CDRL3 amino acid sequences 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, respectively.

[0017] In certain embodiments, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are selected from the group consisting of SEQ ID NOs: 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, 8 6, 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 ... 8, 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.

[0018] In another aspect, the disclosure provides an antibody or isolated antibody comprising a heavy chain variable region comprising complementarity determining regions CDRH1, CDRH2, and CDRH3, and a light chain variable region comprising complementarity determining regions CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences set forth in SEQ ID NOs: 79, 90, 95, 100, 104, and 105, respectively.

[0019] In another aspect, the disclosure provides an antibody or isolated antibody that specifically binds to human LAG-3, comprising a heavy chain variable region comprising complementarity determining regions CDRH1, CDRH2, and CDRH3, and a light chain variable region comprising complementarity determining regions CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences set forth in SEQ ID NOs: 79, 90, 95, 100, 104, and 105, respectively.

[0020] In another aspect, the disclosure provides an antibody or isolated antibody comprising a heavy chain variable region comprising complementarity determining regions CDRH1, CDRH2, and CDRH3, and a light chain variable region comprising complementarity determining regions CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences set forth in SEQ ID NOs: 79, 90, 98, 100, 104, and 105, respectively.

[0021] In another aspect, the disclosure provides an antibody or isolated antibody that specifically binds to human LAG-3, comprising a heavy chain variable region comprising complementarity determining regions CDRH1, CDRH2, and CDRH3, and a light chain variable region comprising complementarity determining regions CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences set forth in SEQ ID NOs: 79, 90, 98, 100, 104, and 105, respectively.

[0022] In certain embodiments, the heavy chain variable region comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 65 or 220. In certain embodiments, the heavy chain variable region 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 the 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.

[0023] In certain embodiments, the light chain variable region comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 73 or 221. In certain embodiments, the light chain variable region 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 the amino acid sequence of SEQ ID NO: 73. 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: 228. In certain embodiments, the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 187.

[0024] In certain embodiments, the heavy chain variable region comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 65 or 220, and the light chain variable region comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 73 or 221. In certain embodiments, the heavy chain variable region comprises 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 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 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.

[0025] In another aspect, the disclosure provides an isolated antibody that specifically binds to human LAG-3, the antibody 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 an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 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.

[0026] In another aspect, the disclosure provides an isolated antibody that specifically binds to human LAG-3, the antibody 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 an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 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.

[0027] In certain embodiments, the antibody comprises a heavy chain variable region comprising the framework regions of the 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. In certain embodiments, the antibody comprises a heavy chain variable region comprising the framework regions of the 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 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 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 NOs: 168-186, and 225-227. In certain embodiments, the antibody comprises a heavy chain variable region having a human-derived framework region. In certain embodiments, the antibody comprises a heavy chain variable framework region that is or is derived from an amino acid sequence encoded by a human gene, and the 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 IGHV1-46*01 (SEQ ID NO: 153), wherein at least one amino acid in the amino acid sequence IGHV1-46*01 (SEQ ID NO: 153) is replaced with an amino acid at an analogous position in a corresponding non-human heavy chain variable framework region.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, where the amino acid positions are designated according to the Kabat numbering system. In certain embodiments, the amino acid substitution is at an amino acid position selected from the group consisting of 4M, 5K, 12V, 23T, 27F, 28N, 29I, 30K, 48I, 69I, 71A, 75S, 76N, 80L, 81Q, and 94T, where the amino acid positions are designated according to the Kabat numbering system. In certain embodiments, 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 positions are designated according to the Kabat numbering system. In certain embodiments, the amino acid substitutions are selected from the group consisting of 4M, 27F, 28N, 29I, 30K, 69I, 71A, and 94T, and the positions of the amino acid substitutions are indicated according to the Kabat numbering system.

[0028] In certain embodiments, the antibody comprises a light chain variable region comprising the framework region of the 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. 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 the amino acid sequence of 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 NOs: 187-191. In certain embodiments, the antibody comprises a light chain variable region having a human-derived framework region. 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, the amino acid sequence being 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 IGKV3-20*01 (SEQ ID NO: 160), wherein at least one amino acid in the amino acid sequence IGKV3-20*01 (SEQ ID NO: 160) is replaced with an amino acid at the analogous position in a corresponding non-human light chain variable framework region. In certain embodiments, the amino acid replacement 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 positions are designated according to the Kabat numbering system.In certain embodiments, the amino acid substitutions are selected from the group consisting of 3L, 22T, 36F, 43S, 47W, 58V, 70S, and 71Y, and the positions of the amino acid substitutions are indicated according to the Kabat numbering system.

[0029] In another aspect, the disclosure 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 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 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 comprising the amino acid sequence of SEQ ID NO: 225. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 168. 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 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 227. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 170.

[0030] In another aspect, the disclosure provides an antibody or isolated antibody that specifically binds to human LAG-3, the 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 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 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 comprising the amino acid sequence of SEQ ID NO: 225. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 168. 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 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 227. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 170.

[0031] In another aspect, the disclosure 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 the amino acid sequence of 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 NOs: 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.

[0032] In another aspect, the disclosure provides an antibody or isolated antibody that specifically binds to human LAG-3, the 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 the amino acid sequence of 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 NOs: 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.

[0033] In another aspect, the disclosure includes heavy chain variable regions and light chain variable regions, the heavy chain variable regions and light chain variable regions being 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 heavy chain variable region and the light chain variable region comprise the amino acid sequence set forth in SEQ ID NOs: 65 and 73, respectively. In certain embodiments, the heavy chain variable region and the light chain variable region comprise the amino acid sequence set forth in SEQ ID NOs: 220 and 73, 65 and 221, or 220 and 221, respectively.

[0034] In another aspect, the disclosure includes 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 are 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 certain embodiments, the amino acid sequences of the heavy chain variable region and the light chain variable region consist of the amino acid sequences set forth in SEQ ID NOs: 65 and 73, respectively. In certain embodiments, the amino acid sequences of the heavy chain variable region and the light chain variable region consist of the amino acid sequences set forth in SEQ ID NOs: 220 and 73, 65 and 221, or 220 and 221, respectively.

[0035] In another aspect, the disclosure provides an antibody or isolated antibody that specifically binds to human LAG-3, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region being 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, The antibody or isolated antibody comprises the amino acid sequence set forth in SEQ ID NOs: 9 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 heavy chain variable region and the light chain variable region comprise the amino acid sequence set forth in SEQ ID NOs: 65 and 73, respectively. In certain embodiments, the heavy chain variable region and the light chain variable region comprise the amino acid sequence set forth in SEQ ID NOs: 220 and 73, 65 and 221, or 220 and 221, respectively.

[0036] In another aspect, the disclosure provides 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 are 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 7 In certain embodiments, the heavy and light chain variable regions comprise the amino acid sequences set forth in SEQ ID NOs: 65 and 73, respectively. In certain embodiments, the heavy and light chain variable regions comprise the amino acid sequences set forth in SEQ ID NOs: 65 and 73, respectively. In certain embodiments, the heavy and light chain variable regions comprise the amino acid sequences set forth in SEQ ID NOs: 220 and 73, 65 and 221, respectively.

[0037] In certain embodiments of any one of the preceding aspects, X in SEQ ID NO:220 is Q, if applicable. In certain embodiments of any one of the preceding aspects, X in SEQ ID NO:220 is pyroglutamic acid, if applicable. In certain embodiments of any one of the preceding aspects, X in SEQ ID NO:221 is E, if applicable. In certain embodiments of any one of the preceding aspects, X in SEQ ID NO:221 is pyroglutamic acid, if applicable. In certain embodiments of any one of the preceding aspects, X in SEQ ID NO:220 is Q and X in SEQ ID NO:221 is E, if applicable. In certain embodiments of any one of the preceding aspects, X in SEQ ID NO:220 is Q and X in SEQ ID NO:221 is pyroglutamic acid, if applicable. In certain embodiments of any one of the preceding aspects, X in SEQ ID NO:220 is pyroglutamic acid and X in SEQ ID NO:221 is E, if applicable. In certain embodiments of any one of the preceding aspects, X in SEQ ID NO:220 is pyroglutamic acid, and X in SEQ ID NO:221 is pyroglutamic acid, if applicable.

[0038] In another aspect, the disclosure 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 comprising the amino acid sequence of SEQ ID NO: 187 or 228.

[0039] In another aspect, the disclosure provides an antibody or isolated antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:168, and a light chain comprising the amino acid sequence of SEQ ID NO:187.

[0040] In another aspect, the disclosure provides an antibody or isolated antibody that specifically binds to human LAG-3, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 168 or 225, and a light chain comprising the amino acid sequence of SEQ ID NO: 187 or 228.

[0041] In another aspect, the disclosure provides an antibody or isolated antibody that specifically binds to human LAG-3, the antibody or isolated antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 168 and a light chain comprising the amino acid sequence of SEQ ID NO: 187.

[0042] In another aspect, the disclosure 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 comprising the amino acid sequence of SEQ ID NO: 187 or 228.

[0043] In another aspect, the disclosure 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.

[0044] In another aspect, the disclosure provides an antibody or isolated antibody that specifically binds to human LAG-3, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 169 or 226, and a light chain comprising the amino acid sequence of SEQ ID NO: 187 or 228.

[0045] In another aspect, the disclosure provides an antibody or isolated antibody that specifically binds to human LAG-3, the 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.

[0046] In another aspect, the disclosure 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 comprising the amino acid sequence of SEQ ID NO: 187 or 228.

[0047] In another aspect, the disclosure 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.

[0048] In another aspect, the disclosure provides an antibody or isolated antibody that specifically binds to human LAG-3, 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.

[0049] In another aspect, the disclosure provides an antibody or isolated antibody that specifically binds to human LAG-3, the 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.

[0050] In another aspect, the disclosure provides an antibody or isolated antibody 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 of the light chain consists of the amino acid sequence set forth in SEQ ID NO: 187 or 228.

[0051] In another aspect, the disclosure provides an antibody or isolated antibody comprising a heavy chain and a light chain, wherein the amino acid sequences of the heavy chain and the light chain consist of the amino acid sequences set forth in SEQ ID NOs: 168 and 187, respectively.

[0052] In another aspect, the 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 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 of the light chain consists of the amino acid sequence set forth in SEQ ID NO: 187 or 228.

[0053] In another aspect, the 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 the light chain consist of the amino acid sequences set forth in SEQ ID NOs: 168 and 187, respectively.

[0054] In another aspect, the disclosure provides an antibody or isolated antibody 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 of the light chain consists of the amino acid sequence set forth in SEQ ID NO: 187 or 228.

[0055] In another aspect, the disclosure provides an antibody or isolated antibody comprising a heavy chain and a light chain, wherein the amino acid sequences of the heavy chain and the light chain consist of the amino acid sequences set forth in SEQ ID NOs: 169 and 187, respectively.

[0056] In another aspect, the 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 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 of the light chain consists of the amino acid sequence set forth in SEQ ID NO: 187 or 228.

[0057] In another aspect, the 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 the light chain consist of the amino acid sequences set forth in SEQ ID NOs: 169 and 187, respectively.

[0058] In another aspect, the disclosure provides an antibody or isolated antibody 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 of the light chain consists of the amino acid sequence set forth in SEQ ID NO: 187 or 228.

[0059] In another aspect, the disclosure provides an antibody or isolated antibody comprising a heavy chain and a light chain, wherein the amino acid sequences of the heavy chain and the light chain consist of the amino acid sequences set forth in SEQ ID NOs: 170 and 187, respectively.

[0060] In another aspect, the 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 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 of the light chain consists of the amino acid sequence set forth in SEQ ID NO: 187 or 228.

[0061] In another aspect, the 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 the light chain consist of the amino acid sequences set forth in SEQ ID NOs: 170 and 187, respectively.

[0062] In certain embodiments of any one of the preceding aspects, X in SEQ ID NO:225 is Q, if applicable. In certain embodiments of any one of the preceding aspects, X in SEQ ID NO:225 is pyroglutamic acid, if applicable. In certain embodiments of any one of the preceding aspects, X in SEQ ID NO:228 is E, if applicable. In certain embodiments of any one of the preceding aspects, X in SEQ ID NO:228 is pyroglutamic acid, if applicable. In certain embodiments of any one of the preceding aspects, X in SEQ ID NO:225 is Q and X in SEQ ID NO:228 is E, if applicable. In certain embodiments of any one of the preceding aspects, X in SEQ ID NO:225 is Q and X in SEQ ID NO:228 is pyroglutamic acid, if applicable. In certain embodiments of any one of the preceding aspects, X in SEQ ID NO:225 is pyroglutamic acid and X in SEQ ID NO:228 is E, if applicable. In certain embodiments of any one of the preceding aspects, X in SEQ ID NO:225 is pyroglutamic acid and X in SEQ ID NO:228 is pyroglutamic acid, if applicable.

[0063] In certain embodiments of any one of the preceding aspects, X in SEQ ID NO:226 is Q, if applicable. In certain embodiments of any one of the preceding aspects, X in SEQ ID NO:226 is pyroglutamic acid, if applicable. In certain embodiments of any one of the preceding aspects, X in SEQ ID NO:228 is E, if applicable. In certain embodiments of any one of the preceding aspects, X in SEQ ID NO:228 is pyroglutamic acid, if applicable. In certain embodiments of any one of the preceding aspects, X in SEQ ID NO:226 is Q and X in SEQ ID NO:228 is E, if applicable. In certain embodiments of any one of the preceding aspects, X in SEQ ID NO:226 is Q and X in SEQ ID NO:228 is pyroglutamic acid, if applicable. In certain embodiments of any one of the preceding aspects, X in SEQ ID NO:226 is pyroglutamic acid and X in SEQ ID NO:228 is E, if applicable. In certain embodiments of any one of the preceding aspects, X in SEQ ID NO:226 is pyroglutamic acid, and X in SEQ ID NO:228 is pyroglutamic acid, if applicable.

[0064] In certain embodiments of any one of the preceding aspects, X in SEQ ID NO:227 is Q, if applicable. In certain embodiments of any one of the preceding aspects, X in SEQ ID NO:227 is pyroglutamic acid, if applicable. In certain embodiments of any one of the preceding aspects, X in SEQ ID NO:228 is E, if applicable. In certain embodiments of any one of the preceding aspects, X in SEQ ID NO:228 is pyroglutamic acid, if applicable. In certain embodiments of any one of the preceding aspects, X in SEQ ID NO:227 is Q and X in SEQ ID NO:228 is E, if applicable. In certain embodiments of any one of the preceding aspects, X in SEQ ID NO:227 is Q and X in SEQ ID NO:228 is pyroglutamic acid, if applicable. In certain embodiments of any one of the preceding aspects, X in SEQ ID NO:227 is pyroglutamic acid and X in SEQ ID NO:228 is E, if applicable. In certain embodiments of any one of the preceding aspects, X in SEQ ID NO:227 is pyroglutamic acid, and X in SEQ ID NO:228 is pyroglutamic acid, if applicable.

[0065] In certain embodiments, the antibody comprises a heavy chain constant region selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, the heavy chain constant region is IgG1. In certain embodiments, the amino acid sequence of IgG1 comprises an N297A mutation and is numbered according to the EU numbering system. In certain embodiments, the antibody is a constant region comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 194. In certain embodiments, the amino acid sequence of IgG1 comprises an N297Q mutation and is numbered according to the EU numbering system. In certain embodiments, the IgG1 is a non-fucosylated IgG1. In certain embodiments, the heavy chain constant region is IgG4. In certain embodiments, the amino acid sequence of IgG4 comprises an S228P mutation and is numbered according to the EU numbering system. In certain embodiments, the antibody is a constant region comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 196.

[0066] In certain embodiments, the antibody comprises a light chain constant region selected from the group consisting of human IgGκ and IgGλ. In certain embodiments, the light chain constant region is IgGκ. 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.

[0067] In another aspect, the disclosure provides an antibody or isolated antibody that cross-competes with an antibody disclosed herein for binding to human LAG-3. In certain embodiments, the disclosure provides an antibody or isolated antibody that cross-competes with an antibody comprising the heavy and light chain variable region amino acid sequences set forth in SEQ ID NOs: 15 and 16, respectively, for binding to human LAG-3. In certain embodiments, the disclosure provides an antibody or isolated antibody that cross-competes 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, for binding to human LAG-3.

[0068] In another aspect, the disclosure provides an antibody or isolated antibody that binds to the same epitope of human LAG-3 as the antibodies disclosed herein. In certain embodiments, the disclosure provides an antibody or isolated antibody that binds to the same epitope of human LAG-3 as an 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 disclosure provides an antibody or isolated antibody that binds to the same epitope of human LAG-3 as 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.

[0069] In another aspect, the disclosure provides an antibody or isolated antibody that binds, e.g., specifically binds, to an epitope of human LAG-3. In certain embodiments, the antibody binds to an epitope located within a region of human LAG-3 consisting of the amino acid sequence of SEQ ID NO:216. In certain embodiments, the antibody binds to an epitope located within a region of human LAG-3 consisting of the amino acid sequence of SEQ ID NO:215. In certain embodiments, the antibody binds to an epitope located within a region of human LAG-3 consisting of the amino acid sequence of SEQ ID NO:214. In certain embodiments, the antibody binds to an epitope located within a region of human LAG-3 consisting of the amino acid sequence of SEQ ID NO:213. In certain embodiments, the antibody binds to an epitope located within a region of human LAG-3 consisting of the amino acid sequence of SEQ ID NO:212. In certain embodiments, the antibody binds to an epitope located within a region of human LAG-3 consisting of the amino acid sequence of SEQ ID NO:211.

[0070] In another aspect, the disclosure provides an antibody or isolated antibody that specifically binds to the same epitope of human LAG-3 as any of the antibodies of the present invention. In certain embodiments, the antibody binds to an epitope located within a region of human LAG-3 consisting of the amino acid sequence of SEQ ID NO: 216. In certain embodiments, the antibody binds to an epitope located within a region of human LAG-3 consisting of the amino acid sequence of SEQ ID NO: 215. In certain embodiments, the antibody binds to an epitope located within a region of human LAG-3 consisting of the amino acid sequence of SEQ ID NO: 214. In certain embodiments, the antibody binds to an epitope located within a region of human LAG-3 consisting of the amino acid sequence of SEQ ID NO: 213. In certain embodiments, the antibody binds to an epitope located within a region of human LAG-3 consisting of the amino acid sequence of SEQ ID NO: 212. In certain embodiments, the antibody binds to an epitope located within a region of human LAG-3 consisting of the amino acid sequence of SEQ ID NO: 211.

[0071] In another aspect, the disclosure provides an antibody that, when bound to a human LAG-3 protein comprising the amino acid sequence of SEQ ID NO:217 or a fragment thereof, 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 in a region consisting of the amino acid sequence set forth in SEQ ID NO:216 in the absence of the antibody, as determined by a hydrogen / deuterium assay. In another aspect, the disclosure provides an antibody that, when bound to a human LAG-3 protein comprising the amino acid sequence of SEQ ID NO:217 or a fragment thereof, reduces hydrogen / deuterium exchange in a region consisting of the amino acid sequence set forth in SEQ ID NO:215 relative to hydrogen / deuterium exchange in a region consisting of the amino acid sequence set forth in SEQ ID NO:215 in the absence of the antibody, as determined by a hydrogen / deuterium assay. In another aspect, the disclosure provides an antibody that, when bound to a human LAG-3 protein comprising the amino acid sequence of SEQ ID NO:217 or a fragment thereof, reduces hydrogen / deuterium exchange in a region consisting of the amino acid sequence set forth in SEQ ID NO:214 relative to hydrogen / deuterium exchange in a region consisting of the amino acid sequence set forth in SEQ ID NO:214 in the absence of the antibody, as determined by a hydrogen / deuterium assay. In another aspect, the disclosure provides an antibody which, when binding to a human LAG-3 protein comprising the amino acid sequence of SEQ ID NO:217 or a fragment thereof, reduces hydrogen / deuterium exchange in a region consisting of the amino acid sequence set forth in SEQ ID NO:213 relative to hydrogen / deuterium exchange in a region consisting of the amino acid sequence set forth in SEQ ID NO:213 in the absence of the antibody, as determined by a hydrogen / deuterium assay. In another aspect, the disclosure provides an antibody which, when binding to a human LAG-3 protein comprising the amino acid sequence of SEQ ID NO:217 or a fragment thereof, reduces hydrogen / deuterium exchange in a region consisting of the amino acid sequence set forth in SEQ ID NO:212 relative to hydrogen / deuterium exchange in a region consisting of the amino acid sequence set forth in SEQ ID NO:212 in the absence of the antibody, as determined by a hydrogen / deuterium assay.In another aspect, the disclosure provides antibodies that, when bound to a human LAG-3 protein comprising the amino acid sequence of SEQ ID NO: 217, or a fragment thereof, reduce hydrogen / deuterium exchange in a region consisting of the amino acid sequence set forth in SEQ ID NO: 211, relative to hydrogen / deuterium exchange in a region consisting of the amino acid sequence set forth in SEQ ID NO: 211 in the absence of the antibody, as determined by a hydrogen / deuterium assay. In some embodiments, the reduction in hydrogen / deuterium exchange is measured using hydrogen-deuterium exchange (HDX), e.g., as described herein in the Examples.

[0072] In another aspect, the disclosure provides an antibody or isolated antibody that specifically binds to the same epitope of human LAG-3 as any of the antibodies of the present invention. In certain embodiments, the antibody, when binding to a human LAG-3 protein comprising the amino acid sequence of SEQ ID NO:217 or a fragment thereof, reduces hydrogen / deuterium exchange in the region consisting of the amino acid sequence set forth in SEQ ID NO:216 relative to hydrogen / deuterium exchange in the region consisting of the amino acid sequence set forth in SEQ ID NO:216 in the absence of the antibody, as determined by hydrogen / deuterium assay. In certain embodiments, the antibody, when binding to a human LAG-3 protein comprising the amino acid sequence of SEQ ID NO:217 or a fragment thereof, reduces hydrogen / deuterium exchange in the region consisting of the amino acid sequence set forth in SEQ ID NO:215 relative to hydrogen / deuterium exchange in the region consisting of the amino acid sequence set forth in SEQ ID NO:215 in the absence of the antibody, as determined by hydrogen / deuterium assay. In certain embodiments, when the antibody binds to a human LAG-3 protein comprising the amino acid sequence of SEQ ID NO:217 or a fragment thereof, it reduces hydrogen / deuterium exchange in the region consisting of the amino acid sequence set forth in SEQ ID NO:214 relative to hydrogen / deuterium exchange in the region consisting of the amino acid sequence set forth in SEQ ID NO:214 in the absence of the antibody, as determined by hydrogen / deuterium assay. In certain embodiments, when the antibody binds to a human LAG-3 protein comprising the amino acid sequence of SEQ ID NO:217 or a fragment thereof, it reduces hydrogen / deuterium exchange in the region consisting of the amino acid sequence set forth in SEQ ID NO:213 relative to hydrogen / deuterium exchange in the region consisting of the amino acid sequence set forth in SEQ ID NO:213 in the absence of the antibody, as determined by hydrogen / deuterium assay. In certain embodiments, when the antibody binds to a human LAG-3 protein comprising the amino acid sequence of SEQ ID NO:217 or a fragment thereof, it reduces hydrogen / deuterium exchange in the region consisting of the amino acid sequence set forth in SEQ ID NO:212 relative to hydrogen / deuterium exchange in the region consisting of the amino acid sequence set forth in SEQ ID NO:212 in the absence of the antibody, as determined by hydrogen / deuterium assay.In certain embodiments, the antibody, when binding to a human LAG-3 protein comprising the amino acid sequence of SEQ ID NO: 217 or a fragment thereof, 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 in a region consisting of the amino acid sequence set forth in SEQ ID NO: 211 in the absence of the antibody, as determined by a hydrogen / deuterium assay. In some embodiments, the reduction in hydrogen / deuterium exchange is measured using hydrogen-deuterium exchange (HDX), e.g., as described herein in the Examples.

[0073] 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 to human LAG-3. In certain embodiments, the antibody inactivates, reduces, or inhibits the activity of human LAG-3. In certain embodiments, the antibody inhibits the binding of human LAG-3 to MHC class II. In certain embodiments, the antibody induces IL-2 production by peripheral blood mononuclear cells (PBMCs) stimulated with Staphylococcal enterotoxin A (SEA). In certain embodiments, the antibody induces TNFα production by tumor infiltrating lymphocytes (TILs) stimulated with anti-CD3 and anti-CD28 antibodies.

[0074] In another aspect, the disclosure provides an antibody or isolated antibody disclosed herein conjugated to a cytotoxic agent.

[0075] In another aspect, the disclosure provides an antibody or isolated antibody as disclosed herein conjugated to a cytostatic agent.

[0076] In another aspect, the disclosure provides an antibody or isolated antibody disclosed herein conjugated to a toxin.

[0077] In another aspect, the present disclosure provides an antibody or isolated antibody as disclosed herein conjugated to a radionuclide.

[0078] In another aspect, the disclosure provides an antibody or an isolated antibody disclosed herein conjugated to a detectable label.

[0079] In another aspect, the disclosure provides an antibody or isolated antibody as disclosed herein, wherein the N-terminal amino acid residue of the heavy chain variable region is pyroglutamic acid (e.g., as a result of post-translational cyclization of the free amino group of the N-terminal E or Q residue of the heavy chain variable region). In another aspect, the disclosure provides an antibody or isolated antibody as disclosed herein, wherein the N-terminal amino acid residue of the heavy chain is pyroglutamic acid (e.g., as a result of post-translational cyclization of the free amino group of the N-terminal E or Q residue of the heavy chain).

[0080] In another aspect, the disclosure provides an antibody or isolated antibody as disclosed herein, wherein the N-terminal amino acid residue of the light chain variable region is pyroglutamic acid (e.g., as a result of post-translational cyclization of the free amino group of the N-terminal E or Q residue of the light chain variable region). In another aspect, the disclosure provides an antibody or isolated antibody as disclosed herein, wherein the N-terminal amino acid residue of the light chain is pyroglutamic acid (e.g., as a result of post-translational cyclization of the free amino group of the N-terminal E or Q residue of the light chain).

[0081] In another aspect, the disclosure provides an antibody or isolated antibody as disclosed herein, wherein the heavy chain is aglycosylated.

[0082] In another aspect, the disclosure provides a pharmaceutical composition comprising an antibody disclosed herein and a pharma- ceutically acceptable carrier or excipient.

[0083] In another aspect, the disclosure provides an isolated polynucleotide encoding a heavy and / or light chain of an antibody disclosed herein. In another aspect, the disclosure provides a vector comprising the polynucleotide. In another aspect, the disclosure provides a recombinant host cell comprising the polynucleotide. In another aspect, the disclosure provides a recombinant host cell comprising the vector. In another aspect, the disclosure provides a method of producing an antibody disclosed herein, 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.

[0084] In one embodiment, the invention relates to 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 medicament.

[0085] In one embodiment, the invention relates to 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 diagnostic.

[0086] In another aspect, the present disclosure provides 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 or pharmaceutical composition disclosed herein. In another aspect, the present disclosure provides a method of treating cancer in a subject, comprising administering to the subject an effective amount of an antibody or pharmaceutical composition disclosed herein. In certain embodiments of the aforementioned methods, the antibody or pharmaceutical composition is administered subcutaneously. In certain embodiments of the aforementioned methods, the antibody or pharmaceutical composition is administered intravenously. In certain embodiments of the aforementioned methods, the antibody or pharmaceutical composition is administered intratumorally. In certain embodiments of the aforementioned methods, the antibody or pharmaceutical composition is delivered to a tumor-draining lymph node. In certain embodiments of the aforementioned methods, the antibody or pharmaceutical composition is administered intra-arterially. In certain embodiments of the aforementioned methods, the antibody or pharmaceutical composition is administered intranasally.

[0087] In one aspect, the invention relates to 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.

[0088] In one aspect, the invention relates to 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.

[0089] In one aspect, the invention relates to 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 of the invention.

[0090] In one aspect, the invention relates to an antibody, polynucleotide, vector, recombinant host cell, and / or pharmaceutical composition of the invention for use in a method for the treatment of cancer.

[0091] In one aspect, the invention relates to an antibody, polynucleotide, vector, recombinant host cell, and / or pharmaceutical composition of the invention for use in a method of treating cancer in a subject.

[0092] In one aspect, the invention relates to an antibody, polynucleotide, vector, recombinant host cell, and / or pharmaceutical composition of the 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 of the invention.

[0093] In one embodiment of the antibody, polynucleotide, vector, recombinant host cell, and / or pharmaceutical composition for use according to the invention, the antibody, polynucleotide, vector, recombinant host cell, and / or pharmaceutical composition is administered subcutaneously or intravenously. In one embodiment of the antibody, polynucleotide, vector, recombinant host cell, and / or pharmaceutical composition for use according to the invention, the antibody, polynucleotide, vector, recombinant host cell, and / or pharmaceutical composition is administered intratumorally or intraarterially. In one embodiment of the antibody, polynucleotide, vector, recombinant host cell, and / or pharmaceutical composition for use according to the invention, the antibody, polynucleotide, vector, recombinant host cell, and / or pharmaceutical composition is administered intranasally.

[0094] In certain embodiments, the aforementioned methods further comprise administering an additional therapeutic agent to the subject. Thus, in one embodiment of the antibodies, polynucleotides, vectors, recombinant host cells, and / or pharmaceutical compositions for use in the methods of the invention, the method further comprises administering an additional therapeutic agent to the subject.

[0095] In one aspect, the invention relates to (a) an antibody, polynucleotide, vector, recombinant host cell, and / or pharmaceutical composition of the invention; and (b) an additional therapeutic agent for use as a medicament.

[0096] In one aspect, the invention relates to (a) an antibody, polynucleotide, vector, recombinant host cell, and / or pharmaceutical composition of the invention; and (b) an additional therapeutic agent for use in a method for treating cancer.

[0097] In one aspect, the invention relates to a pharmaceutical composition, kit, or kit-of-parts comprising (a) an antibody, polynucleotide, vector, recombinant host cell, and / or pharmaceutical composition of the invention, and (b) an additional therapeutic agent.

[0098] In certain embodiments, the additional therapeutic agent is a chemotherapeutic agent, hi certain embodiments, the additional therapeutic agent is a radiation therapeutic agent.

[0099] 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 antagonistic anti-PD-1 antibody, an antagonistic anti-PD-L1 antibody, an antagonistic anti-PD-L2 antibody, an antagonistic anti-CTLA-4 antibody, an antagonistic anti-TIM-3 antibody, an antagonistic anti-LAG-3 antibody, an antagonistic anti-CEACAM1 antibody, an agonist anti-GITR antibody, an agonist anti-OX40 antibody, an antagonistic anti-TIGIT antibody, an agonist anti-CD137 antibody, an antagonistic anti-VISTA antibody, an antagonistic anti-CD73 antibody, and an antagonistic anti-CD96 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.

[0100] 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.

[0101] In certain embodiments, the additional therapeutic agent is an inhibitor of indoleamine-2,3-dioxygenase (IDO). In certain embodiments, the inhibitor is selected from the group consisting of epacadostat, BMS-986205 (also known as F001287, see Example 19 of WO2016 / 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.

[0102] In certain embodiments, the additional therapeutic agent is an inhibitor of ARG, LSD1, CD112, CD112R, or VEGF.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.

[0103] 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 is complexed with a tumor associated antigenic peptide. In certain embodiments, the heat shock protein is gp96 and is complexed with a tumor associated antigenic peptide, and the HSPPC is derived from a tumor obtained from the subject. In certain embodiments, the heat shock protein is a gp96 protein and is complexed with a tumor associated antigenic peptide, and the HSPPC is derived from a tumor obtained from the subject. In certain embodiments, the additional therapeutic agent comprises 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. 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 a peptide-MHC complex. In certain embodiments, the additional therapeutic agent is an adjuvant. In one aspect, the invention relates to (a) an antibody, polynucleotide, vector, recombinant host cell, and / or pharmaceutical composition of the invention, and (b) a vaccine for use as a medicament, e.g. for use in a method for the treatment of cancer, optionally wherein the vaccine comprises a heat shock protein peptide complex (HSPPC) comprising a heat shock protein complexed with an antigenic peptide.In one aspect, the invention relates to a pharmaceutical composition, kit, or kit-of-parts comprising (a) an antibody, polynucleotide, vector, recombinant host cell, and / or pharmaceutical composition of the invention, and (b) a vaccine, optionally wherein the vaccine comprises a heat shock protein peptide complex (HSPPC) comprising a heat shock protein complexed with an antigenic peptide. [Brief description of the drawings]

[0104] [Figure 1A]Figure 1A is a set of histograms showing binding of anti-LAG-3 Fabs to wild-type Jurkat cells or Jurkat cells engineered to express human LAG-3, as measured by flow cytometry. The anti-LAG-3 Fabs tested in this study included P01A12, P01C09, P05E01, P13A04, P13A06, P13B01, P13B02, P13B03, P13B11, P13C06, P13C08, P13C10, P13D04, P13D05, P13E02, P13F01, P13F02, P13F06, P13F 09, P13G01, P13G04, P13G05, P13H05, P14A04, P14B07, P14C04, P14F01, P14F06, P14G01, P14G03, P15B06, P15C02, P15E06, P15F06, P15G05, P16D04, and P16H05. [Figure 1B] Figure IB is a set of histograms showing binding of anti-LAG-3 Fabs to wild-type Jurkat cells or Jurkat cells engineered to express human LAG-3 as measured by flow cytometry. The anti-LAG-3 Fabs tested in this study included P01A12, P01C09, P05E01, P13A04, P13A06, P13B01, P13B02, P13B03, P13B11, P13C06, P13C08, P13C10, P13D04, P13D05, P13E02, P13F01, P13F02, P13F06, P13F 09, P13G01, P13G04, P13G05, P13H05, P14A04, P14B07, P14C04, P14F01, P14F06, P14G01, P14G03, P15B06, P15C02, P15E06, P15F06, P15G05, P16D04, and P16H05. [Figure 1C]Figure 1C is a set of histograms showing binding of anti-LAG-3 Fabs to wild-type Jurkat cells or Jurkat cells engineered to express human LAG-3, as measured by flow cytometry. The anti-LAG-3 Fabs tested in this study included P01A12, P01C09, P05E01, P13A04, P13A06, P13B01, P13B02, P13B03, P13B11, P13C06, P13C08, P13C10, P13D04, P13D05, P13E02, P13F01, P13F02, P13F06, P13F 09, P13G01, P13G04, P13G05, P13H05, P14A04, P14B07, P14C04, P14F01, P14F06, P14G01, P14G03, P15B06, P15C02, P15E06, P15F06, P15G05, P16D04, and P16H05. [Diagram 2] Figures 2A and 2B are graphs showing results from an assay testing the ability of anti-LAG-3 Fab or a negative control Fab not specific for LAG-3 to block binding of cross-linked recombinant LAG-3-6 His to Raji cells expressing MHC class II. Figure 2A is a bar graph showing the percentage of block mediated by the negative control Fab or the anti-LAG-3 Fabs: 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 increasing doses of anti-LAG-3 Fab: P01A12, P13A06, P13B01, P13B02, P13C06, P13C08, P13C10, P13E02, or P14C04, or a negative control Fab. [Diagram 3] A line graph similar to that shown in FIG. 2B plotting the percentage of LAG-3 binding versus increasing doses of full-length chimeric anti-LAG-3 antibodies: P13A06, P13B01, P13B02, P13C06, P13C08, or P13E02, or isotype control antibody. [Figure 4]Graph showing production of IL-2 in human peripheral blood mononuclear cells (PBMCs) upon Staphylococcus enterotoxin A (SEA) stimulation in the absence of any antibody or in the presence of isotype control antibody or chimeric anti-LAG-3 antibody P13B02. [Figure 5A] FIG. 5A is a sequence alignment of the humanized variable regions with the corresponding murine and human germline sequences. FIG. 5A is a sequence alignment comparing the humanized heavy chain variable regions H0-H4 (SEQ ID NOs: 56-60, respectively), the murine antibody P13B02 heavy chain variable region (SEQ ID NO: 15), and the human germline sequences IGHV1-46*01 (SEQ ID NO: 153) and IGHJ1*01 (SEQ ID NO: 200). FIG. 5B is a sequence alignment comparing the humanized light chain variable regions L0-L4 (SEQ ID NOs: 73-77, respectively), the murine antibody P13B02 light chain variable region (SEQ ID NO: 16), and the human germline sequences IGKV3-20*01 (SEQ ID NO: 160) and IGKJ1*01 (SEQ ID NO: 201). Dotted lines represent residues identical to the corresponding residues in H0 (FIG. 5A) or L0 (FIG. 5B). The dashed lines represent the absence of amino acid residues compared to H0 (FIG. 5A) or L0 (FIG. 5B). [Figure 5B] FIG. 5B is a sequence alignment of the humanized variable regions with the corresponding murine and human germline sequences. FIG. 5A is a sequence alignment comparing the humanized heavy chain variable regions H0-H4 (SEQ ID NOs: 56-60, respectively), the murine antibody P13B02 heavy chain variable region (SEQ ID NO: 15), and the human germline sequences IGHV1-46*01 (SEQ ID NO: 153) and IGHJ1*01 (SEQ ID NO: 200). FIG. 5B is a sequence alignment comparing the humanized light chain variable regions L0-L4 (SEQ ID NOs: 73-77, respectively), the murine antibody P13B02 light chain variable region (SEQ ID NO: 16), and the human germline sequences IGKV3-20*01 (SEQ ID NO: 160) and IGKJ1*01 (SEQ ID NO: 201). Dotted lines represent residues identical to the corresponding residues in H0 (FIG. 5A) or L0 (FIG. 5B). The dashed lines represent the absence of amino acid residues compared to H0 (FIG. 5A) or L0 (FIG. 5B). [Figure 6A]Figure 6A is a graph showing binding of anti-LAG-3 antibodies to human T cells activated with Staphylococcus enterotoxin A (SEA) as measured by flow cytometry. Figure 6A is a set of histograms testing chimeric antibody P13B02 (IgG1), and humanized antibodies P13B02-06 (IgG1), P13B02-07 (IgG1), P13B02-16 (IgG1), P13B02-25 (IgG1), P13B02-26 (IgG1), P13B02-27 (IgG1), P13B02-30 (IgG1 G1m17 N297A), and P13B02-30 (IgG4). Figure 6B is a graph showing binding of anti-LAG-3 antibody P13B02-16 (IgG1) or isotype control antibody to activated primary human CD4+ T cells. Median fluorescence intensity (MFI) is plotted against antibody concentration. [Figure 6B] Figure 6B is a graph showing binding of anti-LAG-3 antibodies to human T cells activated with Staphylococcus enterotoxin A (SEA) as measured by flow cytometry. Figure 6A is a set of histograms testing chimeric antibody P13B02 (IgG1), and humanized antibodies P13B02-06 (IgG1), P13B02-07 (IgG1), P13B02-16 (IgG1), P13B02-25 (IgG1), P13B02-26 (IgG1), P13B02-27 (IgG1), P13B02-30 (IgG1 G1m17 N297A), and P13B02-30 (IgG4). Figure 6B is a graph showing binding of anti-LAG-3 antibody P13B02-16 (IgG1) or isotype control antibody to activated primary human CD4+ T cells. Median fluorescence intensity (MFI) is plotted against antibody concentration. [Figure 7A]FIG. 7A is a line graph similar to that shown in FIG. 2B, plotting the percentage of LAG-3 binding versus different doses of isotype control antibody, chimeric antibody P13B02 (IgG1), humanized antibodies P13B02-06 (IgG1), P13B02-07 (IgG1), P13B02-16 (IgG1), P13B02-26 (IgG1), or P13B02-27 (IgG1) (FIG. 7A) or humanized antibody P13B02-30 (IgG1 G1m17 N297A) (FIG. 7B). [Figure 7B] FIG. 7B is a line graph similar to that shown in FIG. 2B, plotting the percentage of LAG-3 binding versus different doses of isotype control antibody, chimeric antibody P13B02 (IgG1), humanized antibodies P13B02-06 (IgG1), P13B02-07 (IgG1), P13B02-16 (IgG1), P13B02-26 (IgG1), or P13B02-27 (IgG1) (FIG. 7A) or humanized antibody P13B02-30 (IgG1 G1m17 N297A) (FIG. 7B). [Figure 8A] Figure 8A is a graph showing the production of IL-2 induced by anti-LAG-3 antibody or isotype control antibody in human peripheral blood mononuclear cells (PBMCs) upon Staphylococcus enterotoxin A (SEA) stimulation. In Figure 8A, the anti-LAG-3 antibody tested is P13B02-30 (IgG1). In Figure 8B, the anti-LAG-3 antibody P13B02-16 (IgG1) 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 antibody ipilimumab (Ipi). [Figure 8B]Figure 8B is a graph showing the production of IL-2 induced by anti-LAG-3 antibody or isotype control antibody in human peripheral blood mononuclear cells (PBMCs) upon Staphylococcus enterotoxin A (SEA) stimulation. In Figure 8A, the anti-LAG-3 antibody tested is P13B02-30 (IgG1). In Figure 8B, the anti-LAG-3 antibody P13B02-16 (IgG1) 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 antibody ipilimumab (Ipi). [Figure 9] Figures 9A and 9B are graphs showing TNFα production in 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 the anti-PD-1 antibody pembrolizumab (Pembro).TILs were isolated from renal cell carcinoma (Figure 9A) or colon carcinoma (Figure 9B) tumors and activated with anti-CD3 / CD28 microbeads. [Figure 10] Figures 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 a sextuplicate dose titration 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) peptide. In the first experiment, antibody concentrations from 0.2 to 50 μg / mL were tested (Figure 10A). In the second experiment, antibody concentrations from 0.1 to 100 μg / mL were tested (Figure 10B). RLU = relative light units of luciferase reporter. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0105] The present disclosure provides antibodies that specifically bind to LAG-3 (e.g., human LAG-3) and antagonize LAG-3 function, e.g., LAG-3-mediated immunosuppression. Pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells making these antibodies, and methods of treating a subject with these antibodies are also provided. The antibodies disclosed herein increase T cell activation in response to an antigen (e.g., a tumor antigen, or an infectious disease antigen), and are therefore particularly useful for treating cancer in a subject, or treating or preventing an infectious disease in a subject. All examples of "isolated antibodies" described herein are further contemplated as antibodies that may, but need not be isolated. All examples of "isolated polynucleotides" described herein are further contemplated as polynucleotides that may, but need not be isolated. All examples of "antibodies" described herein are further contemplated as antibodies that may, but need not be isolated. All examples of "polynucleotides" described herein are further contemplated as polynucleotides that may, but need not be isolated.

[0106] definition As used herein, the terms "about" and "approximately," when used to modify a numerical value or numerical range, indicate a deviation of that value or range of 5%-10% above (e.g., up to 5%-10% above), and 5%-10% below (e.g., up to 5%-10% below) while remaining within the intended meaning of the recited value or range.

[0107] 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 wild-type human LAG-3 gene, e.g., the human LAG-3 protein encoded by GenBank™ Accession No. NM_002286.5. An exemplary immature amino acid sequence of human LAG-3 is provided as SEQ ID NO: 166. Exemplary mature amino acid sequences of human LAG-3 are provided as SEQ ID NO: 167 and SEQ ID NO: 210.

[0108] As used herein, the terms "antibody" and "antibody" include full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules comprising an antibody CDR, VH region, or VL region. Exemplary antibodies include monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, murine antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugate antibodies, antibody drug conjugates, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fvs (scFvs), camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies), and antigen-binding fragments of any of the above. In certain embodiments, the antibodies described herein refer to polyclonal antibody populations. An antibody can be any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG 2a or IgG 2b ). In certain embodiments, the antibodies described herein are IgG antibodies, or classes (e.g., human IgG1 or IgG4) or subclasses thereof. In certain embodiments, the antibodies are humanized monoclonal antibodies. In another particular embodiment, the antibodies are human monoclonal antibodies.

[0109] As used herein, the terms "VH region" and "VL region" refer to a single antibody heavy and light chain variable region, respectively, comprising FR (framework regions) 1, 2, 3, and 4, and CDR (complementarity determining regions) 1, 2, and 3 (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest (NIH Publication No. 91-3242, Bethesda, incorporated herein by reference in its entirety).

[0110] As used herein, the term "CDR" or "complementarity determining region" refers to the discontinuous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest. (1991), as well as by Chothia et al., J. Mol. Biol. 196:901-917 (1987), and by MacCallum et al., J. Mol. Biol. 262:732-745 (1996), all of which are incorporated herein by reference in their entireties, where the definitions include overlapping amino acid residues or subsets of amino acid residues when compared against each other. In certain embodiments, the term "CDR" is a CDR as defined by 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 by Chothia et al., J. Mol. Biol. 196:901-917 (1987). In certain embodiments, the term "CDR" is a CDR as defined by 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).

[0111] As used herein, the term "framework (FR) amino acid residues" refers to those 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 a variable region but are not part of a CDR (e.g., using the Kabat or Chothia definitions of CDRs).

[0112] As used herein, the terms "variable region" or "variable domain" can be used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, generally a portion of a light or heavy chain, typically about 110-125 amino acids in a mature heavy chain and about 90-115 amino acids in a mature light chain from the amino terminus that vary widely in sequence between antibodies and are used in the binding and specificity of a particular antibody to its particular antigen. The more highly conserved regions in the variable domain are called framework regions (FRs), while sequence variability is condensed in regions called complementarity determining regions (CDRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of an antibody to an antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or mouse CDRs and human framework regions (FRs). In certain embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and primate (eg, non-human primate) framework regions (FRs).

[0113] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody.

[0114] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody.

[0115] As used herein, the terms "constant region" or "constant domain" are used interchangeably and have their common meaning in the art. The constant region is the portion of an antibody, e.g., the carboxyl-terminal portion of the light and / or heavy chain, that is not directly involved in binding the antibody to an antigen, but can exhibit various effector functions, such as interaction with Fc receptors (e.g., Fc gamma receptors). The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence relative to the variable domains of the immunoglobulin.

[0116] As used herein, the term "heavy chain", when used in reference to an antibody, can refer to any of the different types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), which result in the IgA, IgD, IgE, IgG, and IgM classes of antibodies (including subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4), respectively, based on the amino acid sequence of the constant domain.

[0117] As used herein, the term "light chain" when used in reference to an antibody can refer to any of the different types, e.g., kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art.

[0118] As used herein, the term "EU numbering system" refers to the EU numbering convention for antibody constant regions as described in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al., Sequences of Proteins of Immunological Interest, USDept. Health and Human Services, 5th edition, 1991, each of which is incorporated herein by reference in its entirety.

[0119] "Binding affinity" generally refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D Affinity can be expressed as the equilibrium dissociation constant (K D ) and the equilibrium association constant (K A ) can be measured and / or expressed in a number of ways known in the art, including, but not limited to, K D But, k off / k on While it is calculated from the quotient of A is k on / k off It is calculated from the quotient of k on For example, k refers to the association rate constant of an antibody to an antigen. off k refers to, for example, the dissociation rate constant of an antibody to an antigen. on and k off can be determined by techniques known to those of skill in the art, such as BIAcore® or KinExA. As used herein, a "low affinity" refers to a larger K D Refers to...

[0120] As used herein, the terms "specifically bind," "specifically recognize," "immunospecifically bind," and "immunospecifically recognize" are similar terms in the context of antibodies and refer to a molecule that binds to an antigen (e.g., an epitope or immune complex) as such binding is understood by one of skill in the art. For example, a molecule that specifically binds to an antigen may bind to other peptides or polypeptides that generally have a lower affinity as determined, for example, by immunoassays, BIAcore®, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art. In certain embodiments, a molecule that immunospecifically binds to an antigen has a K that is at least 2 log (i.e., a factor of 10), 2.5 log, 3 log, 4 log, or greater than the K when the molecule binds to another antigen. A K exceeds A The antigen binds to the antigen having the formula:

[0121] In another specific embodiment, the molecule that specifically binds to the antigen does not cross-react with other proteins under similar binding conditions. In another specific embodiment, the molecule that specifically binds to LAG-3 does not cross-react with other non-LAG-3 proteins. In certain embodiments, provided herein are antibodies that bind to LAG-3 (e.g., human LAG-3) with higher affinity than another non-related antigen. In certain embodiments, provided herein are antibodies that bind to LAG-3 (e.g., human LAG-3) with 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more higher affinity than another non-related antigen, as determined, for example, by radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay. In certain embodiments, 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 an LAG-3 protein, e.g., as measured by radioimmunoassay.

[0122] As used herein, the term "nonfucosylated" or "nonfucosylated," in the context of Fc, refers to the substantial lack of fucose directly or indirectly covalently attached to residue 297 of human IgG1 Fc, or the corresponding residue in a non-IgG1 or non-human IgG1 immunoglobulin, numbered according to the EU numbering system. Thus, in a composition comprising a plurality of nonfucosylated antibodies, at least 70% of the antibodies will be nonfucosylated, directly or indirectly (e.g., via an intervening sugar), at residue 297 of the Fc region of the antibodies, and in some embodiments at least 80%, 85%, 90%, 95%, or 99% will be nonfucosylated, directly or indirectly, at residue 297 of the Fc region.

[0123] As used herein, "epitope" is a term in the art that refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, contiguous amino acids of a polypeptide (linear or contiguous epitope), or an epitope can be, for example, from two or more non-contiguous regions of a polypeptide or polypeptides (conformational, non-linear, non-contiguous, or non-contiguous epitopes). In certain embodiments, the epitope to which an antibody binds can be determined, for example, by NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligo-peptide scanning assays (e.g., CLIPS (Chemical Linkage of Peptides onto Scaffolds) to constrain peptides and map non-contiguous or conformational epitopes), and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be accomplished using any of the methods known in the art (e.g., 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 entireties).Antibody:antigen crystals can be studied using well-known X-ray diffraction techniques and refined using computer software such as X-PLOR (Yale University, 1992, published by Molecular Simulations, Inc., see e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al., U.S. Patent 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. Mutagenesis mapping studies can be accomplished using any method known to those skilled in the art. For a description of mutagenesis techniques, including alanine scanning mutagenesis techniques, see, for example, Champe M et al., (1995) J Biol Chem 270:1388-1394 and Cunningham BC & Wells JA (1989) Science 244:1081-1085, each of which is incorporated herein by reference in its entirety. CLIPS (Chemical Linkage of Peptides onto Scaffolds) is a technique that presents one or more peptides in a structurally constrained configuration to behave as functional mimics of complex protein domains. See, for example, U.S. Publication Nos. US2008 / 0139407 A1, and US2007 / 099240 A1, and U.S. Patent No. 7,972,993, each of which is incorporated herein by reference in its entirety. In certain embodiments, the epitope of the antibody is determined using alanine scanning mutagenesis studies. In certain embodiments, the epitope of the antibody is determined using hydrogen / deuterium exchange with mass spectrometry.In certain embodiments, the epitope of the antibody is determined using CLIPS epitope mapping technology from Pepscan Therapeutics.

[0124] As used herein, the term "epitope located within a region of human LAG-3" consisting of a specific amino acid sequence or set of amino acid residues refers to an epitope that includes one or more of the amino acid residues of the specific region, the specific region including the first and last specific amino acid residues of the region of human LAG-3. In certain embodiments, the epitope includes each one of the amino acid residues located within the specific region. In certain embodiments, one or more additional amino acid residues of human LAG-3 outside the specific region bind to the antibody together with the epitope located within the specific region.

[0125] As used herein, the terms "T cell receptor" and "TCR" are used interchangeably and refer to full-length heterodimeric αβ or γδ TCRs, antigen-binding fragments of full-length TCRs, and molecules comprising the TCR CDRs or variable regions. Examples of TCRs include, but are not limited to, full-length TCRs, antigen-binding fragments of TCRs, soluble TCRs lacking the transmembrane and cytoplasmic regions, single-chain TCRs containing variable regions of TCRs joined by flexible linkers, engineered disulfide bond linked TCR chains, monospecific TCRs, multispecific TCRs (including bispecific TCRs), TCR fusions, human TCRs, humanized TCRs, chimeric TCRs, recombinantly produced TCRs, and synthetic TCRs. The terms encompass wild-type and engineered TCRs (e.g., chimeric TCRs comprising a chimeric TCR chain comprising a first portion from a TCR of a first species and a second portion from a TCR of a second species).

[0126] As used herein, the terms "major histocompatibility complex" and "MHC" are used interchangeably and refer to MHC class I molecules and / or MHC class II molecules.

[0127] As used herein, the term "peptide-MHC complex" refers to an MHC molecule (MHC class I, or MHC class II) in which a peptide is bound to an art-recognized peptide-binding pocket of the MHC.

[0128] As used herein, the terms "treat," "treating," and "treatment" refer to therapeutic or prophylactic measures as described herein. A "treatment" method employs administration of an antibody to a subject having or susceptible to a disease or disorder to prevent, cure, delay, reduce the severity, or ameliorate one or more symptoms of a disease or disorder or a recurrent disease or disorder, or to extend the patient's survival beyond that expected in the absence of such treatment.

[0129] As used herein, the term "effective amount," in the context of administration of a treatment to a subject, refers to the amount of the treatment that achieves a desired prophylactic or therapeutic effect.

[0130] 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.

[0131] The determination of "percent identity" between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be achieved using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for comparing two sequences is the algorithm of Karlin S & Altschul SF (1990) PNAS 87:2264-2268, modified in Karlin S & Altschul SF (1993) PNAS 90:5873-5877, each of which is incorporated herein by reference in its entirety. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul SF et al., (1990) J Mol Biol 215:403, which is incorporated herein by reference in its entirety. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, for example, score=100, wordlength=12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with XBLAST program parameters set, for example, score=50, word length=3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized 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 to detect distant relationships between molecules (supra). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) on the World Wide Web, ncbi.nlm.nih.gov). Another specific, non-limiting example of a mathematical algorithm utilized for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17, which is incorporated herein by reference in its entirety.Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0132] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0133] Anti-LAG-3 antibody In one aspect, the present disclosure provides antibodies that specifically bind to LAG-3 (e.g., human LAG-3) and antagonize LAG-3 function. The amino acid sequences of exemplary antibodies are set forth in Tables 1-7 herein. One of skill in the art will appreciate that, under certain conditions, the N-terminal E or Q amino acid residue can spontaneously convert to pyroglutamic acid by post-translational cyclization of the free amino group to form a lactam. Thus, in certain embodiments, the present disclosure provides an antibody comprising an antibody heavy or light chain variable region disclosed herein (e.g., SEQ ID NOs: 56-72 and 73-77, respectively) or a full-length heavy or light chain disclosed herein (e.g., SEQ ID NOs: 168-186 and 187-191, respectively), in which the N-terminal E or Q amino acid residue is converted to pyroglutamic acid (e.g., as a result of post-translational cyclization of the free amino group of the N-terminal E or Q residue). [Table 1A] [Table 1B] [Table 1C] [Table 1D]

Table 1E

Table 1F

Table 1G

Table 1H

Table 1I

Table 1J

Table 1K

Table 1L

Table 1M

Table 1N

Table 1O

Table 1P

Table 1Q

Table 1R

Table 1S

Table 1T

Table 1U

Table 1V

Table 1W

Table 1X

Table 1Y

Table 1Z

Table 1AA

Table 1BB

Table 1CC

Table 1DD

Table 1EE

Table 1FF

Table 1GG

Table 1HH

Table 1II

Table 1JJ

Table 1KK

Table 1LL

Table 1MM

Table 1NN

Table 1OO

Table 1PP

Table 1QQ

Table 1RR

Table 1SS

Table 1TT

Table 1UU

Table 1VV

Table 1WW

Table 1XX

Table 1YY

Table 1ZZ

Table 1AAA

Table 1BBB

Table 1CCC

Table 1DDD

Table 1EEE

Table 2A

Table 2B

Table 2C

Table 2D

Table 2E

Table 3A

Table 3B

Table 3C

Table 4A

Table 4B

Table 4C

Table 4D

Table 5

Table 6A

Table 6B

Table 6C

Table 7A

[0134] In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), the isolated antibody comprising a VH domain that comprises one, two, or all three of the CDRs of a VH domain set forth in Tables 1, 2, 6, and 7 herein. In certain embodiments, the antibody comprises a CDRH1 of one of the VH domains set forth in Tables 1, 2, 6, and 7. In certain embodiments, the antibody comprises a CDRH2 of one of the VH domains set forth in Tables 1, 2, 6, and 7. In certain embodiments, the antibody comprises a CDRH3 of one of the VH domains set forth in Tables 1, 2, 6, and 7.

[0135] In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), the isolated antibody comprising a VL domain that comprises one, two, or all three of the CDRs of a VL domain disclosed in Tables 1, 3, 6, and 7 herein. In certain embodiments, the antibody comprises a CDRL1 of one of the VL domains disclosed in Tables 1, 3, 6, and 7 herein. In certain embodiments, the antibody comprises a CDRL2 of one of the VL domains disclosed in Tables 1, 3, 6, and 7 herein. In certain embodiments, the antibody comprises a CDRL3 of one of the VL domains disclosed in Tables 1, 3, 6, and 7 herein.

[0136] In certain embodiments, the CDRs of an antibody can be determined 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.

[0137] In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), the antibody comprising the Kabat VH CDRs of a VH disclosed in Tables 1, 6, and 7 herein. In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), the antibody comprising the Kabat VL CDRs of a VL disclosed in Tables 1, 6, and 7 herein. In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), the antibody comprising the Kabat VH CDRs and Kabat VL CDRs of an antibody disclosed in Tables 1, 6, and 7 herein.

[0138] In certain embodiments, the CDRs of an antibody can be determined according to the Chothia numbering scheme, which refers to the positions of the 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 U.S. Patent No. 7,709,226, all of which are incorporated herein by reference in their entireties). Typically, using the Kabat numbering convention, the CDRH1 loop of Chothia is located at heavy chain amino acids 26-32, 33, or 34, the CDRH2 loop of Chothia is located at heavy chain amino acids 52-56, and the CDRH3 loop of Chothia is located at heavy chain amino acids 95-102, while the CDRL1 loop of Chothia is located at light chain amino acids 24-34, the CDRL2 loop of Chothia is located at light chain amino acids 50-56, and the CDRL3 loop of Chothia is located at light chain amino acids 89-97. When numbered using the Kabat numbering convention, the end of the Chothia CDRH1 loop 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 neither 35A nor 35B are present, the loop ends at 32; if only 35A is present, the loop ends at 33; and if both 35A and 35B are present, the loop ends at 34).

[0139] In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising the VH CDRs of Chothia of the VH disclosed in Tables 1, 6, and 7 herein. In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising the VL CDRs of Chothia of the VL disclosed in Tables 1, 6, and 7 herein. In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising the VH CDRs of Chothia and the VL CDRs of Chothia of an antibody disclosed in Tables 1, 6, and 7 herein. In certain embodiments, an antibody that specifically binds to LAG-3 (e.g., human LAG-3) comprises one or more CDRs, where the Chothia and Kabat CDRs have the same amino acid sequence. In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (eg, human LAG-3) and comprises a combination of the Kabat and Chothia CDRs.

[0140] In certain embodiments, the CDRs of an antibody can be determined according to the IMGT numbering system as described in Lefranc MP, (1999) The Immunologist 7:132-136 and Lefranc MP 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, CDHR1 is located at positions 26-35, CDRH2 is located at positions 51-57, CDRH3 is located at positions 93-102, CDRL1 is located at positions 27-32, CDRL2 is located at positions 50-52, and CDRL3 is located at positions 89-97.

[0141] In certain embodiments, the disclosure provides antibodies that specifically bind to LAG-3 (e.g., human LAG-3), e.g., as determined by the IMGT numbering system as described in Lefranc MP (1999) supra and Lefranc MP et al., (1999) supra, and comprise the CDRs of the antibodies disclosed in Tables 1, 6, and 7 herein.

[0142] In certain embodiments, the CDRs of an antibody can be determined according to the AbM numbering scheme, which is a compromise between the Kabat CDRs and Chothia structural loops and refers to the AbM hypervariable regions used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.). In certain embodiments, the present disclosure provides antibodies that specifically bind LAG-3 (e.g., human LAG-3) and comprise the CDRs of the antibodies disclosed in Tables 1, 6, and 7 herein, as determined by the AbM numbering scheme.

[0143] In certain embodiments, the CDRs of an antibody can be determined according to MacCallum RM et al., (1996) J Mol Biol 262:732-745, which is incorporated herein by reference in its entirety. See also, e.g., 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 certain embodiments, the present disclosure provides an antibody that specifically binds to LAG-3 (e.g., human LAG-3) as described in MacCallum RM et al., (1996) (supra), and comprises the CDRs of the antibodies disclosed in Tables 1, 6, and 7 herein.

[0144] In certain embodiments, the disclosure 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 sequences of the CDRH1, CDRH2, and CDRH3 regions 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 comprising the amino acid sequences of the CDRL1, CDRL2, and CDRL3 regions of the VL domain as set forth in any one of claims 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, wherein each CDR is defined according to the Kabat definition, the Chothia definition, a combination of the Kabat definition and the Chothia definition, the IMGT numbering system, the AbM definition, or the MacCallum definition of a CDR.

[0145] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising: (a) CDRH1 comprises the amino acid sequence of DX1YX2X3 (SEQ ID NO:140), X1 is T or N; X2 is I or M, and X3 is H, Y, or D; (b) CDRH2 is X1IDPANX2X3X4X5X6X7PX8X9QX 10 (SEQ ID NO: 142), X1 is E, R, S, or K; X2 is D or G; X3 is N or H; X4 is T or S; X5 is K or H; X6 is Y or F; X7 is D or A; X8 is K or R; X9 is F or L, and X 10 is G or D, (c) CDRH3 comprises an amino acid sequence of YX1X2X3YX4VGGX5DY (SEQ ID NO: 144), X1 is Y, F, or S; X2 is Y or D; X3 is K or R; X4 is D or E, and X5 is F or C; (d) CDRL1 comprises the amino acid sequence of SVSSX1ISSSX2LX3 (SEQ ID NO:147), X1 is S or G; X2 is N or T, and X3 is H or Y; (e) CDRL2 comprises the amino acid sequence of GTSNLAS (SEQ ID NO: 104), and (f) CDRL3 comprises the amino acid sequence of QQWX1X2YPX3T (SEQ ID NO: 149), X1 is S, N, or R; X2 is S, T, or R; and X3 is F, L, H, or W.

[0146] In certain embodiments, CDRH1 comprises the amino acid sequence of DX1YX2X3 (SEQ ID NO: 141), where: X1 is T or N, X2 is I or M, and X3 is H or Y. In certain embodiments, CDRH2 comprises the amino acid sequence of X1IDPANX2X3X4KX5X6PX7FQX8 (SEQ ID NO: 143), where: X1 is E, R, or S, X2 is D or G, X3 is N or H, X4 is T or S, X5 is Y or F, X6 is D or A, X7 is K or R, and X8 is G or D. In certain embodiments, CDRH3 comprises the amino acid sequence of YX1X2X3YDVGGX4DY (SEQ ID NO: 145), where X1 is Y, F, or S, X2 is Y or D, X3 is K or R, and X4 is F or C. In certain embodiments, CDRH3 comprises the amino acid sequence of YYYX1YX2VGGFDY (SEQ ID NO: 146), where X1 is K or R, and X2 is D or E. In certain embodiments, CDRL1 comprises the amino acid sequence of SVSSSISSSNLX1 (SEQ ID NO: 148), where X1 is H or Y. In certain embodiments, CDRL3 comprises the amino acid sequence of QQWX1SYPX2T (SEQ ID NO: 150), where X1 is S, N, or R, and X2 is F, L, or H.

[0147] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising: (a) CDRH1 comprises the amino acid sequence of DTYIH (SEQ ID NO:79); (b) CDRH2 comprises the amino acid sequence of EIDPANDNTKYDPKFQG (SEQ ID NO: 90); (c) CDRH3 comprises an amino acid sequence of YYYX1YX2VGGFDY (SEQ ID NO:146), where: Xi is K or R, and X2 is D or E; (d) CDRL1 comprises the amino acid sequence of SVSSSISSSNLH (SEQ ID NO: 100); (e) CDRL2 comprises the amino acid sequence of GTSNLAS (SEQ ID NO: 104), and (f) CDRL3 comprises the amino acid sequence of QQWSSYPFT (SEQ ID NO: 105),

[0148] In certain embodiments, CDRH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 78 to 82. In certain embodiments, CDRH2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 83 to 93. In certain embodiments, CDRH3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 94 to 99. In certain embodiments, CDRL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 100 to 103. In certain embodiments, CDRL3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 105 to 112.

[0149] In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), wherein the VH domain comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences 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, respectively. In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), wherein the VH domain comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NOs: 79, 90, and 95, respectively. In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), wherein the VH domain comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NOs: 79, 90, and 98, respectively.

[0150] In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), wherein the VL domain comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences 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, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), wherein the VL domain comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 100, 104, and 105, respectively.

[0151] In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3 regions, and a light chain variable region comprising CDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions are selected from the group consisting of SEQ ID NOs: 78, 83, 94, 100, 104, 106, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, , 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, 1 00, 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. In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3 regions, and a light chain variable region comprising CDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions comprise the amino acid sequences set forth in SEQ ID NOs: 79, 90, 95, 100, 104, and 105, respectively.In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3 regions, and a light chain variable region comprising CDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions comprise the amino acid sequences set forth in SEQ ID NOs: 79, 90, 98, 100, 104, and 105, respectively.

[0152] In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising a heavy chain variable region (VH) that comprises one, two, or all three of the VH CDRs of an antibody in Tables 1, 2, 6, and 7. In some embodiments, the antibody comprises one, two, three, or all four of the VH framework regions described herein. In certain embodiments, the antibody comprises one, two, three, or all four of the VH framework regions (FR) described in Table 4 (e.g., one, two, three, or four of the framework regions of a row in Table 4). In certain embodiments, the antibody comprises one, two, three, or all four of the framework regions of the heavy chain variable region sequence of SEQ ID NO: 151 or 222. In certain embodiments, the antibody comprises one, two, three, or all four of the framework regions of the heavy chain variable region sequence of SEQ ID NO: 218 or 223. In certain embodiments, the antibody comprises one, two, three, or four framework regions of a heavy chain variable region sequence 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 framework regions of a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 56-72, and 220.In certain embodiments, the antibody comprises a heavy chain variable framework region that is or is derived from an amino acid sequence encoded by a human gene, the amino acid sequence being selected from the group consisting of IGHV1-46 (e.g., IGHV1-46*01, e.g., having the amino acid sequence of SEQ ID NO: 153), IGHV1-69-2 (e.g., IGHV1-69-2*01, e.g., having the amino acid sequence of SEQ ID NO: 154), IGHV1-3 (e.g., IGHV1-3*01, e.g., having the amino acid sequence of SEQ ID NO: 155), IGHV1-47 (e.g., IGHV1-47*01, e.g., having the amino acid sequence of SEQ ID NO: 156), IGHV1-49 (e.g., IGHV1-49*01, e.g., having the amino acid sequence of SEQ ID NO: 157), IGHV1-50 (e.g., IGHV1-50*01, e.g., having the amino acid sequence of SEQ ID NO: 158), IGHV1-20 (e.g., IGHV1-20*01, e.g., having the amino acid sequence of SEQ ID NO: 159), IGHV1-21 (e.g., IGHV1-21*01, e.g., having the amino acid sequence of SEQ ID NO: 160), IGHV1-22 (e.g., IGHV1-22*01, In certain embodiments, the heavy chain variable framework region derived from said amino acid sequence consists of said amino acid sequence but with up to 20 amino acid substitutions, deletions, and / or insertions, preferably with up to 20 amino acid substitutions. In certain embodiments, the heavy chain variable framework region derived from said amino acid sequence consists of said amino acid sequence, where 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues are replaced with an amino acid found at the analogous position in a corresponding non-human heavy chain variable framework region. In some embodiments, the antibody comprises a heavy chain variable framework region derived from the amino acid sequence of SEQ ID NO: 153, where at least one amino acid in the amino acid sequence of SEQ ID NO: 153 is replaced with an amino acid at the analogous position in a corresponding non-human heavy chain variable framework region. In certain embodiments, the amino acid replacement 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, where the amino acid positions are designated 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, and the position of the amino acid substitution is indicated according to the Kabat numbering system. In another particular 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, and the position of the amino acid substitution is indicated according to 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, and the position of the amino acid substitution is indicated according to the Kabat numbering system.

[0153] In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising a light chain variable region (VH) that comprises one, two, or all three of the VL CDRs of an antibody in Tables 1, 3, 6, and 7. In some embodiments, the antibody comprises one, two, three, or all four of the VL framework regions described herein. In certain embodiments, the antibody comprises one, two, three, or all four of the VL framework regions (FR) described in Table 5 (e.g., one, two, three, or four of the framework regions in one row in Table 5). In certain embodiments, the antibody comprises one, two, three, or all four of the framework regions of the light chain variable region sequence of SEQ ID NO: 152 or 224. In certain embodiments, the antibody comprises one, two, three, or four framework regions of a light chain variable region sequence 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 framework regions of a light chain variable region sequence selected from the group consisting of SEQ ID NOs: 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, the amino acid sequence being selected from the group consisting of IGKV3-20 (e.g., IGKV3-20*01, e.g., having the amino acid sequence of SEQ ID NO: 160), IGKV3D-15 (e.g., IGKV3D-15*01, e.g., having the amino acid sequence of SEQ ID NO: 161), IGKV3-15 (e.g., IGKV3-15*01, e.g., having the amino acid sequence of SEQ ID NO: 161), having the amino acid sequence of SEQ ID NO: 162), IGKV3D-7 (e.g., IGKV3D-7*01, for example having the amino acid sequence of SEQ ID NO: 163), IGKV1-9 (e.g., IGKV1-9*01, for example having the amino acid sequence of SEQ ID NO: 164), and IGKV3-11 (e.g., IGKV3-11*01, for example having the amino acid sequence of SEQ ID NO: 165).In certain embodiments, the light chain variable framework region derived from said amino acid sequence consists of said amino acid sequence but with up to 20 amino acid substitutions, deletions, and / or insertions, preferably with up to 20 amino acid substitutions. In certain embodiments, the light chain variable framework region 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 substituted with an amino acid found at the analogous position in a corresponding non-human light chain variable framework region. In some embodiments, the antibody comprises a light chain variable framework region derived from the amino acid sequence of SEQ ID NO: 160 with at least one amino acid in the amino acid sequence of SEQ ID NO: 160 substituted with an amino acid in the analogous position in a corresponding non-human light chain variable framework region. 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, where the amino acid positions are designated according to 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, where the amino acid substitution positions are designated according to the Kabat numbering system.

[0154] In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising a heavy chain variable region (VH) that comprises one, two, or all three of the VH CDRs of an antibody in Tables 1, 2, 6, and 7 (e.g., the VH CDRs in column 1 of Table 2), and a light chain variable region (VL) that comprises one, two, or all three of the VL CDRs of an antibody in Tables 1, 3, 6, and 7 (e.g., the VL CDRs in column 1 of Table 3). In some embodiments, the antibody comprises the VH framework region and the VL framework region described herein. In certain embodiments, the antibody comprises one, two, three, or all four of the VH framework regions (FRs) set forth in Table 4 (e.g., one, two, three, or four of the framework regions in one row in Table 4) and one, two, three, or all four of the VL framework regions (FRs) set forth in Table 5 (e.g., one, two, three, or four of the framework regions in one row in Table 5).

[0155] In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), wherein the VH domain comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NOs: 79, 90, and 95, or 79, 90, and 98, respectively. In certain embodiments, the antibody comprises one, two, three, or all four of the VH framework regions from the VH of a human or primate antibody. In some embodiments, the antibody comprises the VH framework regions of an antibody set forth in Table 4.

[0156] In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising a VL domain comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 100, 104, and 105, respectively. In certain embodiments, the antibody comprises one, two, three, or all four of the VL framework regions from the VL of a human or primate antibody. In some embodiments, the antibody comprises the VL framework region of an antibody set forth in Table 5.

[0157] In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3 regions, and a light chain variable region comprising CDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions comprise the amino acid sequences set forth in SEQ ID NOs: 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 of the VH framework regions from the VH of a human or primate antibody, and one, two, three, or all four of the VL framework regions from the VL of a human or primate antibody. In some embodiments, the antibody comprises the VH and VL framework regions of an antibody set forth in Tables 4 and 5.

[0158] In certain embodiments, the disclosure 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, the disclosure 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. In certain embodiments, the disclosure 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 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. In certain embodiments, the antibody comprises a heavy chain variable region having an 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 optionally, the amino acid residue at position 1 of the heavy chain variable region is converted to pyroglutamic acid. In certain embodiments, the antibody comprises a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 65. In certain embodiments, the antibody comprises a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 220. In certain embodiments, X in SEQ ID NO: 220 is Q. In certain embodiments, X in SEQ ID NO: 220 is pyroglutamic acid. 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 with the amino acid residue at position 1 of the heavy chain changed to pyroglutamic acid. 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 comprises a heavy chain having 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 pyroglutamic acid. In certain embodiments, the antibody comprises a heavy chain having an amino acid sequence set forth in SEQ ID NO: 169. In certain embodiments, the antibody comprises a heavy chain having an 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 pyroglutamic acid. In certain embodiments, the antibody comprises a heavy chain having an amino acid sequence set forth in SEQ ID NO: 170. In certain embodiments, the antibody comprises a heavy chain having an 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 pyroglutamic acid.

[0159] In certain embodiments, the disclosure 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 disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising 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, 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, and optionally, the amino acid residue at position 1 of the light chain variable region is converted to pyroglutamic acid. 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, X in SEQ ID NO: 221 is E. In certain embodiments, X in SEQ ID NO: 221 is pyroglutamic acid. 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 with the amino acid residue at position 1 of the light chain converted to pyroglutamic acid. 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, X in SEQ ID NO: 228 is E. In certain embodiments, X in SEQ ID NO: 228 is pyroglutamic acid.

[0160] In certain embodiments, the disclosure 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 disclosure 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 disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3) and has a sequence identity of 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, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 200, 210, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 300, 309, 308, 309, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 3 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 heavy chain variable region having an 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 an amino acid sequence set forth in SEQ ID NO: 73, 221, 74, 75, 76, or 77.In certain embodiments, the antibodies are selected from the group consisting of 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 antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequence 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 sequence set forth in SEQ ID NOs: 220 and 221, respectively. In certain embodiments, the X in SEQ ID NO: 220 is Q. In certain embodiments, the X in SEQ ID NO: 220 is pyroglutamic acid. In certain embodiments, the X in SEQ ID NO: 221 is E. In certain embodiments, X in SEQ ID NO:221 is pyroglutamic acid. 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 pyroglutamic acid. In certain embodiments, X in SEQ ID NO:220 is pyroglutamic acid and X in SEQ ID NO:221 is E. In certain embodiments, X in SEQ ID NO:220 is pyroglutamic acid and X in SEQ ID NO:221 is pyroglutamic acid. 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 heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs: 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 pyroglutamic acid. In certain embodiments, the amino acid residue at position 1 of the light chain variable region is converted to pyroglutamic acid. In certain embodiments, the amino acid residue at position 1 of the heavy chain variable region is converted to pyroglutamic acid and the amino acid residue at position 1 of the light chain variable region is converted to pyroglutamic acid.

[0161] In certain embodiments, the disclosure provides 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 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 48 and 51, 49 and 52, 49 and 60, 49 and 61, 49 and 70, 52 and 71, 53 and 72, 54 and 73, 55 and 74, 56 and 75, 57 and 76, 58 and 80, 59 and 91, 60 and 92, 61 and 93, 62 and 94, 63 and 95, 64 and 86, 65 and 87, 66 and 88, 67 and 89, 68 and 96, 69 and 100, 70 and 102, 71 and 84, 72 and 85, 73 and 86, 74 and 87, 75 and 88, 76 and 89, 77 and 97, 78 and 99, 80 and 91, 81 and 92, 82 and and 52, 53 and 8, 38 and 2, or 54 and 55, respectively. In certain embodiments, the disclosure provides 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 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.

[0162] In certain embodiments, the disclosure provides antibodies described herein, e.g., 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, which bind to an epitope of LAG-3 (e.g., an epitope of human LAG-3). In certain embodiments, the disclosure provides antibodies described herein, e.g., 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, Provided are isolated antibodies that bind to an epitope of LAG-3 (e.g., an epitope of human LAG-3) that is the same as or overlaps with an antibody comprising heavy and light chain variable regions of the amino acid sequences set forth in, respectively, 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 certain embodiments, the epitope of the antibody can be determined, for example, by NMR spectroscopy, surface plasmon resonance (BIAcore®) X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping).For X-ray crystallography, crystallization can be accomplished using any of the methods known in the art (e.g., 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 entireties). Antibody:antigen crystals may be studied using well-known X-ray diffraction techniques and refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc. See, e.g., Meth Enzymol (1985) volumes 114&115, eds Wyckoff HW et al., U.S. Patent Application 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 entireties). Mutagenesis mapping studies can be accomplished using any method known to those skilled in the art. For example, see Champe M et al., (1995) (supra), and Cunningham BC & Wells JA (1989) (supra) for a description of mutagenesis techniques, including alanine scanning mutagenesis techniques. In certain embodiments, the epitope of the antibody is determined using alanine scanning mutagenesis studies.Furthermore, antibodies that recognize and bind to the same or overlapping epitopes of LAG-3 (e.g., human LAG-3) can be identified using routine techniques such as immunoassays, for example, by showing the ability of one antibody to block the binding of another antibody to a target antigen, i.e., competitive binding assays. Competitive binding assays can also be used to determine whether two antibodies have similar binding specificity for an epitope. Competitive binding can be determined in an assay in which the immunoglobulin under test inhibits the specific binding of a reference antibody to a common antigen, such as LAG-3 (e.g., human LAG-3). Many types of competitive binding assays are available, such as solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competitive assays (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), solid-phase direct labeling assays, solid-phase direct labeling sandwich assays (see Harlow E & Lane D, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press), solid-phase direct labeling RIA using I-125 label (see Morel GA et al., (1988) Mol Immunol 25(1):7-15), solid-phase direct biotin-avidin EIA (Cheung RC et al., (1988) Mol Immunol 25(1):7-15), solid-phase direct biotin-avidin EIA (Cheung RC et al., (1988) Mol Immunol 25(1):7-15), solid-phase direct labeling assays ... al., (1990) Virology 176:546-52), and direct labeling RIA (Moldenhauer G et al., (1990) Scand J Immunol 32:77-82), all of which are incorporated herein by reference in their entireties. Typically, such assays involve the use of purified antigen (e.g., LAG-3, such as human LAG-3) bound to a solid surface or cells bearing either of these unlabeled test immunoglobulins and a labeled reference immunoglobulin. Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin.Typically, the test immunoglobulin is present in excess. Typically, when a competing antibody is present in excess, it inhibits specific binding of a reference antibody to a common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or more. Competitive binding assays can be configured in a number of different formats, using either labeled antigen or labeled antibodies. In a common version of this assay, the antigen is immobilized on a 96-well plate. The ability of unlabeled antibodies to block binding of the labeled antibody to the antigen is then measured using radioactive or enzyme labels. For further details, see, e.g., Wagener C et al., (1983) J Immunol 130:2308-2315; Wagener C et al., (1984) J Immunol Methods 68:269-274; Kuroki M 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. 386-389, all of which are incorporated herein by reference in their entireties.

[0163] In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), 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, and optionally, the amino acid residue at position 1 of the heavy chain is converted to pyroglutamic acid. 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 pyroglutamic acid. 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 pyroglutamic acid. 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 pyroglutamic acid. 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, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 178.In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 179. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid 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 changed to pyroglutamic acid.

[0164] In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising a light chain comprising the amino acid sequence set forth in SEQ ID NO: 187, 228, 188, 189, 190, or 191, and optionally, the amino acid residue at position 1 of the light chain is converted to pyroglutamic acid. In certain embodiments, the antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 187. In certain embodiments, the antibody comprises a light chain comprising 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 pyroglutamic acid. In certain embodiments, the antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 188. In certain embodiments, the antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 189. In certain embodiments, the antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 190. In certain embodiments, the antibody comprises a light chain comprising 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 changed to pyroglutamic acid.

[0165] In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 168 and a light chain comprising the amino acid sequence of SEQ ID NO: 187. In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising a heavy chain comprising 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 pyroglutamic acid. In certain embodiments, X in SEQ ID NO: 225 is pyroglutamic acid and X in SEQ ID NO: 228 is E. In certain embodiments, X in SEQ ID NO: 225 is pyroglutamic acid and X in SEQ ID NO: 228 is pyroglutamic acid.

[0166] In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), 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 certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 226, and a 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 pyroglutamic acid. In certain embodiments, X in SEQ ID NO: 226 is pyroglutamic acid, and X in SEQ ID NO: 228 is E. In certain embodiments, X in SEQ ID NO: 226 is pyroglutamic acid, and X in SEQ ID NO: 228 is pyroglutamic acid.

[0167] In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), 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 certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 227, and a 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 pyroglutamic acid. In certain embodiments, X in SEQ ID NO: 227 is pyroglutamic acid and X in SEQ ID NO: 228 is E. In certain embodiments, X in SEQ ID NO: 227 is pyroglutamic acid and X in SEQ ID NO: 228 is pyroglutamic acid.

[0168] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 171, and a light chain comprising the amino acid sequence of SEQ ID NO: 187. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising a heavy chain comprising 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 disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising 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 present disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising a heavy 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 present disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising 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 present disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 176, and a light chain comprising the amino acid sequence of SEQ ID NO: 187. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 177, and a light chain comprising the amino acid sequence of SEQ ID NO: 187. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising 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 disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising 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 disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), 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 present disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 181, and a light chain comprising the amino acid sequence of SEQ ID NO: 187. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising 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 disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 183, and a light chain comprising the amino acid sequence of SEQ ID NO: 187. In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 184, and a light chain comprising the amino acid sequence of SEQ ID NO: 187. In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 185, and a light chain comprising the amino acid sequence of SEQ ID NO: 187. In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising a heavy chain comprising 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 pyroglutamic acid. In certain embodiments, the amino acid residue at position 1 of the light chain is converted to pyroglutamic acid. In certain embodiments, the amino acid residue at position 1 of the heavy chain is converted to pyroglutamic acid and the amino acid residue at position 1 of the light chain is converted to pyroglutamic acid.

[0169] Any Ig constant region may be used in the antibodies disclosed herein. In certain embodiments, the Ig region is a constant region of a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class of immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG 2a , and IgG 2b ).

[0170] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), 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 disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 194. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 195. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising a light chain constant region comprising the amino acid sequence of SEQ ID NO: 198. In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), the isolated antibody comprising a light chain constant region comprising the amino acid sequence of SEQ ID NO:219.

[0171] In certain embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., the CH2 domain (residues 231-340 of human IgG1) and / or the CH3 domain (residues 341-447 of human IgG1) and / or hinge region) of an antibody described herein, numbered according to the EU numbering system, to alter one or more functional properties of the antibody, e.g., serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity.

[0172] In certain embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the hinge region (CH1 domain) of the Fc region such that the number of cysteine ​​residues in the hinge region is altered (e.g., increased or decreased), e.g., as described in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of the CH1 domain can be altered, for example, to facilitate assembly of the light and heavy chains or to alter (e.g., increase or decrease) the stability of the antibody.

[0173] In certain embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into an IgG constant domain or an FcRn-binding fragment thereof (preferably an Fc or hinge-Fc domain fragment) to alter (e.g., decrease or increase) the half-life of the antibody in vivo. For examples of mutations that will alter (e.g., decrease or increase) the half-life of an antibody in vivo, see, for example, International Publication Nos. WO02 / 060919, WO98 / 23289, and WO97 / 34631, as well as U.S. 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. In some embodiments, one, two, or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into an IgG constant domain or FcRn-binding fragment thereof (preferably, an Fc or hinge-Fc domain fragment) to decrease the half-life of the antibody in vivo. In other embodiments, one, two, or more amino acid mutations (e.g., substitutions, insertions, or deletions) are introduced into an IgG constant domain or FcRn-binding fragment thereof (preferably, an Fc or hinge-Fc domain fragment) to increase the half-life of the antibody in vivo. In certain embodiments, the antibody 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 the third constant (CH3) domain (residues 341-447 of human IgG1), numbered according to the EU numbering system. In certain embodiments, the IgG1 constant region of the antibodies described herein comprises a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256, numbered according to the EU numbering system. See U.S. Patent No. 7,658,921, which is incorporated herein by reference in its entirety.This type of mutant IgG is referred to as a "YTE mutant" and has been shown to display a four-fold increased half-life compared to the wild-type version of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281:23514-24, 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 at amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU numbering system.

[0174] In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., the CH2 domain (residues 231-340 of human IgG1), and / or the CH3 domain (residues 341-447 of human IgG1), and / or the hinge region, numbered according to the EU numbering system) of an antibody described herein to increase or decrease the affinity of the antibody for an Fc receptor (e.g., an activating Fc receptor) on an effector cell. Mutations in the Fc region of an antibody that decrease or increase the affinity of the antibody for an Fc receptor, and techniques for introducing such mutations into an Fc receptor or fragment thereof, are known to those of skill in the art. Examples of mutations in the Fc receptors of antibodies 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, U.S. Patent No. 6,737,056, and International Publication Nos. WO02 / 060919, WO98 / 23289, and WO97 / 34631, all of which are incorporated by reference herein in their entireties.

[0175] In further embodiments, one, two or more amino acid substitutions are introduced into the IgG constant domain Fc region 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 according to the EU numbering system, can be replaced by a different amino acid residue, such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand for which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. 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 (by point mutation or other means) of the constant region domain can reduce Fc receptor binding of circulating antibodies, thereby increasing tumor localization. For a description of mutations that delete or inactivate constant domains and thereby increase tumor localization, see, e.g., U.S. Patent Nos. 5,585,097 and 8,591,886, each of which is incorporated herein by reference in its entirety. 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 on the Fc region, which may reduce Fc receptor binding (see, e.g., Shields RL et al., (2001) J Biol Chem 276:6591-604, 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 the antibodies described herein: N297A, N297Q, L235A and L237A, L234A and L235A, E233P, L234V, L235A, C236 deletion, P238A, D265A, A327Q, or 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 made in the constant region of the antibodies described herein.

[0176] In certain embodiments, the antibodies described herein comprise an IgG1 constant domain with an N297Q or N297A amino acid substitution numbered according to the EU numbering system. In one embodiment, the antibodies described herein comprise 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 antibodies described herein comprise 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 antibodies described herein at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain numbered according to the EU index numbering are not L, L, and D, respectively. This approach is described in detail in International Publication No. WO14 / 108483, which is incorporated herein by reference in its entirety. In certain embodiments, the amino acids corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, or A, A, and A, respectively.

[0177] In certain embodiments, one or more amino acids selected from amino acid residues 329, 331, and 322, numbered according to the EU numbering system, in the constant region of the antibodies described herein may be replaced by a different amino acid residue, such that the antibody has altered C1q binding and / or reduced or lost complement-dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent No. 6,194,551 (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 the antibodies described herein are altered, thereby altering the ability of the antibody to bind complement. This approach is described further 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 by mutating one or more amino acids (e.g., introducing amino acid substitutions) at the following positions to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for Fcγ receptors: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 70, 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. This approach is further described in International Publication No. WO 00 / 42072, which is incorporated herein by reference in its entirety.

[0178] In certain embodiments, the antibodies described herein comprise a constant region of an IgG4 antibody, in which the serine at amino acid residue 228 of the heavy chain, numbered according to the EU numbering system, is substituted with a proline. In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 196. In certain embodiments, the disclosure provides an isolated antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 197.

[0179] In certain embodiments, any of the constant region mutations or modifications described herein may be introduced into one or both of the heavy chain constant regions of an antibody described herein that has two heavy chain constant regions.

[0180] In certain embodiments, the disclosure provides isolated antibodies that specifically bind to LAG-3 (eg, human LAG-3) and function as antagonists.

[0181] In certain embodiments, the disclosure provides isolated antibodies that specifically bind to LAG-3 (e.g., human LAG-3) and reduce LAG-3 (e.g., human LAG-3) activity 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 of skill in the art compared to LAG-3 (e.g., human LAG-3) activity with no antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind LAG-3 (e.g., human LAG-3)). In certain embodiments, the disclosure provides isolated antibodies that specifically bind to LAG-3 (e.g., human LAG-3) and reduce LAG-3 (e.g., human LAG-3) activity by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold as assessed by methods described herein and / or known to one of skill in the art, compared to LAG-3 (e.g., human LAG-3) activity with no antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind LAG-3 (e.g., human LAG-3)). Non-limiting examples of LAG-3 (e.g., human LAG-3) activity can include LAG-3 (e.g., human LAG-3) signaling, LAG-3 (e.g., human LAG-3) binding to a LAG-3 (e.g., human LAG-3) ligand (e.g., MHC class II), and inhibition of cytokine production (e.g., IL-2, and / or TNF-α). In certain embodiments, the present disclosure provides isolated antibodies that specifically bind to LAG-3 (e.g., human LAG-3) and inactivate, reduce, or inhibit LAG-3 (e.g., human LAG-3) activity. In certain embodiments, reduction of LAG-3 (e.g., human LAG-3) activity is assessed as described in the Examples below.

[0182] In certain embodiments, the disclosure provides isolated antibodies that specifically bind to LAG-3 (e.g., human LAG-3) and reduce LAG-3 (e.g., human LAG-3) binding to its ligand (e.g., 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 assessed by methods described herein (see Examples below) or methods known to one of skill in the art, compared to LAG-3 (e.g., human LAG-3) binding to its ligand (e.g., MHC class II) by no antibody or an unrelated antibody (e.g., an antibody that does not specifically bind LAG-3 (e.g., human LAG-3)). In certain embodiments, the disclosure provides isolated antibodies that specifically bind to LAG-3 (e.g., human LAG-3) and reduce LAG-3 (e.g., human LAG-3) binding to its ligand (e.g., MHC class II) by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold as assessed by methods described herein (see Examples below) or methods known to one of skill in the art, as compared to LAG-3 (e.g., human LAG-3) binding to its ligand (e.g., MHC class II) by no antibody or an unrelated antibody (e.g., an antibody that does not specifically bind LAG-3 (e.g., human LAG-3)).

[0183] In certain embodiments, the disclosure provides isolated antibodies that specifically bind to LAG-3 (e.g., human LAG-3) and increase 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 by methods described herein (see Examples below) or known to one of skill in the art compared to cytokine production with no antibody or an unrelated antibody (e.g., an antibody that does not specifically bind to LAG-3 (e.g., human LAG-3)). In certain embodiments, the disclosure provides isolated antibodies that specifically bind to LAG-3 (e.g., human LAG-3) and increase cytokine production (e.g., IL-2, and / or TNF-α) by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold, as assessed by methods described herein (see Examples, below) or known to one of skill in the art, compared to cytokine production with no antibody or an unrelated antibody (e.g., an antibody that does not specifically bind to LAG-3 (e.g., human LAG-3)).

[0184] In certain embodiments, the disclosure provides an antibody that specifically binds 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-L1 antibody (e.g., avelumab, durvalumab, or atezolizumab), or an anti-CTLA-4 antibody (e.g., ipilimumab), that inhibits IL-2 production as described herein, compared to no antibody or an unrelated antibody (e.g., an antibody that does not specifically bind LAG-3 (e.g., human LAG-3)). In one embodiment, an isolated antibody is provided that increases IL-2 production in human peripheral blood mononuclear cells (PBMCs) in response to Staphylococcus enterotoxin A (SEA) stimulation by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold, as assessed by the method of (see Examples below) or methods known to one of skill in the art.

[0185] In certain embodiments, human peripheral blood mononuclear cells (PBMCs) stimulated with Staphylococcus enterotoxin A (SEA), in the presence of an antibody described herein, specifically bind LAG-3 (e.g., human LAG-3) and exhibit at least about a 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold increased IL-2 production compared to PBMCs stimulated with SEA alone without any antibody or with an irrelevant antibody (e.g., an antibody that does not specifically bind LAG-3 (e.g., human LAG-3)) as assessed by methods described herein (see Examples below) or methods known to one of skill in the art.

[0186] In certain embodiments, the disclosure provides isolated antibodies that specifically bind 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), increase TNFα production in tumor infiltrating lymphocytes (TILs) in response to anti-CD3 antibody and anti-CD28 antibody stimulation by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold compared to TNFα production without an antibody that specifically binds LAG-3 (e.g., human LAG-3), as assessed by methods described herein (see Examples below) or methods known to one of skill in the art. In one embodiment, the TILs are derived from a renal cell carcinoma tumor. In another embodiment, the TILs are derived from a colon cancer tumor.

[0187] In certain embodiments, tumor infiltrating lymphocytes (TILs) stimulated with anti-CD3 and anti-CD28 antibodies in the presence of an antibody described herein specifically bind LAG-3 (e.g., human LAG-3) and increase TNFα production by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold, as assessed by methods described herein (see Examples below) or methods known to one of skill in the art. In one embodiment, the TILs are derived from a renal cell carcinoma tumor. In another embodiment, the TILs are derived from a colon cancer tumor.

[0188] 6.3 Pharmaceutical Compositions Provided herein are compositions (e.g., pharmaceutical compositions) comprising an anti-LAG-3 (e.g., human LAG-3) antibody described herein having a desired degree of purity in a pharma- ceutically 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 employed, and may include buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl, or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, serum albumin, gelatin, or immunoglobulins. These may include buffers such as proteins such as riboflavin, 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 (e.g., Zn-protein complexes), and / or non-ionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0189] In certain embodiments, a composition comprises one or more anti-LAG-3 (e.g., human LAG-3) antibodies disclosed herein, wherein in some of the antibodies, the N-terminal amino acid residue(s) of the heavy and / or light chains are converted to pyroglutamic acid (e.g., as a result of post-translational cyclization of the free amino group of the N-terminal E or Q residue). In certain embodiments, at least 50% (e.g., at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) of the N-terminal amino acid residues of the heavy chains in the composition are converted to pyroglutamic acid. In certain embodiments, less than 50% (e.g., less than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, or 40%) of the N-terminal amino acid residues of the light chains in the composition are converted to pyroglutamic acid. In certain embodiments, at least 50% (e.g., at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) of the N-terminal amino acid residues of the heavy chains in the composition are converted to pyroglutamic acid, and less than 50% (e.g., less than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, or 40%) of the N-terminal amino acid residues of the light chains in the composition are converted to pyroglutamic acid.

[0190] In certain embodiments, the pharmaceutical composition comprises an anti-LAG-3 (e.g., human LAG-3) antibody described herein, and optionally one or more additional prophylactic or therapeutic agents, in a pharma- ceutically acceptable carrier. In certain embodiments, the pharmaceutical composition comprises an effective amount of an antibody described herein, and optionally one or more additional prophylactic or therapeutic agents, in a pharma- ceutically acceptable carrier. In some embodiments, the antibody is the only active ingredient contained in the pharmaceutical composition. The pharmaceutical compositions described herein may be useful for inhibiting LAG-3 (e.g., human LAG-3) activity and treating conditions such as cancer or infectious diseases.

[0191] Pharmaceutically acceptable carriers used in parenteral preparations 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 pharma- ceutically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose, and lactated Ringer's injection. Non-aqueous parenteral vehicles include solidified oils of vegetable origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations can be added to parenteral preparations filled in multi-dose containers, including phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphates and citrates. 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). Sequestering or chelating agents for metal ions include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible vehicles, and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0192] The pharmaceutical composition may 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 contemplated herein. Injectables may be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to 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 composition to be administered may also contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, and other agents, such as, for example, sodium acetate, sorbitan monolaurate, triethanolamine, oleates, and cyclodextrins.

[0193] Preparations for parenteral administration of antibodies include sterile solutions ready for injection, sterile dry soluble products, lyophilized powders to be mixed with a solvent immediately before use, including tablets for subcutaneous injection, sterile suspensions ready for injection, sterile dry insoluble products to be mixed with a vehicle immediately before use, and sterile emulsions. The solutions may be either aqueous or non-aqueous.

[0194] If administered intravenously, suitable carriers include saline or phosphate buffered saline (PBS), as well as solutions containing viscosity enhancing and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol, and mixtures thereof.

[0195] Topical mixtures containing the antibody are prepared as described for local and systemic administration. The resulting mixture may be a solution, suspension, emulsion, etc., and may be formulated as a cream, gel, ointment, emulsion, solution, elixir, lotion, suspension, tincture, paste, foam, aerosol, wash, spray, suppository, bandage, skin patch, or any other formulation suitable for topical administration.

[0196] The anti-LAG-3 (e.g., human LAG-3) antibodies described herein can be formulated as aerosols for local application, such as by inhalation (see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for the delivery of steroids effective in the treatment of inflammatory diseases, particularly asthma, and are incorporated by reference in their entireties). These formulations for administration to the respiratory tract can be in the form of an aerosol or solution for a nebulizer, or as an ultrafine powder for insufflation, alone or in combination with an inert carrier such as lactose. In such cases, the particles of the formulation have a diameter, in one embodiment, of less than 50 microns, in one embodiment, less than 10 microns.

[0197] The anti-LAG-3 (e.g., human LAG-3) antibodies described herein can be formulated for local or topical application, such as topical application to mucous membranes such as the skin and eyes, and for application to the eye, or for intracapsular or intrathecal application, in the form of gels, creams, and lotions. Topical administration is contemplated for transdermal delivery and for administration to the eye or mucous membranes, or for inhalation therapy. Nasal solutions alone or in combination with other pharma- ceutically acceptable excipients can also be administered.

[0198] Transdermal patches, including iontophoretic and electrophoretic devices, are well known to those of skill in the art and can be used to administer antibodies. For example, such patches are disclosed in U.S. Patent Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957, all of which are incorporated herein by reference in their entireties.

[0199] In certain embodiments, the pharmaceutical composition comprising the antibody described herein is a lyophilized powder that can be reconstituted for administration as a solution, emulsion, and other mixture. It can also be reconstituted and formulated as a solid or gel. The lyophilized powder is prepared by dissolving the antibody described herein, or a pharma- ceutically acceptable derivative thereof, in a suitable solvent. In some embodiments, the lyophilized powder is sterile. The solvent may contain excipients that improve the stability or other pharmacological components of the powder or the 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 agents. The solvent may also contain a buffer, such as citrate, sodium or potassium phosphate, or other such buffers known to those of skill in the art, in one embodiment at about neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides the desired formulation. In one embodiment, the resulting solution is apportioned into vials for lyophilization. Each vial contains a single or multiple doses of the compound. The lyophilized powder may be stored under appropriate conditions, such as at about 4° C. to room temperature. Reconstitution of this lyophilized powder with water for injection provides a formulation for use in parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other appropriate carrier. The exact amount depends on the compound selected. Such amounts may be empirically determined.

[0200] The anti-LAG-3 (e.g., human LAG-3) antibodies described herein, and other compositions provided herein, can also be formulated to target to specific tissues, receptors, or other areas of the body of the subject being treated. Many such targeting methods are well known to those of skill in the art. All such targeting methods are contemplated herein for use in the compositions. For non-limiting examples of targeting methods, see, e.g., U.S. Patent Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,071,570 ... See, for example, Nos. 60,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542, and 5,709,874, all of which are incorporated herein by reference in their entireties. In certain embodiments, the antibodies described herein are targeted to tumors.

[0201] Compositions to be used for in vivo administration can be sterile, which is readily accomplished, for example, by filtration through sterile filtration membranes.

[0202] 6.4 Method and Use In another aspect, the present disclosure provides a method of treating a subject with an anti-LAG-3 (e.g., human LAG-3) antibody disclosed herein. Any disease or disorder in a subject that would benefit from inhibition of LAG-3 (e.g., human LAG-3) function can be treated with an anti-LAG-3 (e.g., human LAG-3) antibody disclosed herein. The anti-LAG-3 (e.g., human LAG-3) antibody disclosed herein is particularly useful for inhibiting immune tolerance to tumors, and can therefore be used as immunotherapy for subjects with cancer. For example, in certain embodiments, the present disclosure provides 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 anti-LAG-3 (e.g., human LAG-3) antibody or pharmaceutical composition thereof as disclosed herein. In certain embodiments, the present disclosure provides a method of treating cancer in a subject, comprising administering to the subject an effective amount of an antibody or pharmaceutical composition as disclosed herein.

[0203] Cancers that can be treated with the anti-LAG-3 (e.g., human LAG-3) antibodies or pharmaceutical compositions disclosed herein include, but are not limited to, solid tumors, hematological cancers, leukemia, lymphoma, osteosarcoma, rhabdomyosarcoma, neuroblastoma, renal cancer, renal transitional cell carcinoma, bladder cancer, Wilms' carcinoma, ovarian cancer, pancreatic cancer, breast cancer, (e.g., characterized by mutations in BRCA1 and / or BRCA2), prostate cancer, bone cancer, lung cancer (e.g., non-small cell lung cancer, or small cell lung cancer), gastric cancer, colorectal cancer, and the like. Cancers include cervical cancer, synovial sarcoma, head and neck cancer, squamous cell carcinoma, multiple myeloma, renal cell carcinoma, retinoblastoma, hepatoblastoma, hepatocellular carcinoma, melanoma, rhabdoid tumor of the kidney, Ewing's sarcoma, chondrosarcoma, brain tumor, glioblastoma, meningioma, pituitary adenoma, vestibular schwannoma, primitive neuroectodermal tumor, medulloblastoma, astrocytoma, anaplastic astrocytoma, oligodendroglioma, ependymoma, choroid plexus papilloma, polycythemia vera, thrombocythemia, idiopathic myofibrosis, soft tissue sarcoma, thyroid cancer, endometrial cancer, carcinoid cancer, liver cancer, breast cancer, and gastric cancer. In certain embodiments, the cancer is a metastatic cancer, e.g., of the various above.

[0204] In certain embodiments, the cancer is a solid tumor, a hematological cancer (e.g., leukemia, lymphoma, myeloma), and metastatic lesions thereof. In one embodiment, the cancer is a solid tumor. Examples of solid tumors include malignant lesions, such as sarcomas and carcinomas (e.g., adenocarcinomas) of various organ systems, such as those affecting the lung, breast, lymphatic system, gastrointestinal tract or colon, reproductive and genitourinary organs (e.g., kidney, urothelium, bladder cells), pharynx, CNS (e.g., brain, neural or glial cells), skin (e.g., melanoma), head and neck (e.g., head and neck squamous cell carcinoma (HNCC)), and pancreas. For example, melanoma, colon cancer, gastric cancer, rectal cancer, renal cell carcinoma, breast cancer (e.g., breast cancer that does not express one, two or all of the estrogen receptor, the progesterone receptor or Her2 / neu, e.g., triple negative breast cancer), liver cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC) (e.g., NSCLC with squamous and / or non-squamous histology) or small cell lung cancer), prostate cancer, head and neck cancer (e.g., HPV+ squamous cell carcinoma), small intestine cancer, and esophageal cancer.

[0205] In one embodiment, the cancer is hematological cancer, for example, leukemia, lymphoma, or myeloma.In one embodiment, the cancer is leukemia, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute myeloblastic leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), chronic lymphocytic leukemia (CLL), or hairy cell leukemia. In one embodiment, the cancer is a lymphoma, such as B cell lymphoma, diffuse large B cell lymphoma (DLBCL), activated B cell-like (ABC) diffuse large B cell lymphoma, germinal center B cell (GCB) diffuse large B cell lymphoma, mantle cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, relapsed non-Hodgkin lymphoma, refractory non-Hodgkin lymphoma, relapsed follicular non-Hodgkin lymphoma, Burkitt lymphoma, small lymphocytic lymphoma, follicular lymphoma, lymphoplasmacytic lymphoma, or extranodal marginal zone lymphoma. In one embodiment, the cancer is a myeloma, such as multiple myeloma.

[0206] In certain embodiments, the present disclosure provides a method for preventing or treating an infectious disease in a subject, comprising administering to the subject an effective amount of an anti-LAG-3 (e.g., human LAG-3) antibody or pharmaceutical composition thereof as disclosed herein. In one embodiment, a method for preventing and / or treating an infection (e.g., viral infection, bacterial infection, fungal infection, protozoal infection, or parasitic infection) is provided herein. The infection prevented and / or treated according to the method may be caused by an infectious agent identified herein. In certain embodiments, the anti-LAG-3 (e.g., human LAG-3) antibody or composition thereof described herein is the only active agent administered to the subject. In some embodiments, the anti-LAG-3 (e.g., human LAG-3) antibody or composition thereof described herein is used in combination with an anti-infective intervention (e.g., anti-viral, anti-bacterial, anti-fungal, or anti-helminthic) for the treatment of an infectious disease.

[0207] Infectious diseases that can be treated and / or prevented by the anti-LAG-3 (e.g., human LAG-3) antibodies or pharmaceutical compositions disclosed herein are caused by infectious agents including, but not limited to, bacteria, fungi, protozae, and viruses. In certain embodiments, the infectious diseases treated and / or prevented by the anti-LAG-3 (e.g., human LAG-3) antibodies or pharmaceutical compositions disclosed herein are caused by viruses. Viral diseases or infections that can be prevented and / or treated in accordance with the methods described herein include, but are not limited to, those caused by hepatitis A, hepatitis B, hepatitis C, influenza (e.g., influenza A or influenza B), chickenpox, adenovirus, herpes simplex type I (HSV-I), herpes simplex type II (HSV-II), cowpox, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papillomavirus, papovavirus, cytomegalovirus, echinovirus, arbovirus, hantavirus, coxsackievirus, mumps virus, measles virus, rubella virus, poliovirus, smallpox virus, Epstein-Barr virus, human immunodeficiency virus type I (HIV-I), human immunodeficiency virus type II (HIV-II), and viral disease agents such as meningitis, encephalitis, dengue fever, or smallpox.

[0208] Bacterial infections that may be prevented and / or treated include infections caused by Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Proteus vulgaris, Staphylococcus viridans, and Pseudomonas aeruginosa. Bacterial diseases caused by bacteria (e.g., Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Proteus vulgaris, Staphylococcus viridans, and Pseudomonas aeruginosa) that can be prevented and / or treated in accordance with the methods described herein include, but are not limited to, mycobacteria rickettsia, mycoplasma, neisseria, S. pneumonia, Borrelia burgdorferi (Lyme disease), Bacillus antracis (anthrax), tetanus, Streptococcus, Staphylococcus, mycobacterium, pertissus, cholera, plague, diphtheria, chlamydia, S. aureus, and Legionella.

[0209] Protozoal diseases or 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, trypanosoma, or malaria. Parasitic diseases or 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.

[0210] Fungal diseases or infections that may be prevented and / or treated in accordance with the methods described herein include, but are not limited to, Candida infections, zygomycosis, Candida mastitis, progressive disseminated trichosporonosis with occult trichosporonemia, disseminated candidiasis, pulmonary paracoccidioidomycosis, pulmonary aspergillosis, Pneumocystis carinii pneumonia, cryptococcal meningitis, coccidioidal meningoencephalitis, and cerebrospinal vasculitis, Aspergillus niger infection, Fusarium keratitis, sinus fungal disease, Aspergillus fumigatus endocarditis, tibial dyschondroplasia, Candida glabrata vaginitis, oropharyngeal candidiasis, X-linked chronic granulomatous disease, tinea pedis, cutaneous candidiasis, fungal placentitis, disseminated trichosporonosis, allergic bronchopulmonary aspergillosis, fungal keratitis, Cryptococcus neoformans infection, fungal peritonitis, Curvularia geniculata infection, staphylococcal endophthalmitis, sporotrichosis, and dermatophytosis.

[0211] In certain embodiments, the present disclosure provides a method for preventing or treating a nervous system disease or disorder in a subject, comprising administering to the subject an effective amount of an anti-LAG-3 (e.g., human LAG-3) antibody or a pharmaceutical composition thereof as disclosed herein. In some embodiments, the nervous system disease or disorder is a synucleinopathy. In some embodiments, the nervous system disease or disorder is Parkinson's disease.

[0212] In certain embodiments, the methods further include 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 (e.g., azacytidine). In certain embodiments, the checkpoint targeting agent is selected from the group consisting of an antagonistic anti-CTLA-4 antibody, an antagonistic anti-PD-L1 antibody, an antagonistic anti-PD-L2 antibody, an antagonistic anti-PD-1 antibody, an antagonistic anti-TIM-3 antibody, an antagonistic anti-LAG-3 antibody, an antagonistic anti-CEACAM1 antibody, an agonist anti-LAG-3 (e.g., human LAG-3) antibody, an agonist anti-OX40 antibody, an antagonistic anti-TIGIT antibody, an agonist anti-CD137 antibody, an antagonistic anti-VISTA antibody, an antagonistic anti-CD73 antibody, and an antagonistic anti-CD96 antibody.

[0213] In one embodiment, the invention relates to an antibody and / or pharmaceutical composition of the invention for use in a method of the invention, the method further comprising administering an additional therapeutic agent to a subject. In one embodiment, the invention relates to (a) an antibody and / or pharmaceutical composition of the invention, and (b) an additional therapeutic agent for use as a medicament. In one embodiment, the invention relates to (a) an antibody and / or pharmaceutical composition of the invention, and (b) an additional therapeutic agent for use in a method of treating cancer. In a further embodiment, the invention relates to a pharmaceutical composition, kit, or kit of parts comprising (a) an antibody and / or pharmaceutical composition of the 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.

[0214] In certain embodiments, anti-PD-1 antibodies are used in the methods disclosed herein. In certain 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 Mediimmune. 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.

[0215] Further non-limiting examples of anti-PD-1 antibodies that can be used in the treatment methods disclosed herein are disclosed in the following patents and patent applications, which are incorporated by reference in their entireties: U.S. Pat. No. 6,808,710, U.S. Pat. No. 7,332,582, U.S. Pat. No. 7,488,802, U.S. Pat. No. 8,008,449, U.S. Pat. No. 8,114,845, U.S. Pat. No. 8,168,757, U.S. Pat. No. 8,354,509, U.S. Pat. No. 8,686,119, U.S. Pat. No. 8,735,553, U.S. Pat. No. 8,747,847, U.S. Pat. No. 8,779,105, U.S. Pat. No. 8,927,697, U.S. Pat. No. 8,993,731, U.S. Pat. No. 9,102,727, U.S. Pat. No. 9,205,148, U.S. Publication No. 2013 / 0202623. A1, U.S. Publication No. 2013 / 0291136 A1, U.S. Publication No. 2014 / 0044738 A1, U.S. Publication No. 2014 / 0356363 A1, U.S. Publication No. 2016 / 0075783 A1, and PCT Publication Nos. WO2013 / 033091 A1, WO2015 / 036394 A1, WO2014 / 179664 A2, WO2014 / 209804 A1, WO2014 / 206107 A1, WO2015 / 058573 A1, WO2015 / 085847 A1, and WO2015 / 200119 A1, PCT Publication No. 2016 / 015685 A1, and PCT Publication No. WO2016 / 020856 A1.

[0216] 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 Mediimmune. 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.

[0217] Non-limiting examples of anti-PD-L1 antibodies that may be used in the treatment methods disclosed herein are disclosed in the following patents and patent applications, which are incorporated by reference in their entireties: U.S. Patent No. 7,943,743; U.S. Patent No. 8,168,179; U.S. Patent No. 8,217,149; U.S. Patent No. 8,552,154; U.S. Patent No. 8,779,108; U.S. Patent No. 8,981,063; U.S. Patent No. 9,175,082; U.S. Publication No. 2010 / 0203056 A1; U.S. Publication No. 2003 / 0232323 A1; U.S. Publication No. 2013 / 0323249 A1; U.S. Publication No. 2014 / 0341917 A1; U.S. Publication No. 2014 / 0044738 A1; U.S. Publication No. 2015 / 0203580 A1, U.S. Publication No. 2015 / 0225483 A1, U.S. Publication No. 2015 / 0346208 A1, U.S. Publication No. 2015 / 0355184 A1, and PCT Publication Nos. WO2014 / 100079 A1, WO2014 / 022758 A1, WO2014 / 055897 A2, WO2015 / 061668 A1, WO2015 / 109124 A1, WO2015 / 195163 A1, WO2016 / 000619 A1, and WO2016 / 030350 A1.

[0218] 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 Mediimmune.

[0219] Non-limiting examples of anti-CTLA-4 antibodies that can be used in the treatment methods disclosed herein are disclosed in the following patents and patent applications, which are incorporated by reference in their entireties: U.S. Patent No. 6,984,720, U.S. Patent No. 7,411,057, U.S. Patent No. 7,034,121, U.S. Patent No. 8,697,845, U.S. Publication No. US2009 / 0123477 A1, U.S. Publication No. US2014 / 0105914 A1, U.S. Publication No. US2013 / 0267688 A1, U.S. Publication No. US2016 / 0145355 A1, PCT Publication No. WO2014 / 207064 A1, and PCT Publication No. WO2016 / 015675 A1.

[0220] In certain embodiments, an anti-LAG-3 (e.g., human LAG-3) antibody disclosed herein is administered to a subject in combination with a compound that targets an 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, e.g., 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 certain embodiments, the anti-LAG-3 (e.g., human LAG-3) antibody disclosed herein is administered to a subject in combination with an IDO inhibitor for treating cancer. The IDO inhibitor described herein for use in treating cancer is present in a pharmaceutical composition in a solid dosage form, such as a tablet, pill, or capsule, the pharmaceutical composition comprising an IDO inhibitor and a pharma- ceutically acceptable excipient. Thus, the antibody described herein and the IDO inhibitor described herein can be administered separately, sequentially, or simultaneously as separate dosage forms. In one embodiment, the antibody is administered parenterally and the IDO inhibitor is administered orally. In certain 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. WO2010 / 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.

[0221] In certain embodiments, the present disclosure 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 inhibitor of indoleamine-2,3-dioxygenase (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.

[0222] In certain embodiments, the anti-LAG-3 (e.g., human LAG-3) antibodies disclosed herein are administered to a subject in combination with another anti-cancer agent. Exemplary anti-cancer agents include antibody therapeutics such as trastuzumab (Herceptin), antibodies against co-stimulatory or co-inhibitory molecules such as CTLA-4, CD137, and PD-1, and antibodies against cytokines such as IL-10 and TGF-β.

[0223] In certain embodiments, the additional therapeutic agent is an inhibitor of JAK, PI3K delta, BRD, PI3K gamma, or Axl / Mer. In certain embodiments, the additional therapeutic agent is an inhibitor of JAK, including JAK1 and / or JAK2. In certain embodiments, the additional therapeutic agent is an inhibitor of PI3K delta. In certain embodiments, the additional therapeutic agent is an inhibitor of BRD. In certain embodiments, the additional therapeutic agent is an inhibitor of PI3K gamma. In certain embodiments, the additional therapeutic agent is an inhibitor of Axl / Mer.

[0224] Further examples of anti-cancer agents include those that block immune cell trafficking, such as antagonists to chemokine receptors, including CCR2 and CCR4, and those that augment the immune system, such as adjuvants or adoptive T cell transfer.

[0225] One or more additional immune checkpoint modulators can be used in combination with the anti-LAG-3 (e.g., human LAG-3) antibodies disclosed herein to treat any disease, disorder, or condition described herein, e.g., a TAM-associated disease, disorder, or condition. Exemplary immune checkpoint modulators include modulators for 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, VISTA, PD-1, PD-L1, and PD-L2. In some embodiments, the immune checkpoint molecule is a costimulatory checkpoint molecule selected from CD27, CD28, CD40, ICOS, OX40, GITR, and CD137. In some embodiments, the immune checkpoint molecule is a co-inhibitory checkpoint molecule selected from A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, PD-1, TIM-3, and VISTA. In some embodiments, the anti-LAG-3 (e.g., human LAG-3) antibodies disclosed herein can be used in combination with one or more agents selected from a KIR inhibitor, a TIGIT inhibitor, a LAIR1 inhibitor, a CD160 inhibitor, a 2B4 inhibitor, and a TGFR beta inhibitor.

[0226] In some embodiments, the modulator of an immune checkpoint molecule is an antagonistic anti-PD1 antibody, an antagonistic anti-PD-L1 antibody, or an antagonistic anti-CTLA-4 antibody.

[0227] In some embodiments, the modulator of the immune checkpoint molecule is an agonist of GITR, such as an agonist anti-GITR antibody. In some embodiments, the agonist anti-GITR antibody is TRX518 or MK-4166.

[0228] In some embodiments, the modulator of the immune checkpoint molecule is an agonist of OX40, such as an agonist anti-OX40 antibody or an OX40L fusion protein. In some embodiments, the agonist anti-OX40 antibody is MEDI0562. In some embodiments, the OX40L fusion protein is MEDI6383.

[0229] The anti-LAG-3 (e.g., human LAG-3) antibodies disclosed herein can be used in combination with one or more agents for treating a disease, such as cancer. In some embodiments, the agent is an alkylating agent, a proteasome inhibitor, a corticosteroid, or an immunomodulatory agent. Examples of alkylating agents 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).

[0230] In certain embodiments, the anti-LAG-3 (e.g., human LAG-3) antibody disclosed herein is administered to a subject in combination with a vaccine. The vaccine can be, for example, a peptide vaccine, a DNA vaccine, or an RNA vaccine. In certain embodiments, the vaccine is a heat shock protein-based tumor vaccine or a heat shock protein-based pathogen vaccine. In certain embodiments, the anti-LAG-3 (e.g., human LAG-3) antibody 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. As a result of heat shock or other forms of stress, such as exposure to toxins, oxidative stress, or glucose deprivation, their expression can be potently induced to much higher levels. Five families have been classified according to molecular weight: HSP-110, -90, -70, -60, and -28. HSPs deliver immunogenic peptides through the cross-presentation pathway in antigen-presenting cells (APCs) such as macrophages and dendritic cells (DCs), resulting in T cell activation. HSPs function as chaperone carriers of tumor-associated antigenic peptide-forming complexes capable of inducing tumor-specific immunity. Upon release from dying tumor cells, HSP-antigen complexes are taken up by antigen-presenting cells (APCs) and the antigens are processed into peptides that bind to MHC class I and class II molecules, leading to the activation of anti-tumor CD8+ and CD4+ T cells. The immunity elicited by HSP complexes derived from tumor preparations is specifically directed to the unique antigenic peptide repertoire expressed by each subject's cancer. Thus, in one embodiment, the invention relates to (a) an antibody and / or pharmaceutical composition of the invention, and (b) a vaccine for use as a medicament, e.g., for use in a method for treating cancer. In one embodiment, the invention relates to a pharmaceutical composition, kit, or kit-of-parts comprising (a) an antibody and / or pharmaceutical composition of the invention, and (b) a vaccine. In one embodiment, the vaccine is a heat shock protein-based tumor vaccine.In one embodiment, the vaccine is a heat shock protein-based pathogen vaccine.

[0231] Heat shock protein peptide complexes (HSPPCs) are protein-peptide complexes consisting of heat shock proteins non-covalently complexed with antigenic peptides. HSPPCs elicit both innate and adaptive immune responses. In certain embodiments, the antigenic peptide(s) exhibit antigenicity against the cancer being treated. HSPPCs are efficiently captured by APCs via membrane receptors (mainly CD91) or by binding to Toll-like receptors. Internalization of HSPPCs leads to functional maturation of APCs with chemokine and cytokine production, resulting in activation of natural killer cells (NK), monocytes, and Th1 and Th2 mediated immune responses. In certain embodiments, 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, the HSPPC comprises hsc70, hsp70, hsp90, hsp110, grp170, gp96, calreticulin, or a combination of two or more thereof.

[0232] In certain embodiments, the heat shock protein peptide complex (HSPPC) comprises a recombinant heat shock protein (e.g., hsp70 or hsc70) or a peptide-binding domain thereof complexed with a recombinant antigenic peptide. The recombinant heat shock protein can be produced by recombinant DNA technology, for example, using the human hsc70 sequence as described in Dworniczak and Mirault, Nucleic Acids Res. 15:5181-5197 (1987), and Genbank Accession Nos. P11142 and / or Y00371, each of which is incorporated herein by reference in its entirety. In certain embodiments, the Hsp70 sequence is as described in Hunt and Morimoto Proc. Natl. Acad. Sci. USA 82(19), 6455-6459 (1985), and Genbank Accession Nos. P0DMV8 and / or M11717, each of which is incorporated herein by reference in its entirety. Antigenic peptides can also be prepared by recombinant DNA methods known in the art.

[0233] In certain embodiments, the antigenic peptide comprises modified amino acid. In certain embodiments, the modified amino acid comprises post-translational modification. In certain embodiments, the modified amino acid comprises a mimic of post-translational modification. In certain embodiments, the modified amino acid is Tyr, Ser, Thr, Arg, Lys, or His phosphorylated at side chain hydroxyl or amine. In certain embodiments, the modified amino acid is a mimic of Tyr, Ser, Thr, Arg, Lys, or His amino acid phosphorylated at side chain hydroxyl or amine.

[0234] In certain embodiments, the anti-LAG-3 (e.g., human LAG-3) antibodies disclosed herein are administered to a subject in combination with a heat shock protein peptide complex (HSPPC), such as heat shock protein peptide complex-96 (HSPPC-96), to treat cancer. HSPPC-96 comprises a 96 kDa heat shock protein (Hsp), gp96, complexed with an antigenic peptide. HSPPC-96 is a cancer immunotherapy produced from the subject's tumor and contains the antigenic "fingerprint" of the cancer. In certain embodiments, this fingerprint contains unique antigens that are only present in the specific cancer cells of that particular subject, and injection of the vaccine is intended to stimulate the subject's immune system to recognize and attack any cells that have the specific cancer fingerprint. Thus, in one embodiment, the invention relates to the antibodies and / or pharmaceutical compositions of the invention in combination with heat shock protein peptide complexes (HSPPC) for use as medicines and / or for use in methods of treating cancer.

[0235] In certain embodiments, HSPPCs, such as HSPPC-96, are produced from the tumor tissue of the subject. In certain embodiments, HSPPCs (e.g., HSPPC-96) are produced from the tumor of the type of cancer or metastasis thereof to be treated. In another particular embodiment, HSPPCs (e.g., HSPPC-96) are autologous to the subject to be treated. In certain embodiments, the tumor tissue is non-necrotic tumor tissue. In certain embodiments, at least 1 gram (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 non-necrotic tumor tissue is used to produce the vaccine regimen. In certain embodiments, after surgical resection, the non-necrotic tumor tissue is frozen before use in vaccine preparation. In some embodiments, HSPPCs, such as HSPPC-96, are isolated from tumor tissue by purification techniques, filtered, and prepared for injectable vaccines. In certain embodiments, a subject is administered 6-12 doses of HSPPC, e.g., HSPPC-96. In such embodiments, the doses of HSPPC, e.g., HSPPC-96, can be administered weekly for the first 4 doses, then every 2 weeks for 2-8 additional doses.

[0236] Further examples of HSPPCs that can be used in accordance with the methods described herein are disclosed in the following patents and patent applications: U.S. Patent Nos. 6,391,306, 6,383,492, 6,403,095, 6,410,026, 6,436,404, 6,447,780, 6,447,781, and 6,610,659, all of which are incorporated by reference herein in their entireties.

[0237] In certain embodiments, the anti-LAG-3 antibodies disclosed herein are administered to a subject in combination with an adjuvant. Various adjuvants can be used depending on the treatment situation. Non-limiting examples of suitable adjuvants include, but are not limited to, complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), Montanide ISA (incomplete Sepic adjuvant), Ribi adjuvant system (RAS), Titer Max, muramyl peptides, Syntex adjuvant formulation (SAF), alum (aluminum hydroxide and / or aluminum phosphate), aluminum salt adjuvants, Gerbu® adjuvant, nitrocellulose-absorbed antigens, encapsulated or entrapped antigens, 3 De-O-acylated monophosphoryl lipid A (3 D-MPL), immunostimulatory oligonucleotides, Toll-like receptor (TLR) ligands, mannan-binding lectin (MBL) ligands, STING agonists, saponins, Quil A, immune stimulating complexes such as QS-21, QS-7, ISCOMATRIX, and others. Other adjuvants include CpG oligonucleotides and double-stranded RNA molecules, such as poly(A), poly(U). Combinations of the above adjuvants may also be used. See, for example, U.S. Patent Nos. 6,645,495, 7,029,678, and 7,858,589, all of which are incorporated herein by reference in their entirety. In one embodiment, the adjuvant used herein is QS-21 STIMULON.

[0238] In certain embodiments, the 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. Thus, in one embodiment, the present invention relates to an antibody and / or pharmaceutical composition of the present invention in combination with an additional therapeutic agent comprising a TCR for use as a medicament and / or for use in a method of treating cancer.

[0239] In certain embodiments, the 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.

[0240] In certain embodiments, the anti-LAG-3 antibody disclosed herein is administered to a subject in combination with a TCR mimetic antibody.In certain embodiments, the TCR mimetic antibody is an antibody that specifically binds to a peptide-MHC complex.For non-limiting examples of TCR mimetic antibodies, see, for example, U.S. Patent No. 9,074,000, and U.S. Publication No. US2009 / 0304679 A1, and U.S. Publication No. US2014 / 0134191 A1.All of these are incorporated herein by reference in their entirety.

[0241] The anti-LAG-3 (e.g., human LAG-3) antibody and additional therapeutic agent (e.g., chemotherapeutic agent, radiotherapeutic agent, checkpoint targeting agent, IDO inhibitor, vaccine, adjuvant, soluble TCR, cells expressing a TCR, cells expressing a chimeric antigen receptor, and / or a TCR mimetic antibody) can be administered separately, sequentially, or simultaneously in separate dosage forms. In one embodiment, the anti-LAG-3 (e.g., human LAG-3) antibody is administered parenterally and the IDO inhibitor is administered orally.

[0242] The antibodies or pharmaceutical compositions described herein can be delivered to a subject by a variety of routes, including, but 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, by use of an inhaler or nebulizer and a formulation that includes an aerosolizing agent for use as a spray. In certain embodiments, the antibodies or pharmaceutical compositions described herein are delivered subcutaneously or intravenously. In certain embodiments, the antibodies or pharmaceutical compositions described herein are delivered intratumorally. In certain embodiments, the antibodies or pharmaceutical compositions described herein are delivered intraarterially. In certain embodiments, the antibodies or pharmaceutical compositions described herein are delivered to a tumor-draining lymph node. In certain embodiments, the antibodies or pharmaceutical compositions described herein are delivered intranasally.

[0243] The amount of the antibody or composition that will be effective in the treatment and / or prevention of the condition will depend on the nature of the disease, and can be determined by standard clinical techniques.

[0244] The exact dosage used in the composition also depends on the route of administration and the severity of the infection or disease caused thereby, and should be determined according to the doctor's judgment and each subject's circumstances.For example, the effective dosage may also vary depending on the means of administration, the target site, the physiological condition of the patient (including age, weight and health), whether the patient is human or animal, other medicines administered, or whether the treatment is preventive or therapeutic.Usually, the patient is human, but non-human mammals, including transgenic mammals, can also be treated.The treatment dosage is optimally titrated to optimize safety and efficacy.

[0245] The LAG-3 (e.g., human LAG-3) antibodies described herein can also be used to assay LAG-3 (e.g., human LAG-3) protein levels in biological samples using classical immunohistological methods known to those of skill in the art, including immunoassays such as enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, or Western blotting. Suitable antibody assay labels are known in the art and include enzyme labels such as glucose oxidase, iodine ( 125 I, 121 I), Carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), Indium ( 121 In), and technetium ( 99 Labels include radioisotopes such as 3Tc), 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 the anti-LAG-3 (e.g., human LAG-3) antibody described herein can be labeled and used in combination with the anti-LAG-3 (e.g., human LAG-3) antibody to detect LAG-3 (e.g., human LAG-3) protein levels. Thus, in one embodiment, the present invention relates to the use of the antibodies of the present invention for in vitro detection of LAG-3 (e.g., human LAG-3) protein in a biological sample. In a further embodiment, the present invention relates to the use of the anti-LAG-3 antibodies of the present invention for assessing and / or detecting LAG-3 (e.g., human LAG-3) protein levels in a biological sample in vitro, where the anti-LAG-3 antibody is optionally conjugated to a radionuclide or detectable label and / or carries a label as described herein and / or uses immunohistological methods.

[0246] Assaying for expression levels of LAG-3 (e.g., human LAG-3) protein is intended to include qualitatively or quantitatively measuring or estimating the level of LAG-3 (e.g., human LAG-3) protein in a first biological sample, either directly (e.g., by determining or estimating absolute protein levels) or relatively (e.g., by comparing with disease-related protein levels in a second biological sample). The expression level of LAG-3 (e.g., human LAG-3) polypeptide in a first biological sample can be measured or estimated and compared to a standard LAG-3 (e.g., human LAG-3) protein level, which is taken from a second biological sample obtained from an individual without the disorder or determined by averaging levels from a population of individuals without the disorder. As understood in the art, once a "standard" LAG-3 (e.g., human LAG-3) polypeptide level is known, it can be repeatedly used as a standard for comparison. Therefore, in a further embodiment, the present invention relates to an in vitro method of assessing and / or detecting LAG-3 protein levels, e.g., human LAG-3 protein levels, in a biological sample, comprising qualitatively or quantitatively measuring or estimating the levels of LAG-3 protein, e.g., human LAG-3 protein, in a biological sample by immunohistological methods.

[0247] As used herein, the term "biological sample" refers to any biological sample, cell line, tissue, or other source of cells potentially expressing LAG-3 (e.g., human LAG-3) obtained from a subject. Methods for obtaining tissue biopsies and body fluids from animals (e.g., humans) are known in the art. Biological samples include peripheral blood mononuclear cells.

[0248] The anti-LAG-3 (e.g., human LAG-3) antibodies described herein can be used for prognostic, diagnostic, monitoring, and screening applications, including in vitro and in vivo applications based on the present description, known and standard to those skilled in the art. Prognostic, diagnostic, monitoring, and screening assays and kits for in vitro assessment and evaluation of immune system status and / or immune response can be used to predict, diagnose, and monitor to evaluate patient samples known to have or suspected to have immune system dysfunction, or for expected or desired immune system, antigen, or vaccine responses. Assessment and evaluation of immune system status and / or immune response is also useful to determine the suitability of a patient for clinical trials of a drug against different drugs or antibodies, or for administration of a particular chemotherapeutic, radiotherapeutic, or antibody. This type of prognostic and diagnostic monitoring and evaluation has already been used in practice with antibodies against the HER2 protein (HercepTest™, Dako) in breast cancer, and this assay is also used to evaluate patients for antibody therapy with Herceptin®. In vivo applications include directed cell therapy and radioimaging of immune system modulation and immune responses. Thus, in one embodiment, the present invention relates to an anti-LAG-3 antibody and / or pharmaceutical composition of the present invention for use as a diagnostic. In one embodiment, the present invention relates to an anti-LAG-3 antibody and / or pharmaceutical composition of the present invention for use in a method for predicting, diagnosing, and / or monitoring a subject with or suspected of having an immune system dysfunction and / or with respect to an expected or desired immune system response, antigen response, or vaccine response. In another embodiment, the present invention relates to the use of an anti-LAG-3 antibody of the present invention for predicting, diagnosing, and / or monitoring a subject with or suspected of having an immune system dysfunction and / or with respect to an expected or desired immune system response, antigen response, or vaccine response by in vitro assessment and / or detection of human LAG-3 protein levels in a biological sample of the subject.

[0249] In one embodiment, anti-LAG-3 (e.g., human LAG-3) antibodies can be used in immunohistochemistry of biopsy samples. In another embodiment, anti-LAG-3 (e.g., human LAG-3) antibodies can be used to detect LAG-3 (e.g., human LAG-3) levels, or levels of cells containing LAG-3 (e.g., human LAG-3) on their membrane surface, which may be associated with certain disease symptoms. The anti-LAG-3 (e.g., human LAG-3) antibodies described herein may carry a detectable or functional label. When a fluorescent label is used, a combination of both currently available microscopes and fluorescence activated cell sorter analysis (FACS) or method procedures known in the art may be utilized to identify and quantify specific binding members. The anti-LAG-3 (e.g., human LAG-3) antibodies described herein may carry a fluorescent label. Exemplary fluorescent labels include, for example, reactive and conjugated probes such as aminocoumarins, fluorescein, and Texas Red, Alexa Fluor dyes, Cy dyes, and DyLight dyes. Anti-LAG-3 (e.g., human LAG-3) antibodies can be isotopic 3 H, 14 C. 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 67 Cu, 90 Y, 99 Tc, 111 In, 117 Lu, 121 I, 124 I, 125 I, 131 I, 198 Au, 211 At, 213 Bi, 225 Ac, and 186The label may carry a radioactive label such as Re. When a radioactive label is used, currently available counting procedures known in the art may be utilized to identify and quantify the specific binding of anti-LAG-3 (e.g., human LAG-3) antibodies to LAG-3 (e.g., human LAG-3). In the case where the label is an enzyme, detection may be achieved by any of the currently available colorimetric, spectrophotometric, fluorospectroscopic, amperometric, or gasometric techniques known in the art. This may be achieved by contacting the sample or control sample with an anti-LAG-3 (e.g., human LAG-3) antibody under conditions that allow the formation of a complex between the antibody and LAG-3 (e.g., human LAG-3). Any complexes formed between the antibody and LAG-3 (e.g., human LAG-3) are detected and compared in the sample and the control. In view of the specific binding of the antibodies described herein to LAG-3 (e.g., human LAG-3), the antibodies can be used to specifically detect LAG-3 (e.g., human LAG-3) expression on the surface of cells. The antibodies described herein can also be used to purify LAG-3 (e.g., human LAG-3) via immunoaffinity purification. Also included herein are assay systems that can be prepared in the form of test kits, for example, for quantitative analysis of the extent of the presence of LAG-3 (e.g., human LAG-3) or LAG-3 (e.g., human LAG-3) / LAG-3 (e.g., human LAG-3) ligand complexes. The systems, or test kits, kits, or kits of parts, may include a labeled component, e.g., a labeled antibody, and one or more additional immunochemical reagents.

[0250] 6.5 Polynucleotides, Vectors, and Methods for Producing Anti-LAG-3 Antibodies In another aspect, provided herein are polynucleotides comprising a nucleotide sequence encoding an antibody or fragment thereof (e.g., a light chain variable region and / or a heavy chain variable region) described herein that specifically binds to a LAG-3 (e.g., human LAG-3) antigen, and vectors, e.g., vectors comprising such polynucleotides for recombinant expression in host cells (e.g., E. coli and mammalian cells). Provided herein are polynucleotides comprising a nucleotide sequence encoding any of the heavy and / or light chains of the antibodies provided herein, and vectors comprising such polynucleotide sequences, e.g., expression vectors for their efficient expression in host cells, e.g., mammalian cells.

[0251] As used herein, an "isolated" polynucleotide or nucleic acid molecule is one that is separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule (e.g., mouse or human). Furthermore, an "isolated" nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular material or culture medium if produced by recombinant techniques, or may be substantially free of chemical precursors or other chemicals if chemically synthesized. For example, the term "substantially free" includes preparations of polynucleotides or nucleic acid molecules that have less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (particularly less than about 10%) of other materials, e.g., cellular material, culture medium, other nucleic acid molecules, chemical precursors, and / or other chemicals. In certain embodiments, the nucleic acid molecule(s) encoding the antibodies described herein are isolated or purified.

[0252] In certain aspects, provided herein are polynucleotides comprising nucleotide sequences encoding antibodies that immunospecifically bind to a LAG-3 (e.g., human LAG-3) polypeptide, including the amino acid sequences described herein, as well as antibodies that compete with such antibodies (e.g., in a dose-dependent manner) for binding to a LAG-3 (e.g., human LAG-3) polypeptide or bind to the same epitope as such antibodies.

[0253] In certain aspects, provided herein is a polynucleotide comprising a nucleotide sequence encoding a light chain or a heavy chain of an antibody described herein. The polynucleotide can comprise a nucleotide sequence encoding a light chain comprising the VL FRs and CDRs of an antibody described herein (see, e.g., Tables 1, 3, 5, 6, and 7), or a nucleotide sequence encoding a heavy chain comprising the VH FRs and CDRs of an antibody described herein (see, e.g., Tables 1, 2, 4, 6, and 7).

[0254] Also provided herein are polynucleotides encoding anti-LAG-3 (e.g., human LAG-3) antibodies that are optimized, for example, by codon / RNA optimization, substitution with a heterologous signal sequence, and elimination of mRNA instability elements. Thus, methods for generating optimized nucleic acids encoding anti-LAG-3 (e.g., human LAG-3) antibodies or fragments thereof (e.g., light chain, heavy chain, VH domain, or VL domain) for recombinant expression by introducing codon changes in mRNA and / or eliminating inhibitory regions can be performed by adopting the optimization methods described in, for example, U.S. Patent Nos. 5,965,726, 6,174,666, 6,291,664, 6,414,132, and 6,794,498, all of which are incorporated herein by reference in their entirety. For example, potential splice sites and instability elements (e.g., A / T or A / U rich elements) within the RNA can be mutated without changing the amino acids encoded by the nucleic acid sequence to increase the stability of the RNA for recombinant expression. The alterations take advantage of the degeneracy of the genetic code, for example, by using alternative codons for the same amino acid. In some embodiments, it may be desirable to change one or more codons to encode, for example, a conservative variation of the original amino acid with a similar amino acid and similar chemical structure and properties and / or function. Such methods can increase expression of an anti-LAG-3 (e.g., human LAG-3) antibody or fragment thereof by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more compared to expression of an anti-LAG-3 (e.g., human LAG-3) antibody encoded by a non-optimized polynucleotide.

[0255] In certain embodiments, an optimized polynucleotide sequence encoding an anti-LAG-3 (e.g., human LAG-3) antibody or fragment thereof (e.g., VL domain and / or VH domain) described herein can hybridize to an antisense (e.g., complementary) polynucleotide of a non-optimized polynucleotide sequence encoding an anti-LAG-3 (e.g., human LAG-3) or fragment thereof (e.g., VL domain and / or VH domain) described herein. In certain embodiments, an optimized nucleotide sequence encoding an anti-LAG-3 (e.g., human LAG-3) antibody or fragment thereof described herein hybridizes under high stringency conditions to an antisense polynucleotide of a non-optimized polynucleotide sequence encoding an anti-LAG-3 (e.g., human LAG-3) antibody or fragment thereof described herein. In certain embodiments, an optimized nucleotide sequence encoding an anti-LAG-3 (e.g., human LAG-3) antibody or fragment thereof described herein hybridizes to an antisense polynucleotide of a non-optimized polynucleotide sequence encoding an anti-LAG-3 (e.g., human LAG-3) antibody or fragment thereof described herein under high stringency, intermediate, or low stringency hybridization conditions. Information regarding hybridization conditions is described in U.S. Patent Application Publication No. US2005 / 0048549 (e.g., paragraphs 72-73), which is incorporated herein by reference in its entirety.

[0256] Polynucleotides can be obtained and the nucleotide sequence of the polynucleotides can be determined by any method known in the art. The nucleotide sequences encoding the antibodies described herein, such as those described in Tables 1, 6, and 7, and modified versions of these antibodies, can be determined using methods known in the art, i.e., nucleotide codons known to encode specific amino acids are assembled in such a way as to generate nucleic acids encoding the antibodies. Such polynucleotides encoding antibodies can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier G et al., (1994), BioTechniques 17:242-6, the entirety of which is incorporated herein by reference), which briefly includes the synthesis of overlapping polynucleotides that include portions of the sequences encoding the antibodies, annealing and ligating these oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

[0257] Alternatively, polynucleotides encoding the antibodies described herein can be generated from nucleic acid from a suitable source (e.g., a hybridoma) using methods known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from a hybridoma cell producing the antibody of interest. Such PCR amplification methods can be used to obtain nucleic acid comprising sequences encoding the light and / or heavy chains of the antibody. Such PCR amplification methods can be used to obtain nucleic acid comprising sequences encoding the light and / or heavy chain variable regions of the antibody. The amplified nucleic acid can be cloned into a vector for expression in a host cell and for further cloning, e.g., to generate chimeric and humanized antibodies.

[0258] If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, nucleic acid encoding an immunoglobulin can be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from any tissue or cell that expresses the antibody (e.g., a hybridoma cell selected to express an antibody described herein), or nucleic acid isolated therefrom, preferably poly A+ RNA), for example, by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for the particular gene sequence, to identify a cDNA clone from the cDNA library that encodes the antibody. The amplified nucleic acid generated by PCR can then be cloned into a replicable cloning vector using any method known in the art.

[0259] DNA encoding the anti-LAG-3 (e.g., human LAG-3) antibodies described herein can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of anti-LAG-3 (e.g., human LAG-3) antibodies). Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into an expression vector, which can then be transfected into host cells such as E. coli cells, monkey COS cells, Chinese Hamster Ovary (CHO) cells (e.g., CHO cells from CHO GS System™ (Lonza)), or myeloma cells that do not produce immunoglobulin protein, to obtain the synthesis of anti-LAG-3 (e.g., human LAG-3) antibodies in the recombinant host cells.

[0260] To generate a full antibody, the VH or VL sequence can be amplified in the scFv clone using PCR primers that contain the VH or VL nucleotide sequence, a restriction site, and flanking sequences to protect the restriction site. Using cloning techniques known to those skilled in the art, the PCR amplified VH domain can be cloned into a vector expressing a heavy chain constant region, e.g., human gamma 4 constant region, and the PCR amplified VL domain can be cloned into a vector expressing a light chain constant region, e.g., human kappa or lambda constant region. In certain embodiments, the vector for expressing the VH or VL domain contains an EF-1α promoter, a secretion signal, a cloning site for the variable domain, a constant domain, and a selection marker such as neomycin. The VH and VL domains can also be cloned into one vector expressing the necessary constant region. The heavy chain conversion vector and the light chain conversion vector are then co-introduced into a cell line to generate a stable or transient type cell line expressing a full-length antibody, e.g., IgG, using techniques known to those skilled in the art.

[0261] The DNA can also be modified, for example, by substituting the coding sequences for human heavy and light chain constant domains for the murine sequences, or by covalently joining to the immunoglobulin coding sequences all or part of the coding sequence for a non-immunoglobulin polypeptide.

[0262] Also provided are polynucleotides that hybridize under high, intermediate, or low stringency hybridization conditions to polynucleotides encoding the antibodies described herein. In certain embodiments, the polynucleotides described herein also hybridize under high, intermediate, or low stringency hybridization conditions to polynucleotides encoding the VH and / or VL domains provided herein.

[0263] Hybridization conditions are described in the art and are known to those skilled in the art. For example, hybridization under stringent conditions can include hybridization with filter-bound DNA in 6× sodium chloride / sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC / 0.1% SDS at about 50-65° C., and hybridization under highly stringent conditions can include hybridization with filter-bound nucleic acid in 6×SSC at about 45° C., followed by one or more washes in 0.1×SSC / 0.2% SDS at about 68° C. Hybridization under other stringent hybridization conditions is known to those of skill in the art and is described, for example, in Ausubel FM et al., eds., (1989) Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, pages 6.3.1-6.3.6 and 2.10.3, which are incorporated herein by reference in their entirety.

[0264] In certain aspects, provided herein are cells (e.g., host cells) that express (e.g., recombinantly) the antibodies described herein that specifically bind to LAG-3 (e.g., human LAG-3) and related polynucleotides and expression vectors. Provided herein are vectors (e.g., expression vectors) that include a polynucleotide that includes a nucleotide sequence encoding an anti-LAG-3 (e.g., human LAG-3) antibody or fragment for recombinant expression in a host cell, preferably a mammalian cell. Also provided herein are host cells that include such vectors for recombinantly expressing an anti-LAG-3 (e.g., human LAG-3) antibody (e.g., human or humanized antibody) described herein. In certain aspects, provided herein are methods for producing the antibodies described herein, including expressing such antibodies from a host cell.

[0265] Recombinant expression of an antibody described herein (e.g., a full-length antibody, a heavy and / or light chain of the antibody, or a single chain antibody described herein) that specifically binds to LAG-3 (e.g., human LAG-3) involves the construction of an expression vector containing a polynucleotide encoding the antibody. Once a polynucleotide encoding an antibody molecule, the heavy and / or light chain of an antibody described herein, or a fragment thereof (e.g., heavy and / or light chain variable regions) is obtained, a vector for the production of the antibody molecule can be produced by recombinant DNA technology using techniques known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody or antibody fragment (e.g., light or heavy chain) encoding nucleotide sequence are described herein. Methods known to those skilled in the art can be used to construct expression vectors containing the antibody or antibody fragment (e.g., light or heavy chain) encoding sequence and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are replicable vectors comprising a nucleotide sequence encoding the antibody molecule described herein, the heavy or light chain of the antibody, the heavy or light chain variable domain of the antibody or a fragment thereof, or the heavy or light chain CDRs, operably linked to a promoter. Such vectors can, for example, comprise a nucleotide sequence encoding the constant region of the antibody molecule (see, for example, International Publication Nos. WO86 / 05807 and WO89 / 01036, and U.S. Patent No. 5,122,464, which are incorporated herein by reference in their entirety), and the variable region of the antibody can be cloned into such vectors for expression of the entire heavy chain, the entire light chain, or both the entire heavy and light chains.

[0266] The expression vector can be transformed into a cell (e.g., a host cell) by conventional techniques, and the resulting cell can be cultured by conventional techniques to produce an antibody or fragment thereof described herein. Thus, provided herein is a host cell containing a polynucleotide encoding an antibody or fragment thereof described herein, or a heavy or light chain thereof, or a fragment thereof, or a single chain antibody described herein, operably linked to a promoter for expression of such sequences in the host cell. In certain embodiments, for expression of a double chain antibody, vectors encoding both the heavy and light chains can be individually co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below. In certain embodiments, the host cell contains a vector comprising a polynucleotide encoding both the heavy and light chains of an antibody or fragment thereof described herein. In certain embodiments, the host cell contains two different vectors, a first vector comprising a polynucleotide encoding the heavy chain or heavy chain variable region of an antibody or fragment thereof described herein, and a second vector comprising a polynucleotide encoding the light chain or light chain variable region of an antibody or fragment thereof described herein. In other embodiments, the first host cell comprises a first vector comprising a polynucleotide encoding a heavy chain or heavy chain variable region of an antibody or fragment thereof described herein, and the second host cell comprises a second vector comprising a polynucleotide encoding a light chain or light chain variable region of an antibody described herein. In certain embodiments, the heavy chain / heavy chain variable region expressed by the first cell associates with the light chain / light chain variable region of the second cell to form an anti-LAG-3 (e.g., human LAG-3) antibody described herein. In certain embodiments, a population of host cells comprising such a first host cell and such a second host cell is provided herein.

[0267] In certain embodiments, 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 (e.g., human LAG-3) antibody described herein, and a second vector comprising a polynucleotide encoding the heavy chain / heavy chain variable region of an anti-LAG-3 (e.g., human LAG-3) antibody described herein.

[0268] A variety of host-expression vector systems can be utilized to express the antibody molecules described herein (see, e.g., U.S. Patent No. 5,807,715, incorporated herein by reference in its entirety). Such host-expression systems represent vehicles in which coding sequences of interest may be produced and subsequently purified, but also represent cells which, when transformed or transfected with the appropriate nucleotide coding sequences, may express the antibody molecules described herein in situ. These include microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody coding sequences, yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing the antibody coding sequences, insect cell lines infected with recombinant viral expression vectors (e.g., baculovirus) containing the antibody coding sequences, plant cell lines (e.g., green algae such as Chlamydomonas reinhardtii) infected with recombinant viral expression vectors (e.g., Cauliflower Mosaic Virus, CaMV, Tobacco Mosaic Virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the antibody coding sequences, or mammalian cell lines (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter). 293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH 3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, and BMT10 cells). In certain embodiments, the cells for expressing the antibodies described herein are CHO cells, e.g., CHO cells from the CHO GS System™ (Lonza).In certain embodiments, the heavy and / or light chains of the antibodies produced by CHO cells may have an N-terminal glutamine or glutamic acid residue replaced by pyroglutamic acid. In certain embodiments, the cells for expressing the antibodies described herein are human cells, e.g., human cell lines. In certain embodiments, the mammalian expression vector is pOptiVEC™ or pcDNA3.3. In certain embodiments, bacterial cells, such as Escherichia coli, or eukaryotic cells (e.g., mammalian cells), particularly for the expression of whole recombinant antibody molecules, are used for the expression of the recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary (CHO) cells in combination with a vector such as the major intermediate-early gene promoter element from human cytomegalovirus are effective expression systems 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 certain embodiments, expression of the nucleotide sequence encoding the antibodies described herein that specifically bind to LAG-3 (e.g., human LAG-3) is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0269] In bacterial systems, many expression vectors can be advantageously selected depending on the intended use for expressing the antibody molecule. For example, when a large amount of such antibody is produced for the production of pharmaceutical compositions of the antibody molecule, a vector that induces the expression of a high level of a fusion protein product that is easily purified may be desirable. Such vectors include, but are not limited to, E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983) EMBO J 2:1791-1794), pIN vector (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, in which the antibody coding sequence can be individually ligated into the vector in frame with the lac Z coding region to produce a fusion protein. All of these are incorporated herein by reference in their entirety. For example, pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. pGEX vectors are designed to contain thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0270] In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV), for example, can be used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence can be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0271] In mammalian host cells, many viral-based expression systems are available. When adenovirus is used as an expression vector, the antibody coding sequence of interest can be ligated into an adenovirus transcription / translation control complex, e.g., the 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 non-essential regions of the viral genome (e.g., regions El or E3) will yield recombinant viruses that are viable in infected hosts and capable of expressing the antibody molecule (see, e.g., Logan J & Shenk T (1984) PNAS 81(12):3655-9, incorporated herein by reference in its entirety). Specific initiation signals may also be required for efficient translation of the inserted antibody coding sequence. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bitter G et al., (1987) Methods Enzymol. 153:516-544, incorporated herein by reference in its entirety).

[0272] In addition, a host cell strain can be chosen which modulates the expression of the inserted sequences or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have unique and specific mechanisms for post-translational processing and modification of proteins and gene products. An appropriate cell line or host system can be selected to ensure the correct modification and processing of the expressed foreign protein. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of 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 mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10, and HsS78Bst cells. In certain embodiments, the anti-LAG-3 (e.g., human LAG-3) antibodies described herein are produced in mammalian cells, such as CHO cells.

[0273] In certain embodiments, the antibodies described herein have reduced or no fucose content. Such antibodies can be produced using techniques known to those skilled in the art. For example, the antibodies can be expressed in cells that are defective or lack the ability to fucosylate. In a specific example, a cell line with knockout of both alleles of α1,6-fucosyltransferase can be used to produce antibodies with 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.

[0274] For long-term, high-yield production of recombinant proteins, stable expression can be used. For example, cell lines can be engineered that stably express the anti-LAG-3 (e.g., human LAG-3) antibodies described herein. In certain embodiments, the cells provided herein stably express the light chain / light chain variable region and heavy chain / heavy chain variable region that associate to form the antibodies described herein.

[0275] In certain embodiments, rather than using expression vectors containing viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression regulatory elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. After introduction of the foreign DNA / polynucleotide, engineered cells can be grown in rich medium for 1-2 days and then switched to selective medium. The selectable marker in the recombinant plasmid confers resistance to the selection and allows the cells to grow to stably integrate the plasmid into their chromosomes and form foci that can be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that express the anti-LAG-3 (e.g., human LAG-3) antibodies or fragments thereof described herein. Such engineered cell lines can be particularly useful in screening and evaluation of compositions that interact directly or indirectly with the antibody molecule.

[0276] Many 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 (Lowy I et al., (1980) Cell 22(3):817-23) genes, which can be used in tk-, hgprt-, or aprt- cells, respectively, all of which are incorporated herein by reference in their entirety. Also, antimetabolite resistance can be used based on selection for 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 the 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 & Felgner 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 entireties.Methods commonly known in the art of recombinant DNA technology can be routinely applied to select the desired recombinant clones, and such methods are described, for example, in Ausubel FM 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 Chapters 12 and 13 of Dracopoli NC et al., (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994), Colbere-Garapin F et al., (1981) J Mol Biol 150:1-14, which are incorporated herein by reference in their entireties.

[0277] The expression level 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). If the vector-based marker that expresses the antibody is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the number of copies of the marker gene. Since the amplified region will be associated with the antibody gene, production of the antibody will also increase (Crouse GF et al., (1983) Mol Cell Biol 3:257-66, which is incorporated herein by reference in its entirety).

[0278] A host cell can be co-introduced 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 can contain identical selectable markers that allow equal expression of heavy and light chain polypeptides. A host cell can be co-introduced with different amounts of two or more expression vectors. For example, a host cell can be transfected with any one of the following ratios of first expression vector to 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.

[0279] Alternatively, a single vector capable of encoding and expressing both heavy and light chain polypeptides can be used. In such a situation, the light chain should be placed before the heavy chain to avoid excessive amounts of non-toxic heavy chain (Proudfoot NJ (1986) Nature 322:562-565, and Koehler G (1980) PNAS 77:2197-2199, each of which is incorporated herein by reference in its entirety). The coding sequences for the heavy and light chains can comprise cDNA or genomic DNA. Expression vectors can be monocistronic or multicistronic. Multicistronic nucleic acid constructs can encode 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, or within the range of 2-5, 5-10, or 10-20 genes / nucleotide sequences. For example, a bicistronic nucleic acid construct can include, in the following order: a promoter, a first gene (e.g., the heavy chain of an antibody described herein), and a second gene, (e.g., the light chain of an antibody described herein). In such an expression vector, transcription of both genes can be driven by a promoter, while translation of the mRNA from the first gene can be by a cap-dependent scanning mechanism and translation of the mRNA from the second gene can be by a cap-independent mechanism, e.g., via an IRES.

[0280] Once the antibody molecules described herein have been produced by recombinant expression, they can be purified by any method known in the art for the purification of immunoglobulin molecules, such as, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for specific antigens following Protein A, and size exclusion column chromatography), centrifugation, absorption differential solubility, or by any other standard technique for the purification of proteins. Additionally, the antibodies described herein may be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0281] In certain embodiments, the antibodies described herein are isolated or purified. Generally, an isolated antibody is one that is substantially free of other antibodies having different antigen specificities than the isolated antibody. For example, in certain embodiments, preparations of antibodies described herein are substantially free of cellular material and / or chemical precursors. The phrase "substantially free of cellular material" includes preparations of antibodies in which the antibody is separated from cellular components of cells, either isolated or recombinantly produced. Thus, antibodies that are substantially free of cellular material include preparations of antibodies that have less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of heterologous proteins (also referred to herein as "contaminating proteins") and / or variants, e.g., different post-translational modifications of antibodies or other different versions of antibodies (e.g., antibody fragments). When the antibody is recombinantly produced, it is also generally substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the protein preparation. When the antibody is produced by chemical synthesis, it is generally substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals involved in the synthesis of the protein. Thus, such preparations of the antibody have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest. In certain embodiments, the antibodies described herein are isolated or purified.

[0282] Antibodies or fragments thereof that specifically bind to LAG-3 (e.g., human LAG-3) can be produced by any method known in the art for the synthesis of antibodies, for example, by chemical synthesis or by recombinant expression techniques. The methods described herein employ, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields that are within the skill of the art. These techniques are described, for example, in and explained in detail in the references cited herein.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 FM et al. 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 by reference in their entireties.

[0283] In certain embodiments, the antibodies described herein are antibodies (e.g., recombinant antibodies) that are prepared, expressed, generated, or isolated by any means, including, for example, synthetic production, genetic manipulation of DNA sequences. In certain embodiments, such antibodies comprise sequences (e.g., DNA sequences or amino acid sequences) that do not naturally occur within the antibody germline repertoire of an animal or mammal (e.g., human) in vivo.

[0284] In one aspect, provided herein is a method of making an antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising culturing a cell or host cell described herein. In one embodiment, the method is performed in vitro. In certain aspects, provided herein is a method of making an antibody that specifically binds to LAG-3 (e.g., human LAG-3), comprising expressing (e.g., recombinantly expressing) the antibody using a cell or host cell described herein (e.g., a cell or host cell comprising a polynucleotide encoding an antibody described herein). In certain embodiments, the cell is an isolated cell. In certain embodiments, an exogenous polynucleotide has been introduced into the cell. In certain embodiments, the method further comprises purifying the antibody obtained from the cell or host cell.

[0285] Methods for producing polyclonal antibodies are known in the art (see, e.g., Chapter 11 of Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., eds., John Wiley and Sons, New York, incorporated herein by reference in its entirety).

[0286] Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques, including those known in the art and taught, for example, in Harlow E & Lane D, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988), Hammerling GJ et al., Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, NY, 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 through hybridoma technology. For example, monoclonal antibodies can be produced by recombinant techniques from host cells exogenously expressing an antibody or fragment thereof described herein, e.g., the light chain and / or heavy chain of such an antibody.

[0287] In certain embodiments, a "monoclonal antibody," as used herein, is an antibody produced by a single cell (e.g., a hybridoma or host cell producing recombinant antibody), which specifically binds to LAG-3 (e.g., human LAG-3), e.g., as determined by ELISA or other antigen-binding or competitive binding assays known in the art or in the examples provided herein. In certain embodiments, a monoclonal antibody may be a chimeric or humanized antibody. In certain embodiments, a monoclonal antibody is a monovalent antibody or a multivalent (e.g., bivalent) antibody. In certain embodiments, a monoclonal antibody is a monospecific or multispecific antibody (e.g., bispecific antibody). The monoclonal antibodies described herein may be made, e.g., by hybridoma methods described in Kohler G & Milstein C (1975) Nature 256:495, or may be isolated, e.g., from a phage library, e.g., using techniques described herein. Other methods for preparation of clonal cell lines and the monoclonal antibodies expressed thereby are known in the art (see, e.g., Chapter 11 of Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., supra).

[0288] Methods for producing and screening specific antibodies using hybridoma technology are routine and 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 elicit lymphocytes that produce or can produce antibodies that will specifically bind to the protein used for immunization (e.g., LAG-3 (e.g., human LAG-3)). Alternatively, lymphocytes may be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding JW (Ed), Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986), which is incorporated herein by reference in its entirety). Additionally, animals can be immunized using RIMMS (repeated immunization multiple sites) technology (Kilpatrick KE et al., (1997) Hybridoma 16:381-9, incorporated herein by reference in its entirety).

[0289] In some embodiments, a mouse (or other animal such as a rat, monkey, donkey, pig, sheep, hamster, or dog) can be immunized with an antigen (e.g., LAG-3 (e.g., human LAG-3)) and upon detection of an immune response, e.g., detection of antibodies specific to the antigen in the mouse serum, the mouse pancreas is harvested and pancreatic cells are isolated. The pancreatic cells are then fused by known techniques with any suitable myeloma cells, e.g., cells from cell line SP20 available from the American Type Culture Collection (ATCC®) (Manassas, VA), to form hybridomas. Hybridomas are selected and cloned by limiting dilution. In certain embodiments, lymph nodes of the immunized mouse are harvested and fused with NS0 myeloma cells.

[0290] The hybridoma cells thus prepared are preferably seeded and grown in a suitable culture medium containing one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine-guanine-phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomas contains hypoxanthine, aminopterin, and thymidine (HAT medium), which will inhibit the growth of HGPRT-deficient cells.

[0291] A specific embodiment uses myeloma cells that fuse effectively, support stable high-level antibody production by selected antibody-producing cells, and are sensitive to media such as HAT medium. Among these, the myeloma cell lines are mouse myelomas such as NS0 cell lines or those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center (San Diego, CA, USA), and SP-2 or X63-Ag8.653 cells available from the American Type Culture Collection (Rockville, MD, 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.51-63 (Marcel Dekker, Inc., New York, 1987), each of which is incorporated herein by reference in its entirety).

[0292] The culture medium in which the hybridoma cells are growing is assayed for production of monoclonal antibodies directed against LAG-3 (e.g., human LAG-3). The binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by methods known in the art, for example, by immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0293] After hybridoma cells that produce antibodies of the desired specificity, affinity, and / or activity are identified, the clones may be subcloned by limiting dilution methods and grown by 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 medium. In addition, hybridoma cells may be grown in vivo as ascites tumors in animals.

[0294] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0295] The antibodies described herein include antibody fragments that recognize specific LAG-3 (e.g., human LAG-3) and can be generated by any technique known to one of skill in the art. For example, the Fab and F(ab')2 fragments described herein can be generated 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 the two identical arms of an antibody molecule and contains an intact light chain paired with the VH and CH1 domains of the heavy chain. The F(ab')2 fragment contains the two antigen-binding arms of an antibody molecule linked by disulfide bonds in the hinge region.

[0296] Additionally, the antibodies described herein can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are exposed on the surface of phage particles carrying the polynucleotide sequences encoding them. In particular, DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (e.g., human or mouse cDNA libraries of infected tissues). The DNA encoding the VH and VL domains are recombined together with an scFv linker by PCR and cloned into a phagemid vector. The vector is electroporated into E. coli, which is infected with helper phage. The phages used in these methods are typically filamentous phages, including fd and M13, and the VH and VL domains are usually recombinantly fused to either phage gene III or gene VIII. Phage expressing antigen-binding domains that bind to a particular antigen can be selected or identified with the antigen, for example, with labeled antigen or antigen bound or captured to a solid surface or bead.Examples of phage display methods that can be used to generate the antibodies described herein include those described in Brinkman U et al., (1995) J Immunol Methods 182:41-50; 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 57:191-280, PCT Publication No. PCT / GB91 / 001134, International Publication Nos. WO90 / 02809, WO91 / 10737, WO92 / 01047, WO92 / 18619, WO93 / 11236, WO95 / 15982, WO95 / 20401, and WO97 / 13844, and U.S. Pat. Nos. 5,698,426, 5,222,527, and 5,312,626. Nos. 3,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108, all of which are incorporated herein by reference in their entirety.

[0297] As described above, after phage selection, the antibody coding region from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen-binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, for example, as described below. Techniques can also be used to recombinantly produce antibody fragments, such as Fab, Fab', and F(ab')2 fragments, using methods known in the art, such as those disclosed in PCT Publication No. WO92 / 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.

[0298] In certain embodiments, to generate whole antibodies, PCR primers containing the VH or VL nucleotide sequence, a restriction site, and flanking sequences to protect the restriction site can be used to amplify the VH or VL sequence from a template, e.g., an scFv clone. Using cloning techniques known to those skilled in the art, the PCR amplified VH domain can be cloned into a vector expressing a VH constant region, and the PCR amplified VL domain can be cloned into a vector expressing a VL constant region, e.g., a human kappa or lambda constant region. The VH and VL domains can also be cloned into one vector expressing the necessary constant regions. The heavy chain conversion vector and the light chain conversion vector are then co-introduced into a cell line to generate a stable or transient type cell line expressing a full-length antibody, e.g., an IgG, using techniques known to those skilled in the art.

[0299] A chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody can contain 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 U.S. 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.

[0300] A humanized antibody is capable of binding to a given antigen and comprises a framework region having substantially the amino acid sequence of a human immunoglobulin and a CDR having substantially the amino acid sequence of a non-human immunoglobulin (e.g., a murine immunoglobulin). In certain embodiments, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically a portion of a human immunoglobulin. The antibody may also comprise the CH1, hinge, 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 by a variety of techniques, including CDR grafting (EP 239400, WO 91 / 09967, and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (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 (U.S. Pat. No. 5,565,332), as well as, for example, U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, International Publication No. WO93 / 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. 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 entireties. See also U.S. Application Publication No. US2005 / 0042664 A1 (February 24, 2005), which is incorporated herein by reference in its entirety.

[0301] Methods for making multispecific (e.g., bispecific) antibodies have been described, see, e.g., U.S. Patent Nos. 7,951,917, 7,183,076, 8,227,577, 5,837,242, 5,989,830, 5,869,620, 6,132,992, and 8,586,713, all of which are incorporated by reference herein in their entireties.

[0302] Single domain antibodies, e.g., antibodies lacking light chains, can be produced by methods 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, U.S. Patent No. 6,005,079, and International Publication Nos. WO94 / 04678, WO94 / 25591, and WO01 / 44301, all of which are incorporated herein by reference in their entirety.

[0303] Furthermore, antibodies that specifically bind to the LAG-3 (e.g., human LAG-3) antigen can then be utilized to generate anti-idiotypic antibodies that "mimic" the antigen, using techniques known to those of skill in the art. See, e.g., 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.

[0304] In certain embodiments, an antibody described herein that binds to the same epitope of LAG-3 (e.g., human LAG-3) as an anti-LAG-3 (e.g., human LAG-3) antibody described herein is a human antibody. In certain embodiments, an antibody described herein that competitively inhibits (e.g., in a dose-dependent manner) the binding of any one of the antibodies described herein to LAG-3 (e.g., human LAG-3) is a human antibody. Human antibodies can be produced using any method known in the art. For example, transgenic mice that are incapable of expressing functional endogenous immunoglobulins but can express human immunoglobulin genes can be used. In particular, human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable regions, constant regions, and diversity regions can be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes can be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, J. HHomozygous deletion of the region prevents endogenous antibody production. The 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. The transgenic mice are immunized in the usual manner with a selected antigen, e.g., all or a portion of an antigen (e.g., LAG-3 (e.g., human LAG-3)). Monoclonal antibodies directed against the antigen can be obtained from the immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation and subsequently undergo class switching and somatic mutation. Thus, using such technology, it is possible to produce therapeutically useful IgG, IgA, IgM, and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg N & Huszar D (1995) Int Rev Immunol 13:65-93, which is incorporated herein by reference in its entirety. For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., International Publication Nos. WO 98 / 24893, WO 96 / 34096, and WO 96 / 33735, as well as U.S. Patent Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598, all of which are incorporated herein by reference in their entirety. Examples of mice capable of producing human antibodies include the Xenomouse™ (Abgenix, Inc., U.S. Pat. Nos. 6,075,181 and 6,150,184), HuAb-Mouse™ (Mederex, Inc. / Gen Pharm, U.S. Pat. Nos. 5,545,806 and 5,569,825), TranschromoMouse™ (Kirin), and KM Mouse™ (Medarex / Kirin).

[0305] Human antibodies that specifically bind to LAG-3 (e.g., human LAG-3) can be produced by various methods known in the art, including the above-mentioned phage display method using antibody libraries derived from human immunoglobulin sequences.See also U.S. Patent Nos. 4,444,887, 4,716,111, and 5,885,793, as well as International Publication Nos. WO98 / 46645, WO98 / 50433, WO98 / 24893, WO98 / 16654, WO96 / 34096, WO96 / 33735, and WO91 / 10741.All of these are incorporated herein by reference in their entirety.

[0306] 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 determine those that secrete human monoclonal antibodies that specifically bind to a target antigen (e.g., LAG-3 (e.g., human LAG-3)). Such methods are known and described in the art, see, e.g., 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.

[0307] 6.6 Kits Also provided herein are kits comprising one or more of the antibodies or pharmaceutical compositions or conjugates thereof described herein. In certain embodiments, provided herein are pharmaceutical packs or kits comprising one or more containers filled with one or more of the components of the pharmaceutical compositions described herein, such as one or more antibodies provided herein. In some embodiments, the kits contain the pharmaceutical compositions described herein and any prophylactic or therapeutic agents, such as those described herein. In certain embodiments, the kits may contain T cell mitogens, such as, for example, phytohemagglutinin (PHA) and / or phorbol myristate acetate (PMA), or TCR complex stimulating antibodies, such as anti-CD3 and anti-CD28 antibodies. Optionally, associated with such container(s) may include a notice in a format prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use, or sale for administration to humans.

[0308] Also provided herein are kits that can be used in the above methods. In one embodiment, the kits include an antibody, preferably a purified antibody, described herein in one or more containers. In...

Claims

1. 1. An isolated antibody that specifically binds to human LAG-3, comprising a heavy chain variable region comprising complementarity determining regions CDRH1, CDRH2, and CDRH3, and a light chain variable region comprising complementarity determining regions CDRL1, CDRL2, and CDRL3; CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are SEQ ID NOs: 78, 83, 94, 100, 104, and 105; 78, 85, 95, 100, 104, and 105; 78, 86, 96, 100, 104, and 105; 78, 86, 97, 102, 104, and 112; 78, 91, 94, 100, 104, and 107; 79, 89, 95, 100, 104, and 105; 80, 85, 96, 100, 104, and 105; or 81, 87, 96, 100, 104, and 105.

2. 2. The isolated antibody of claim 1 , wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 151, 222, 218, or 223, and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 152 or 224.

3. 2. The isolated antibody of claim 1 , wherein the heavy chain variable region and the light chain variable region comprise the 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, 17 and 18, 19 and 8, or 54 and 55, respectively.

4. The antibody, (a) comprising a heavy chain variable framework region that is or is derived from an amino acid sequence encoded by a human gene, said amino acid sequence being 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); and / or 2. The isolated antibody of claim 1, wherein the antibody comprises a light chain variable framework region that is an amino acid sequence encoded by or derived from a human gene, and the amino acid sequence is selected from the group consisting of IGKV3-20*01 (SEQ ID NO: 160), 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).

5. The antibody, (a) comprising a heavy chain variable framework region derived from the amino acid sequence IGHV1-46*01 (SEQ ID NO: 153), wherein at least one amino acid in the amino acid sequence IGHV1-46*01 (SEQ ID NO: 153) is replaced with an amino acid at an analogous position in a corresponding non-human heavy chain variable framework region, wherein the amino acid replacement 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, and wherein the amino acid replacement is selected from the group consisting of 4M, 5K, 12V, 23T, 27F, 28N, 29I, 30K, 48I, 69I, 71A, 75S, 76N, 80L, 81Q, and 94T; and / or (b) comprising a light chain variable framework region derived from the amino acid sequence IGKV3-20*01 (SEQ ID NO: 160), wherein at least one amino acid in the amino acid sequence IGKV3-20*01 (SEQ ID NO: 160) is replaced with an amino acid at an analogous position in a corresponding non-human light chain variable framework region, wherein the amino acid replacement is at an amino acid position selected from the group consisting of 3, 22, 36, 43, 47, 58, 70, and 71, and wherein the amino acid replacement is selected from the group consisting of 3L, 22T, 36F, 43S, 47W, 58V, 70S, and 71Y; The isolated antibody of claim 4 , wherein the positions of the amino acid substitutions are indicated according to the Kabat numbering system.

6. The antibody is a human IgG １ , IgG ２ , IgG ３ , IgG ４ , IgA １ , and IgA ２ and wherein the heavy chain constant region is selected from the group consisting of: (a) IgG containing the N297A mutation １ a heavy chain constant region, said heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 194; (b) IgG containing the N297Q mutation １ A heavy chain constant region; (c) Non-fucosylated IgG １ a heavy chain constant region; and / or (d) IgG containing the S228P mutation ４ a heavy chain constant region, said heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 196; 2. The isolated antibody of claim 1, wherein the amino acid residues are numbered according to the EU numbering system.

7. 2. The isolated antibody of claim 1, wherein the antibody comprises a human kappa light chain constant region or a human lambda light chain constant region, and the human kappa light chain constant region comprises the amino acid sequence of SEQ ID NO: 198 or 219.

8. The antibody, (a) is a humanized antibody, a murine antibody, or a chimeric antibody; (b) is antagonistic to human LAG-3; (c) inactivating, reducing or inhibiting the activity of human LAG-3; and / or (d) the isolated antibody of claim 1, which inhibits the binding of human LAG-3 to MHC class II.

9. The isolated antibody of claim 1 conjugated to a cytotoxic agent, a cytostatic agent, a toxin, a radionuclide, or a detectable label.

10. A pharmaceutical composition comprising the antibody of claim 1 and a pharma- ceutically acceptable carrier or excipient.

11. (a) the heavy chain variable region and the light chain variable region of the antibody of claim 1; (b) the heavy and light chains of the antibody of claim 1; or (c) any one of the amino acid sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 14, 17, 19, or 54, and any one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 18, or 55. An isolated polynucleotide encoding the

12. A vector comprising the polynucleotide of claim 11.

13. (a) the polynucleotide of claim 11; (b) the vector according to claim 12; (c) an isolated polynucleotide encoding the heavy chain variable region of the antibody of claim 1, and an isolated polynucleotide encoding the light chain variable region of the antibody of claim 1; (d) an isolated polynucleotide encoding the heavy chain of the antibody of claim 1, and an isolated polynucleotide encoding the light chain of the antibody of claim 1; or (e) a vector comprising a polynucleotide encoding the heavy chain variable region or the heavy chain of the antibody according to claim 1, and a vector comprising a polynucleotide encoding the light chain variable region or the light chain of the antibody according to claim 1. A recombinant host cell comprising:

14. 1. A method for producing an antibody that binds to human LAG-3, comprising: The method comprising culturing the host cell of claim 13 so that the polynucleotide is expressed and the antibody is produced.

15. 11. Use of the pharmaceutical composition of claim 10 in the manufacture of a medicament for increasing T cell activation in response to an antigen in a subject.

16. 11. Use of the pharmaceutical composition of claim 10 in the manufacture of a medicament for the treatment of cancer in a subject.

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