EA043100B1 - ANTIBODIES AGAINST TIM-3 AND METHODS OF THEIR APPLICATION - Google Patents

Description

1. Cross-references to related applications

The present application claims priority on U.S. Provisional Application No. 62/342,610, filed May 27, 2016; and U.S. Provisional Application No. 62/420276, filed November 10

2016, each of which is incorporated herein by reference in its entirety.

2. Field of technology

The invention relates to antibodies that specifically bind to TIM-3 (eg human TIM-3) and methods of using the same.

3. State of the art

T-cell immunoglobulin and mucin domain 3 (TIM-3) is a type I membrane protein of the immunoglobulin (Ig) superfamily. It contains an extracellular Ig variable domain-like (IgV) domain, an extracellular mucin-like domain, and a cytoplasmic domain with six conserved tyrosine residues (Monney et al. (2002) Nature 415:536-41). TIM-3 is expressed on activated T helper type 1 (Th1) and CD8+ T lymphocytes (Tc1), some macrophages (Monney et al. (2002) Nature 415:536-41), activated natural killer (NK) cells (Ndhlovu et al (2012) Blood 119 (16):3734-43) and IL-17-producing Th17 cells (Nakae et al. (2007) J. Leukoc. Biol. 81: 1258-68).

Studies have shown that TIM-3 function inhibits T cell, myeloid cell and NK cell mediated responses and promotes immunological tolerance. For example, TIM-3 IgV peptide fused to an immunoglobulin domain that binds and neutralizes TIM-3 ligands caused Th1 cell hyperproliferation and release of Th1 cytokines in immunized mice (Sabatos et al. (2003) Nat. Immunol. 4:1102-10) . Thus, in vivo administration of an anti-TIM-3 antibody increased the pathological severity of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis (Monney et al. (2002) Nature 415:536-41). In addition, TIM-3 expression is upregulated in CD8+ T cells in cancer patients. For example, approximately 30% of NY-ESO1-specific CD8+ T cells in patients with advanced melanoma were upregulated in TIM-3 expression (Fourcade et al. (2010) J. Exp. Med. 207:2175-86).

Given the apparent role of human TIM-3 in modulating the immune response, therapeutic agents designed to antagonize TIM-3 signaling are very promising for the treatment of diseases that involve TIM-3 mediated immune suppression.

4. Brief description of the invention

The present invention provides antibodies that specifically bind to TIM-3 (eg human TIM-3) and antagonize TIM-3 function, eg TIM-3 mediated immune suppression. Also provided are pharmaceutical compositions containing said antibodies, nucleic acids encoding said antibodies, expression vectors and host cells for producing said antibodies, and methods of treating an individual with said antibodies. Antibodies as described herein are particularly suitable for increasing T cell activation in response to an antigen (e.g., tumor antigen or infectious disease antigen) and/or reducing Treg-mediated immune suppression, and accordingly for treating cancer in an individual, or to treat or prevent an infectious disease in an individual.

Accordingly, according to one aspect of the present invention, there is provided an antibody or isolated antibody comprising a heavy chain variable region comprising the complementarity determining regions CDRH1, CDRH2 and CDRH3 and a light chain variable region comprising the complementarity determining regions CDRL1, CDRL2 and CDRL3, wherein:

(a) CDRH1 contains the amino acid sequence XiX^X^XjS (SEQ ID NO: 48), where

X1 is R, S, A, G, K, M or T,

X ₂ is Q, S, A, G, R or T,

X ₃ is N, Y, G or Q,

X4 is A or Q, and

X ₅ represents W, M, A, S or T;

(b) CDRH2 contains the amino acid sequence WVSAISGSGGSTY (SEQ ID NO: 2);

(c) CDRH3 contains the amino acid sequence AKGGDYGGNYFD (SEQ ID NO: 3);

(d) CDRL1 contains the amino acid sequence X1ASQSVX ₂ SSYLA (SEQ ID NO: 52), where

X1 is R or G, and

X ₂ is absent or represents S;

(e) CDRL2 contains the amino acid sequence X1ASX2RAT (SEQ ID NO: 53), where

X1 is D or G and X ₂ is N, S or T; and (f) CDRL3 contains the amino acid sequence QQYGSSPX1T (SEQ ID NO: 54), where X1 is L or I.

According to another aspect of the present invention, there is provided an antibody or isolated antibody that specifically binds to human TIM-3, comprising a heavy chain variable region containing complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region containing complementarity determining regions CDRL1, CDRL2 and CDRL3,

- 1 043100 while:

(a) CDRH1 contains the amino acid sequence X ₁ X ₂ X ₃ X ₄ X ₅ S (SEQ ID NO: 48), where

X ₁ is R, S, A, G, K, M or T,

X2 is Q, S, A, G, R or T,

X ₃ represents N, Y, G or Q,

X ₄ is A or Q, and

X ₅ is W, M, A, S or T;

(b) CDRH2 contains the amino acid sequence WVSAISGSGGSTY (SEQ ID NO: 2);

(c) CDRH3 contains the amino acid sequence AKGGDYGGNYFD (SEQ ID NO: 3);

(d) CDRL1 contains the amino acid sequence X1ASQSVX2SSYLA (SEQ ID NO: 52), where

X ₁ is R or G, and

X2 is absent or is S;

(e) CDRL2 contains the amino acid sequence X1ASX2RAT (SEQ ID NO: 53), where

X ₁ is D or G and X ₂ is N, S or T; and (f) CDRL3 contains the amino acid sequence QQYGSSPX1T (SEQ ID NO: 54), where X ₁ is L or I.

According to another aspect of the present invention, there is provided an antibody, or an isolated antibody, comprising a heavy chain variable region comprising the complementarity determining regions CDRH1, CDRH2 and CDRH3 and a light chain variable region comprising the complementarity determining regions CDRL1, CDRL2 and CDRL3, said antibody being internalized upon binding with cells expressing human TIM-3, and CDRH3 contains the amino acid sequence AKGGDYGGNYFD (SEQ ID NO: 3).

According to another aspect of the present invention, there is provided an antibody or isolated antibody that specifically binds to human TIM-3, comprising a heavy chain variable region comprising the complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising the complementarity determining regions CDRL1, CDRL2 and CDRL3, wherein said antibody is internalized upon binding to cells expressing human TIM-3, and CDRH3 contains the amino acid sequence AKGGDYGGNYFD (SEQ ID NO: 3).

According to certain implementation options:

(a) CDRH1 contains the amino acid sequence X1X ₂ X ₃ X ₄ X ₅ S (SEQ ID NO: 48), where

X _i represents R, S, A, G, K, M or T,

X2 is Q, S, A, G, R or T,

X3 is N, Y, G or Q,

X4 is A or Q, and

X5 is W, M, A, S or T;

(b) CDRH2 contains the amino acid sequence WVSAISGSGGSTY (SEQ ID NO: 2);

(c) CDRL1 contains the amino acid sequence X1ASQSVX2SSYLA (SEQ ID NO: 52), where

X _i is R or G, and

X ₂ is absent or represents S;

(d) CDRL2 contains the amino acid sequence X1ASX2RAT (SEQ ID NO: 53), where

X _i is D or G, and

X ₂ represents N, S or T; and (e) CDRL3 contains the amino acid sequence QQYGSSPX1T (SEQ ID NO: 54), where X ₁ is L or I.

In some embodiments, CDRH1 contains the amino acid sequence X1X2NAWS (SEQ ID NO: 49), where: X ₁ is R or A; and X ₂ is Q or R. In some embodiments, CDRH1 comprises the amino acid sequence X ₁ X ₂ GQX ₃ S (SEQ ID NO: 50), where: X ₁ is K, M, or G; X ₂ is A or S; and X ₃ is S or T. In some embodiments, CDRH1 comprises the amino acid sequence X1X2QQAS (SEQ ID NO: 51), where X ₁ is S, R, T, or G; and X ₂ is A, S, T, or G. In some embodiments, CDRH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 4-12.

In some embodiments, CDRL1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-16. In some embodiments, the CDRL2 contains an amino acid sequence selected from the group consisting of the sequences of SEQ ID NOs: 17-21. In some embodiments, the CDRL3 contains an amino acid sequence selected from the group consisting of SEQ ID NOS: 22 and 23.

In some embodiments, CDRH1, CDRH2, and CDRH3 comprise the amino acid sequences CDRH1, CDRH2, and CDRH3, respectively, of SEQ ID NOs: 1, 2, and 3; 4, 2 and 3; 5, 2 and 3; 6, 2 and 3; 7, 2 and 3; 8, 2 and 3; 9, 2 and 3; 10, 2 and 3; 11, 2 and 3; or 12, 2 and 3.

In some embodiments, CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences of CDRL1, CDRL2, and CDRL3, respectively, the sequences of SEQ ID NOS: 13, 17, and

22; 14, 17 and 22; 15, 18 and 22; 14, 19 and 22; 14, 20 and 22; 14, 21 and 22; 16, 20 and 22; or 14, 17 and 23.

In some embodiments, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences of SEQ ID NOs: 1, 2, 3, 14, 21, and 22; 4, 2, 3, 14, 21 and 22; 5, 2, 3, 14, 21 and 22; 6, 2, 3, 14, 21 and 22; 7, 2, 3, 14, 21 and 22; 8, 2, 3, 14, 21 and 22; 9, 2, 3, 14, 21 and 22; 10, 2, 3, 14, 21 and 22; 11, 2, 3, 14, 21 and 22; or 12, 2, 3, 14, 21 and 22, respectively.

According to another aspect of the present invention, an antibody or isolated antibody is provided, comprising a heavy chain variable region comprising the complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising the complementarity determining regions CDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2, CDRH3 , CDRL1, CDRL2 and CDRL3 contain the amino acid sequences of SEQ ID NOs: 1, 2, 3, 14, 21 and 22, respectively.

According to another aspect of the present invention, there is provided an antibody or isolated antibody that specifically binds to human TIM-3, comprising a heavy chain variable region containing complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region containing complementarity determining regions CDRL1, CDRL2 and CDRL3, while CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 contain the amino acid sequences of SEQ ID NOs: 1, 2, 3, 14, 21 and 22, respectively.

According to another aspect of the present invention, an antibody or isolated antibody is provided, comprising a heavy chain variable region comprising the complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising the complementarity determining regions CDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2, CDRH3 , CDRL1, CDRL2 and CDRL3 contain the amino acid sequences of SEQ ID NOs: 5, 2, 3, 14, 21 and 22, respectively.

According to another aspect of the present invention, there is provided an antibody or isolated antibody that specifically binds to human TIM-3, comprising a heavy chain variable region comprising the complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising the complementarity determining regions CDRL1, CDRL2 and CDRL3, while CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 contain the amino acid sequences of SEQ ID NOs: 5, 2, 3, 14, 21 and 22, respectively.

According to another aspect of the present invention, an antibody or isolated antibody is provided, comprising a heavy chain variable region containing the complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region containing the complementarity determining regions CDRL1, CDRL2 and CDRL3, while CDRH1, CDRH2, CDRH3 , CDRL1, CDRL2 and CDRL3 contain the amino acid sequences of SEQ ID NOs: 9, 2, 3, 14, 21 and 22, respectively.

According to another aspect of the present invention, there is provided an antibody or isolated antibody that specifically binds to human TIM-3, comprising a heavy chain variable region containing complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region containing complementarity determining regions CDRL1, CDRL2 and CDRL3, while CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 contain the amino acid sequences of SEQ ID NOs: 9, 2, 3, 14, 21 and 22, respectively.

According to another aspect of the present invention, an antibody or isolated antibody is provided, comprising a heavy chain variable region containing the complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region containing the complementarity determining regions CDRL1, CDRL2 and CDRL3, while CDRH1, CDRH2, CDRH3 , CDRL1, CDRL2 and CDRL3 contain the amino acid sequences of SEQ ID NOs: 1, 2, 3, 15, 18 and 22, respectively.

According to another aspect of the present invention, there is provided an antibody or isolated antibody that specifically binds to human TIM-3, comprising a heavy chain variable region comprising the complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region comprising the complementarity determining regions CDRL1, CDRL2 and CDRL3, while CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 contain the amino acid sequences of SEQ ID NOs: 1, 2, 3, 15, 18 and 22, respectively.

In some embodiments, said antibody is internalized upon binding to cells expressing human TIM-3.

According to another aspect of the present invention, an antibody or isolated antibody is provided that specifically binds to human TIM-3, said antibody being internalized upon binding to cells expressing human TIM-3.

In some embodiments, a lower percentage of cells expressing human TIM-3 survive in the presence of said antibody than in the presence of pab1944w (IgG1 N297A) in an assay comprising the following steps: (a) seeding cells expressing human TIM-3 with dense 3 043100 2x 10 ⁴ cells per well in a tissue culture plate; (b) adding 1111 ng/ml aHFc-NC-DM1 and 1111 ng/ml the indicated antibody or pab1944w (IgG1 N297A) to a final volume of 100 μl/well; (c) incubation at 37°C and 5% CO ₂ for 72 h; (d) measuring the survival of cells expressing human TIM-3; and (e) calculating percent relative cell survival compared to untreated cells expressing human TIM-3. In some embodiments, the percentage of cell survival in the presence of said antibody is at least 50% lower than the percentage of cell survival in the presence of pab1944w (IgG1 N297A). In some embodiments, said cells expressing human TIM-3 are Kasumi-3 cells. In some embodiments, said cells expressing human TIM-3 are Kasumi-3 (ATCC® CRL-2725™) cells. In some embodiments, said cells expressing human TIM-3 are Jurkat cells engineered to express human TIM-3.

In some embodiments, a lower percentage of cells expressing human TIM-3 survive in the presence of said antibody than in the presence of Hum11 (IgG ₄ S228P) in an assay comprising the following steps: (a) seeding cells expressing human TIM-3 at a density 2χ 10 ⁴ cells per well in a tissue culture plate; (b) adding 1111 ng/ml aHFc-NC-DM1 and 1111 ng/ml the indicated antibody or Hum11 (IgG ₄ S228P) to a final volume of 100 μl/well; (c) incubation at 37°C and 5% CO ₂ for 72 h; (d) measuring the survival of cells expressing human TIM-3; and (e) calculating percent relative cell survival compared to untreated cells expressing human TIM-3. In some embodiments, the percentage of cell survival in the presence of said antibody is at least 50% lower than the percentage of cell survival in the presence of Hum11 (IgG ₄ S228P). In some embodiments, said cells expressing human TIM-3 are Kasumi-3 cells. In some embodiments, said cells expressing human TIM-3 are Kasumi-3 cells (ATCC® CRL-2725™). In some embodiments, said cells expressing human TIM-3 are Jurkat cells engineered to express human TIM-3.

In some embodiments, said antibody comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 55. In some embodiments, said 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 the amino acid sequence selected from the group consisting of the sequences of SEQ ID NO: 24-35. In some embodiments, said heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 24-35. In some embodiments, said heavy chain variable region comprises an amino acid sequence, sequence SEQ ID NO: 25. In some embodiments, said heavy chain variable region comprises an amino acid sequence, sequence SEQ ID NO: 28. In some embodiments, said heavy chain variable region comprises amino acid sequence, sequence SEQ ID NO: 32. In some embodiments, the N-terminal glutamate (E) residue of the heavy chain variable region of an antibody as described herein is replaced with a pyroglutamate (pE) residue.

In some embodiments, said antibody comprises a light chain variable region comprising an amino acid sequence of SEQ ID NO: 56. In some embodiments, said antibody comprises a light chain variable region comprising an amino acid sequence of at least 75, 80, 85, 90 , 95 or 100% identical to the amino acid sequence selected from the group consisting of the sequences of SEQ ID NO: 36-47. In some embodiments, said light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 36-47. In some embodiments, said light chain variable region comprises an amino acid sequence, sequences of SEQ ID NO: 46. In some embodiments, the N-terminal glutamate (E) residue of the light chain variable region of an antibody as described herein is replaced with a pyroglutamate (pE) residue.

According to another aspect of the present invention, there is provided an antibody or an isolated antibody comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24-35. In some embodiments, said heavy chain variable region comprises an amino acid sequence, sequence SEQ ID NO: 25. In some embodiments, said heavy chain variable region comprises an amino acid sequence, sequence SEQ ID NO: 28. In some embodiments, said heavy chain variable region comprises amino acid sequence, sequence SEQ ID NO: 32. According to some

- 4 043100 embodiments, said antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 58. In some embodiments, said antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 61. In some embodiments, said antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 65. In some embodiments, said antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 70, 71, 72, 73, 74, or 75. In some embodiments, N The α-terminal glutamate residue (E) of the heavy chain of an antibody as described herein is replaced by a pyroglutamate residue (pE).

According to another aspect of the present invention, there is provided an antibody or isolated antibody that specifically binds to human TIM-3, comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24-35. In some embodiments, said heavy chain variable region comprises an amino acid sequence, sequence SEQ ID NO: 25. In some embodiments, said heavy chain variable region comprises an amino acid sequence, sequence SEQ ID NO: 28. In some embodiments, said heavy chain variable region comprises amino acid sequence, sequence SEQ ID NO: 32. In some embodiments, said antibody comprises a heavy chain comprising an amino acid sequence, sequence SEQ ID NO: 58. In some embodiments, said antibody comprises a heavy chain comprising an amino acid sequence, sequence SEQ ID NO: 61. In some embodiments, said antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 65. In some embodiments, said antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 70, 71, 72, 73, 74 or 75.

According to another aspect of the present invention, there is provided an antibody or isolated antibody comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of the sequences of SEQ ID NOs: 36-47. In some embodiments, said light chain variable region comprises an amino acid sequence of SEQ ID NO: 46. In some embodiments, said antibody comprises a light chain comprising an amino acid sequence of SEQ ID NO: 69. In some embodiments, said antibody comprises a light chain containing the amino acid sequence, the sequences of SEQ ID NO: 76 or 77. In some embodiments, the N-terminal glutamate (E) residue of the light chain of an antibody as described herein is replaced by a pyroglutamate (pE) residue.

According to another aspect of the present invention, there is provided an antibody or isolated antibody that specifically binds to human TIM-3, comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 36-47. In some embodiments, said light chain variable region comprises an amino acid sequence, sequence SEQ ID NO: 46. In some embodiments, said antibody comprises a light chain comprising an amino acid sequence, sequence SEQ ID NO: 69. In some embodiments, said antibody comprises a light chain containing the amino acid sequence, the sequences of SEQ ID NO: 76 or 77.

According to another aspect of the present invention, there is provided an antibody or an isolated antibody comprising a heavy chain variable region and a light chain variable region, characterized in that said heavy chain variable region and said light chain variable region, respectively, comprise the amino acid sequences of SEQ ID NO: 24 and 36; 24 and 38; 26 and 42; 24 and 42; 24 and 46; 24 and 43; 26 and 43; 26 and 46; 26 and 41; 24 and 41; 25 and 39; 24 and 47; 25 and 40; 26 and 47; 25 and 37; 25 and 45; 25 and 44; 25 and 46; 25 and 42; 25 and 41; 25 and 43; 25 and 47; 27 and 46; 28 and 46; 29 and 46; 30 and 46; 31 and 46; 32 and 46; 33 and 46; 34 and 46; or 35 and 46. In some embodiments, said heavy chain variable region and said light chain variable region, respectively, comprise the amino acid sequences of SEQ ID NOs: 25 and 46. In some embodiments, said heavy chain variable region and said light chain variable region the chains respectively comprise the amino acid sequences of SEQ ID NOs: 28 and 46. In some embodiments, said heavy chain variable region and said light chain variable region, respectively, comprise the amino acid sequences of SEQ ID NOs: 32 and 46. In some embodiments, embodiments, the N-terminal glutamate (E) residue of the heavy chain variable region of an antibody as described herein is replaced with a pyroglutamate (pE) residue; and/or the N-terminal glutamate residue (E) of the light chain variable region of said antibody is replaced by a pyroglutamate residue (pE).

According to another aspect of the present invention, an antibody or isolated antibody is provided,

- 5 043100 containing a heavy chain variable region and a light chain variable region, characterized in that the amino acid sequences of said heavy chain variable region and said light chain variable region, respectively, consist of the amino acid sequences shown in the sequences of SEQ ID NOs: 24 and 36; 24 and 38; 26 and 42; 24 and 42; 24 and 46; 24 and 43; 26 and 43; 26 and 46; 26 and 41; 24 and 41; 25 and 39; 24 and 47; 25 and 40; 26 and 47; 25 and 37; 25 and 45; 25 and 44; 25 and 46; 25 and 42; 25 and 41; 25 and 43; 25 and 47; 27 and 46; 28 and 46; 29 and 46; 30 and 46; 31 and 46; 32 and 46; 33 and 46; 34 and 46; or 35 and 46. In some embodiments, the amino acid sequences of said heavy chain variable region and said light chain variable region, respectively, consist of the amino acid sequences shown in SEQ ID NOs: 25 and 46. In some embodiments, the amino acid sequences of said variable region heavy chain and said light chain variable region, respectively, consist of the amino acid sequences shown in SEQ ID NOs: 28 and 46. In some embodiments, the amino acid sequences of said heavy chain variable region and said light chain variable region, respectively, consist of the sequences amino acids shown in SEQ ID NOs: 32 and 46.

According to another aspect of the present invention, there is provided an antibody or isolated antibody that specifically binds to human TIM-3, comprising a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region and said light chain variable region, respectively, comprise amino acid sequences, sequences of SEQ ID NOs: 24 and 36; 24 and 38; 26 and 42; 24 and 42; 24 and 46; 24 and 43; 26 and 43; 26 and 46; 26 and 41; 24 and 41; 25 and 39; 24 and 47; 25 and 40; 26 and 47; 25 and 37; 25 and 45; 25 and 44; 25 and 46; 25 and 42; 25 and 41; 25 and 43; 25 and 47; 27 and 46; 28 and 46; 29 and 46; 30 and 46; 31 and 46; 32 and 46; 33 and 46; 34 and 46; or 35 and 46. In some embodiments, said heavy chain variable region and said light chain variable region, respectively, comprise the amino acid sequences of SEQ ID NOs: 25 and 46. In some embodiments, said heavy chain variable region and said light chain variable region the chains respectively comprise the amino acid sequences of SEQ ID NOS: 28 and 46. In some embodiments, said heavy chain variable region and said light chain variable region, respectively, comprise the amino acid sequences of SEQ ID NOS: 32 and 46.

According to another aspect of the present invention, there is provided an antibody or isolated antibody that specifically binds to human TIM-3, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of said heavy chain variable region and said light chain variable region, respectively, are from the amino acid sequences shown in the sequences of SEQ ID NOs: 24 and 36; 24 and 38; 26 and 42; 24 and 42; 24 and 46; 24 and 43; 26 and 43; 26 and 46; 26 and 41; 24 and 41; 25 and 39; 24 and 47; 25 and 40; 26 and 47; 25 and 37; 25 and 45; 25 and 44; 25 and 46; 25 and 42; 25 and 41; 25 and 43; 25 and 47; 27 and 46; 28 and 46; 29 and 46; 30 and 46; 31 and 46; 32 and 46; 33 and 46; 34 and 46; or 35 and 46. In some embodiments, the amino acid sequences of said heavy chain variable region and said light chain variable region, respectively, consist of the amino acid sequences shown in SEQ ID NOs: 25 and 46. In some embodiments, the amino acid sequences of said variable region heavy chain and said light chain variable region, respectively, consist of the amino acid sequences shown in SEQ ID NOs: 28 and 46. In some embodiments, the amino acid sequences of said heavy chain variable region and said light chain variable region, respectively, consist of the sequences amino acids shown in SEQ ID NOs: 32 and 46.

According to another aspect of the present invention, there is provided an antibody or an isolated antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 58 and a light chain comprising the amino acid sequence of SEQ ID NO: 69.

According to another aspect of the present invention, there is provided an antibody or isolated antibody that specifically binds to human TIM-3, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 58 and a light chain containing the amino acid sequence of SEQ ID NO: 69.

According to another aspect of the present invention, there is provided an antibody or an isolated antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 61 and a light chain comprising the amino acid sequence of SEQ ID NO: 69.

According to another aspect of the present invention, there is provided an antibody or isolated antibody that specifically binds to human TIM-3, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 61 and a light chain containing the amino acid sequence of SEQ ID NO: 69.

According to another aspect of the present invention, there is provided an antibody or an isolated antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 65 and a light chain comprising the amino acid sequence of SEQ ID NO: 69.

- 6 043100

According to another aspect of the present invention, there is provided an antibody or isolated antibody that specifically binds to human TIM-3, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 65 and a light chain containing the amino acid sequence of SEQ ID NO: 69.

In some embodiments, the N-terminal glutamate (E) residue of the heavy chain of an antibody as described herein is replaced with a pyroglutamate (pE) residue; and/or the N-terminal glutamate residue (E) of the light chain of said antibody is replaced by a pyroglutamate residue (pE).

In some embodiments, said antibody comprises a heavy chain variable region comprising an amino acid sequence derived from a human IGHV3-23 germline sequence. In some embodiments, said antibody comprises a light chain variable region comprising an amino acid sequence derived from a human germline sequence selected from the group consisting of IGKV1-27, IGKV3-11, IGKV3-20, and IGKV3D-20.

According to another aspect of the present invention, there is provided an antibody or isolated antibody that specifically binds to human TIM-3, comprising a heavy chain variable region comprising an amino acid sequence derived from the human IGHV3-23 germline sequence and a light chain variable region comprising an amino acid sequence, derived from a human germline sequence selected from the group consisting of IGKV1-27, IGKV3-11, IGKV3-20 and IGKV3D-20.

In some embodiments, said antibody comprises a heavy chain constant region selected from the group consisting of human IgG1, IgG ₂ , IgG ₃ , IgG ₄ , IgA1 and IgA ₂ . In some embodiments, said heavy chain constant region is IgG1. In some embodiments, said IgGi amino acid sequence contains the N297A mutation according to the EU numbering system. In some embodiments, said antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, said IgG1 amino acid sequence contains the N297Q mutation according to the EU numbering system. In some embodiments, said IgG _i is a non-fucosylated IgG _i . In some embodiments, said heavy chain constant region is IgG ₄ . In some embodiments, said IgG ₄ amino acid sequence contains the S228P mutation according to the EU numbering system. In some embodiments, said antibody comprises a heavy chain constant region comprising an amino acid sequence of SEQ ID NO: 74.

In some embodiments, said antibody comprises a light chain constant region selected from the group consisting of human IgGK and IgGλ. In some embodiments, said light chain constant region is an IgGK. In some embodiments, said antibody comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 76. In some embodiments, said light chain constant region is IqGλ.

According to another aspect of the present invention, an antibody or isolated antibody is provided that cross-competes for binding to human TIM-3 with an antibody as described herein. In some embodiments, the present invention provides an antibody or isolated antibody that cross-competes for binding to human TIM-3 with an antibody comprising the heavy and light chain variable region amino acid sequences, SEQ ID NOS: 55 and 56, respectively. In some embodiments, the present invention provides an antibody or isolated antibody that cross-competes for binding to human TIM-3 with an antibody comprising the heavy and light chain variable region amino acid sequences, SEQ ID NOS: 25 and 46, respectively. In some embodiments, the present invention provides an antibody or isolated antibody that cross-competes for binding to human TIM-3 with an antibody comprising the heavy and light chain variable region amino acid sequences, SEQ ID NOS: 28 and 46, respectively. In some embodiments, the present invention provides an antibody or isolated antibody that cross-competes for binding to human TIM-3 with an antibody comprising heavy and light chain variable region amino acid sequences, SEQ ID NOs: 32 and 46, respectively.

According to another aspect of the present invention, an antibody or isolated antibody is provided that binds to the same human TIM-3 epitope as an antibody as described herein. In some embodiments, the present invention provides an antibody or isolated antibody that binds to the same human TIM-3 epitope as an antibody comprising the amino acid sequences of the heavy and light chain variable regions, SEQ ID NOS: 55 and 56, respectively. According to some embodiments of this

- 7 043100 of the invention provides an antibody or isolated antibody that binds to the same epitope of human TIM-3 as an antibody containing the amino acid sequences of the heavy and light chain variable regions, the sequences of SEQ ID NOS: 25 and 46, respectively. In some embodiments, the present invention provides an antibody or isolated antibody that binds to the same human TIM-3 epitope as an antibody comprising the amino acid sequences of the heavy and light chain variable regions, SEQ ID NOS: 28 and 46, respectively. In some embodiments, the present invention provides an antibody or isolated antibody that binds to the same human TIM-3 epitope as an antibody comprising the amino acid sequences of the heavy and light chain variable regions, SEQ ID NOS: 32 and 46, respectively.

According to another aspect of the present invention, there is provided an antibody or isolated antibody that specifically binds to human TIM-3, characterized in that said antibody specifically binds to the TIM-3 protein variant with the amino acid sequence of SEQ ID NO: 101 with a lower affinity compared to binding with the wild-type protein TIM-3 with the amino acid sequence of SEQ ID NO: 79.

According to another aspect of the present invention, an antibody or isolated antibody is provided that specifically binds to the same human TIM-3 epitope as any antibody of the present invention. In one embodiment, said antibody specifically binds to the TIM-3 protein variant of the amino acid sequence of SEQ ID NO: 101 with lower affinity compared to binding to the wild-type TIM-3 protein of the amino acid sequence of SEQ ID NO: 79.

According to another aspect of the present invention, there is provided an antibody or isolated antibody that specifically binds to human TIM-3, characterized in that said antibody does not specifically bind to the TIM-3 protein variant of the amino acid sequence of SEQ ID NO: 101.

According to another aspect of the present invention, an antibody or isolated antibody is provided that specifically binds to the same human TIM-3 epitope as any antibody of the present invention. In one embodiment, said antibody does not specifically bind to the TIM-3 protein variant with the amino acid sequence of SEQ ID NO: 101.

According to another aspect of the present invention, an antibody or isolated antibody is provided that specifically binds to human TIM-3, characterized in that the binding between said antibody and the TIM-3 protein variant with the amino acid sequence of SEQ ID NO: 101 is substantially weakened relative to the binding between said antibody and the wild-type TIM-3 protein with the amino acid sequence of SEQ ID NO: 79.

According to another aspect of the present invention, an antibody or isolated antibody is provided that specifically binds to the same human TIM-3 epitope as any antibody of the present invention. In one embodiment, the binding between said antibody and the TIM-3 protein variant with the amino acid sequence of SEQ ID NO: 101 is substantially weakened relative to the binding between the indicated antibody and the wild-type TIM-3 protein with the amino acid sequence of SEQ ID NO: 79.

According to another aspect of the present invention, there is provided an antibody or isolated antibody that specifically binds to human TIM-3, characterized in that said antibody exhibits, compared to binding to wild-type TIM-3 protein with the amino acid sequence of SEQ ID NO: 79, reduced binding or no binding to the TIM-3 protein variant with the amino acid sequence of SEQ ID NO: 101.

According to another aspect of the present invention, an antibody or isolated antibody is provided that specifically binds to the same human TIM-3 epitope as any antibody of the present invention. In one embodiment, said antibody exhibits, compared to binding to the wild-type TIM-3 protein of the amino acid sequence of SEQ ID NO: 79, reduced binding or no binding to the TIM-3 protein variant of the amino acid sequence of SEQ ID NO: 101.

According to another aspect of the present invention, an antibody or isolated antibody is provided that binds, eg, specifically binds, to a human TIM-3 epitope. In some embodiments, said antibody binds to residue 40 of SEQ ID NO: 79.

According to another aspect of the present invention, an antibody or isolated antibody is provided that specifically binds to the same human TIM-3 epitope as any antibody of the present invention. In some embodiments, said antibody binds to residue 40 of SEQ ID NO: 79.

According to another aspect of the present invention, an antibody or isolated antibody is provided that binds, eg, specifically binds, to a human TIM-3 epitope. In some embodiments, said antibody binds to an epitope located within the human TIM-3 region, consisting of the amino acid sequence shown in the sequence

- 8 043100 SEQ ID NO: 93. In some embodiments, said antibody binds to an epitope located within the human TIM-3 region, consisting of the amino acid sequence shown in SEQ ID NO: 94. In some embodiments, said antibody binds with an epitope located within the region of human TIM-3, consisting of the amino acid sequence shown in the sequence of SEQ ID NO: 95. In some embodiments, said antibody binds to an epitope located within the region of human TIM-3, consisting of the amino acid sequence, shown in SEQ ID NO: 96. In some embodiments, said antibody binds to an epitope located within the human TIM-3 region, consisting of the amino acid sequence shown in SEQ ID NO: 97. In certain embodiments, said antibody binds to an epitope located within the human TIM-3 region, consisting of the amino acid sequence shown in SEQ ID NO: 98. In some embodiments, said antibody binds to an epitope located within the human TIM-3 region, consisting of the amino acid sequence shown in in SEQ ID NO: 99. In some embodiments, said antibody binds to an epitope located within the human TIM-3 region, consisting of the amino acid sequence shown in SEQ ID NO: 100.

According to another aspect of the present invention, an antibody or isolated antibody is provided that specifically binds to the same human TIM-3 epitope as any antibody of the present invention. In some embodiments, said antibody binds to an epitope located within the human TIM-3 region, consisting of the amino acid sequence shown in SEQ ID NO: 93. In some embodiments, said antibody binds to an epitope located within the TIM-3 region. human, consisting of the amino acid sequence presented in the sequence of SEQ ID NO: 94. In some embodiments, the specified antibody binds to an epitope located within the region of human TIM-3, consisting of the amino acid sequence presented in the sequence of SEQ ID NO: 95. According to In some embodiments, said antibody binds to an epitope located within the human TIM-3 region, consisting of the amino acid sequence shown in SEQ ID NO: 96. In some embodiments, said antibody binds to an epitope located within the human TIM-3 region. consisting of the amino acid sequence shown in SEQ ID NO: 97. In some embodiments, said antibody binds to an epitope located within the human TIM-3 region, consisting of the amino acid sequence shown in SEQ ID NO: 98. According to some in embodiments, said antibody binds to an epitope located within a human TIM-3 region, consisting of the amino acid sequence shown in SEQ ID NO: 99. In some embodiments, said antibody binds to an epitope located within a human TIM-3 region, consisting of the amino acid sequence shown in SEQ ID NO: 100.

According to another aspect of the present invention, there is provided an antibody which, when bound to a human TIM-3 protein or fragment thereof containing the amino acid sequence of SEQ ID NO: 102, reduces hydrogen-deuterium exchange in the region consisting of the amino acid sequence shown in SEQ ID NO: 102. NO: 93, relative to hydrogen-deuterium exchange in the region consisting of the amino acid sequence shown in the sequence of SEQ ID NO: 93, in the absence of said antibody, as assessed using a hydrogen/deuterium assay. According to another aspect of the present invention, there is provided an antibody which, when bound to a human TIM-3 protein or fragment thereof containing the amino acid sequence of SEQ ID NO: 102, reduces hydrogen-deuterium exchange in the region consisting of the amino acid sequence shown in SEQ ID NO: 102. NO: 94, relative to hydrogen-deuterium exchange in the region consisting of the amino acid sequence shown in the sequence of SEQ ID NO: 94, in the absence of said antibody, as assessed using a hydrogen/deuterium assay. According to another aspect of the present invention, there is provided an antibody which, when bound to a human TIM-3 protein or fragment thereof containing the amino acid sequence of SEQ ID NO: 102, reduces hydrogen-deuterium exchange in the region consisting of the amino acid sequence shown in SEQ ID NO: 102. NO: 95, relative to hydrogen-deuterium exchange in the region consisting of the amino acid sequence shown in the sequence of SEQ ID NO: 95, in the absence of said antibody, as assessed using a hydrogen/deuterium assay. According to another aspect of the present invention, there is provided an antibody which, when bound to a human TIM-3 protein or fragment thereof containing the amino acid sequence of SEQ ID NO: 102, reduces hydrogen-deuterium exchange in a region consisting of the amino acid sequence shown in SEQ ID NO: 96, regarding the hydrogen-deuterium exchange in the region consisting of

- 9 043100 the amino acid sequence shown in the sequence of SEQ ID NO: 96, in the absence of the specified antibody, as assessed using a hydrogen/deuterium assay. According to another aspect of the present invention, there is provided an antibody which, when bound to a human TIM-3 protein or fragment thereof containing the amino acid sequence of SEQ ID NO: 102, reduces hydrogen-deuterium exchange in the region consisting of the amino acid sequence shown in SEQ ID NO: 102. NO: 97, relative to hydrogen-deuterium exchange in the region consisting of the amino acid sequence shown in the sequence of SEQ ID NO: 97, in the absence of said antibody, as assessed using a hydrogen/deuterium assay. According to another aspect of the present invention, there is provided an antibody which, when bound to a human TIM-3 protein or fragment thereof containing the amino acid sequence of SEQ ID NO: 102, reduces hydrogen-deuterium exchange in the region consisting of the amino acid sequence shown in SEQ ID NO: 102. NO: 98, relative to hydrogen-deuterium exchange in the region consisting of the amino acid sequence shown in the sequence of SEQ ID NO: 98, in the absence of said antibody, as assessed using a hydrogen/deuterium assay. In some embodiments, said reduction in hydrogen-deuterium exchange is measured using hydrogen-deuterium exchange (HDX), for example, as described herein in the examples section.

According to another aspect of the present invention, an antibody or isolated antibody is provided that specifically binds to the same human TIM-3 epitope as any antibody of the present invention. In some embodiments, said antibody, when bound to a human TIM-3 protein or fragment thereof containing the amino acid sequence of SEQ ID NO: 102, reduces hydrogen-deuterium exchange in the region consisting of the amino acid sequence shown in SEQ ID NO: 93 , relative to hydrogen-deuterium exchange in the region consisting of the amino acid sequence shown in the sequence of SEQ ID NO: 93, in the absence of said antibody, as assessed using a hydrogen/deuterium assay. In some embodiments, said antibody, when bound to a human TIM-3 protein or fragment thereof containing the amino acid sequence of SEQ ID NO: 102, reduces hydrogen-deuterium exchange in the region consisting of the amino acid sequence shown in SEQ ID NO: 94 , relative to hydrogen-deuterium exchange in the region consisting of the amino acid sequence shown in the sequence of SEQ ID NO: 94, in the absence of said antibody, as assessed using a hydrogen/deuterium assay. In some embodiments, said antibody, when bound to a human TIM-3 protein or fragment thereof containing the amino acid sequence of SEQ ID NO: 102, reduces hydrogen-deuterium exchange in the region consisting of the amino acid sequence shown in SEQ ID NO: 95 , relative to hydrogen-deuterium exchange in the region consisting of the amino acid sequence shown in the sequence of SEQ ID NO: 95, in the absence of said antibody, as assessed using a hydrogen/deuterium assay. In some embodiments, said antibody, when bound to a human TIM-3 protein or fragment thereof containing the amino acid sequence of SEQ ID NO: 102, reduces hydrogen-deuterium exchange in the region consisting of the amino acid sequence shown in SEQ ID NO: 96 , relative to hydrogen-deuterium exchange in the region consisting of the amino acid sequence shown in the sequence of SEQ ID NO: 96, in the absence of said antibody, as assessed using a hydrogen/deuterium assay. In some embodiments, said antibody, when bound to a human TIM-3 protein or fragment thereof containing the amino acid sequence of SEQ ID NO: 102, reduces hydrogen-deuterium exchange in the region consisting of the amino acid sequence shown in SEQ ID NO: 97 , relative to hydrogen-deuterium exchange in the region consisting of the amino acid sequence shown in the sequence of SEQ ID NO: 97, in the absence of said antibody, as assessed using a hydrogen/deuterium assay. In some embodiments, said antibody, when bound to a human TIM-3 protein or fragment thereof containing the amino acid sequence of SEQ ID NO: 102, reduces hydrogen-deuterium exchange in the region consisting of the amino acid sequence shown in SEQ ID NO: 98 , relative to hydrogen-deuterium exchange in the region consisting of the amino acid sequence shown in the sequence of SEQ ID NO: 98, in the absence of said antibody, as assessed using a hydrogen/deuterium assay. In some embodiments, said reduction in hydrogen-deuterium exchange is measured using hydrogen-deuterium exchange (HDX), for example, as described herein in the examples section.

According to another aspect of the present invention, an antibody or isolated antibody is provided that binds, e.g., specifically binds to the same human TIM-3 epitope as any antibody of the present invention, said epitope being determined using hydrogen-deuterium exchange (HDX), e.g. , as described in the examples section, using a Pepscan assay, eg, as described in the examples section, or using an alanine scan, eg, as described in the examples section.

In some embodiments, said antibody comprises a human IgG heavy chain constant region that is a wild-type human IgG heavy chain constant region variant, wherein said human IgG heavy chain constant region variant binds to the human Fc-gamma receptor with an affinity lower than compared with the binding affinity of the wild-type human IgG heavy chain constant region to the human Fc-gamma receptor. In some embodiments, said human Fc-gamma receptor is selected from the group consisting of FcyRI, FcyRII, and FcyRIII. In some embodiments, said human IgG heavy chain constant region variant is an IgG1 constant region containing the N297A mutation.

In some embodiments, said antibody is a human antibody. In some embodiments, said antibody is antagonistic to human TIM-3. In some embodiments, said antibody deactivates, reduces, or inhibits human TIM-3 activity. In some embodiments, said antibody inhibits the binding of human TIM-3 to phosphatidylserine. In some embodiments, said antibody induces the production of IFNy by staphylococcal enterotoxin A (SEA)-stimulated peripheral blood mononuclear cells (PBMCs). In some embodiments, said antibody induces the production of IFNγ or TNFa by tumor infiltrating lymphocytes (TILs) stimulated with anti-CD3 and anti-CD28 antibodies.

In some embodiments, said antibody is internalized upon binding to cells expressing human TIM-3.

According to another aspect of the present invention, an antibody or isolated antibody as described herein conjugated to a cytotoxic agent is provided.

According to another aspect of the present invention, an antibody or isolated antibody as described herein conjugated to a cytostatic agent is provided.

According to another aspect of the present invention, an antibody or isolated antibody as described herein conjugated to a toxin is provided.

According to another aspect of the present invention, an antibody or isolated antibody as described herein is conjugated to a radionuclide.

According to another aspect of the present invention, an antibody or isolated antibody as described herein is conjugated to a detectable label.

According to another aspect of the present invention, there is provided a pharmaceutical composition comprising an antibody as described herein and a pharmaceutically acceptable carrier or excipient.

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

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

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

According to another aspect of the present invention, there is provided a method for increasing T cell activation in response to an antigen in an individual, said method comprising administering to said individual an effective amount of an antibody or pharmaceutical composition as described herein. According to another aspect of the present invention, there is provided a method of treating cancer in an individual, comprising administering to said individual an effective amount of an antibody or pharmaceutical composition as described herein. In certain embodiments of the methods described above, said antibody or said pharmaceutical composition is administered subcutaneously. In certain embodiments of the methods described above, said antibody or said pharmaceutical composition is administered intravenously. In certain embodiments of the methods described above, said antibody or said pharmaceutical composition is administered into a tumor. In certain embodiments of the methods described above, said antibody or said pharmaceutical composition is delivered to a tumor-draining lymph node. In certain embodiments of the methods described above, said antibody or said pharmaceutical composition is administered intra-arterially.

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

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

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

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

According to one aspect, the present invention relates to an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition according to the present invention for use in a method of treating cancer in an individual.

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

In one embodiment of an antibody, polynucleotide, vector, recombinant host cell, and/or pharmaceutical composition for use according to the present invention, said antibody, polynucleotide, vector, recombinant host cell, and/or pharmaceutical composition is administered subcutaneously or intravenously. In another embodiment of an antibody, polynucleotide, vector, recombinant host cell, and/or pharmaceutical composition for use according to the present invention, said antibody, polynucleotide, vector, recombinant host cell, and/or pharmaceutical composition is administered intratumorally or intra-arterially.

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

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

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

According to one aspect, the present invention relates to a pharmaceutical composition, kit or composite kit containing (a) an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition according to the present invention and (b) an additional therapeutic agent.

In some embodiments, said additional therapeutic agent is a chemotherapeutic agent. In some embodiments, said additional therapeutic agent is a radiotherapeutic agent.

In some embodiments, said additional therapeutic agent is a checkpoint targeting agent. In some embodiments, said checkpoint-targeting agent is selected from the group consisting of an antibody

- 12 043100 PD-1 antagonist, PD-L1 antagonist antibody, PD-L2 antagonist antibody, CTLA-4 antagonist antibody, TIM-3 antagonist antibody, LAG-3 antagonist antibody, antibodies an anti-CEACAM1 antagonist, an anti-CD137 agonist antibody, an anti-TIGIT antagonist antibody, an anti-VISTA antagonist antibody, an anti-GITR agonist antibody, and an anti-OX40 agonist antibody. In some embodiments, said additional therapeutic agent is an anti-PD-1 antibody. In some embodiments, said anti-PD-1 antibody is pembrolizumab. In some embodiments, said anti-PD-1 antibody is nivolumab.

In some embodiments, said additional therapeutic agent is an indolamine-2,3-dioxygenase (IDO) inhibitor. In some embodiments, said inhibitor is selected from the group consisting of epacadostat, F001287, indoxymod, and NLG919. In some embodiments, said inhibitor is epacadostat. In some embodiments, said inhibitor is F001287. In some embodiments, said inhibitor is indoxymod. In some embodiments, said inhibitor is NLG919.

In some embodiments, said additional therapeutic agent is a vaccine. In some embodiments, said vaccine comprises a peptide-heat shock protein complex (HSPPC) comprising a heat shock protein in complex with an antigenic peptide. In some embodiments, said heat shock protein is hsc70 and is complexed with a tumor-associated antigenic peptide. In some embodiments, said heat shock protein is a gp96 protein and is complexed with a tumor-associated antigenic peptide, wherein said HSPPC is derived from an individual's tumor. In some embodiments, said additional therapeutic agent comprises a TCR. In some embodiments, said additional therapeutic agent is a soluble TCR. In some embodiments, said additional therapeutic agent is a TCR expressing cell. In some embodiments, said additional therapeutic agent is a cell expressing a chimeric antigen receptor. In some embodiments, said additional therapeutic agent is an antibody that specifically binds to a peptide-MHC complex. In some embodiments, said additional therapeutic agent is an adjuvant. According to one aspect, the present invention relates to (a) an antibody, polynucleotide, vector, recombinant host cell and/or pharmaceutical composition according to the present invention; and (b) a vaccine for use as a medicament, for example for use in a method of treating cancer, optionally characterized in that said vaccine comprises a peptide-heat shock protein complex (HSPPC) comprising a heat shock protein complexed with an antigenic peptide. According to one aspect, the present invention relates to a pharmaceutical composition, kit or composite kit containing (a) an antibody, a polynucleotide, a vector, a recombinant host cell and/or a pharmaceutical composition according to the present invention and (b) a vaccine, optionally characterized in that said vaccine contains a complex of peptide and heat shock protein (HSPPC), containing a heat shock protein in complex with an antigenic peptide.

5. Brief description of the drawings

In FIG. 1 is a group of bar graphs showing the binding of anti-TIM-3 antibodies pab2085 (IgG1) and pab2088 (IgG1) or an isotype control antibody to wild-type mouse 1624-5 or 1624-5 cells engineered to express human TIM-3, as assessed by using flow cytometry.

In FIG. 2A and 2B are a pair of graphs showing the binding of anti-TIM-3 antibodies pab2085 (IgG1) (Fig. 2A) and pab2088 (IgG1) (Fig. 2B) to recombinant human TIM-1-His (rhTIM-1-His), recombinant Human TIM-4-His (rhTIM-4 His), recombinant human TIM-3-His (rhTIM-3 His), recombinant human TIM-3-Fc (rhTIM-3-Fc), and recombinant Javanese TIM-3-Fc macaque (rcmTIM-3-Fc) as assessed using the Luminex assay. The graphs show the dependence of the values of the median fluorescence intensity (MFI) on the concentrations of antibodies.

In FIG. 3A, 3B, 3C and 3D are a group of histograms showing the binding of anti-TIM-3 antibodies or an isotype control antibody to mouse 1624-5 cells engineered to express human TIM-3 (FIGS. 3A and 3B) or cynomolgus TIM-3 ( Fig. 3C and 3D), as assessed using flow cytometry. Anti-TIM-3 antibodies tested in this study include pab2173, pab2174, pab2175, pab2176, pab2177, pab2178, pab2179, pab2180, pab2181, pab2182, pab2183, pab2184, pab2185, pab2186, pab2187, pab2188, pab2189, pab2190, pab2191 and pab2192, all of which contain an IgG1 Fc region.

In FIG. 4 is a graph showing the binding of anti-TIM-3 antibody pab2085, light chain optimized variants (pab2184, pab2186, pab2187, pab2188, pab2189, pab2190, pab2191 and pab2192) or isotype control antibody to primary human CD8+ T cells, activated 043100 with anti-CD3 and anti-CD28 antibodies measured using flow cytometry.

Light chain optimized variants contain an IgG1 Fc region variant. The graphs show the dependence of MFI values on a number of antibody concentrations tested.

In FIG. 5 is a graph showing the binding of anti-TIM-3 antibody pab2188 (IgG1 variant) or isotype control antibody to cynomolgus primary CD11b+ myeloid cells measured using flow cytometry. The graphs show the dependence of MFI values on antibody concentrations.

In FIG. 6A and 6B are graphs showing the percent binding between irradiated phosphatidylserine-expressing mouse WR19L lymphoma cells and recombinant human TIM-3-Fc (FIG. 6A) or recombinant cynomolgus TIM-3-Fc (FIG. 6B) in the presence of a titrated dose of antibody against TIM-3 or IgG1 isotype control antibody. Anti-TIM-3 antibodies tested in this study are pab2085 (IgG1) and pab2188 (IgG1 variant).

In FIG. 7 is a bar graph showing IFN-y production induced by anti-TIM-3 antibodies or isotype control IgG1 antibody in combination with anti-PD-1 antibody pembrolizumab in human peripheral blood mononuclear cells (PBMCs) upon stimulation with staphylococcus enterotoxin A (SEA). Anti-TIM-3 antibodies tested in this study include optimized light chain variants pab2175 (IgG1), pab2176 (IgG1), pab2180 (IgG1), pab2182 (IgG1), pab2183 (IgG1 variant), pab2184 (IgG1 variant), pab2186 (IgG1 variant), pab2187 (IgG1 variant), pab2188 (IgG1 variant), pab2189 (IgG1 variant), pab2190 (IgG1 variant), pab2191 (IgG1 variant), and pab2192 (IgG1 variant).

In FIG. 8A, 8B, 8C, 8D, 8E, and 8F are a group of bar graphs showing IFN-y production induced by anti-TIM-3 antibody pab2188w (N297A IgG1) or isotype control IgG1 antibody N297A, alone or in combination with anti-PD-1 antibody pembrolizumab. , in human PBMC from six different donors after SEA stimulation. The protocol used for this study, shown in FIG. 8A-8F was a modified protocol used in the study shown in FIG. 7.

In FIG. 9A, 9B, 9C, 9D, 9E and 9F are graphs or histograms showing the binding of anti-TIM-3 antibodies to cells expressing TIM-3. In FIG. 9A, 9B, 9E, and 9F are plots of MFI values versus a range of antibody concentrations tested. In FIG. 9C and 9D are a group of histograms showing the binding of anti-TIM-3 antibodies to TIM-3 expressing cells. TIM-3 antibodies tested include pab2188w (IgG1 N297A), AM-1 (IgG1 N297A), AM-2 (IgG1 N297A), AM-3 (IgG1 N297A), AM-4 (IgG1 N297A), AM-5 (IgG1 N297A), AM-6 (IgG1 N297A), AM-7 (IgG ₁ N297A), AM-8 (IgG ₁ N297A) and AM-9 (IgG ₁ N297A). Cells tested were Jurkat cells ectopically expressing human TIM-3 (Fig. 9A), Kasumi-3, a human acute myeloid leukemia cell line endogenously expressing TIM-3 (Fig. 9B), human CD8+ T cells stimulated with staphylococcal enterotoxin A (SEA ) (FIG. 9C), SEA stimulated cynomolgus monkey CD8+ T cells (FIG. 9D), and primary human CD14+ myeloid cells (FIG. 9E) and cynomolgus monkey (FIG. 9F).

In FIG. 10A, 10B, 10C and 10D are graphs showing the binding of anti-TIM-3 antibodies or N297A isotype control IgG1 antibody to recombinant human TIM-3 His (rhTIM-3 His), recombinant cynomolgus TIM-3 Fc (rcmTIM-3 Fc). , recombinant mouse TIM-3 Fc (rmTIM-3 Fc), recombinant human TIM-1 His (rhTIM-1 His), recombinant human TIM-4 His (rhTIM-4 His), recombinant human OX40 His (rhOX40 His), recombinant Human GITR-Fc (rhGITR Fc), recombinant human DR3 Fc (rhDR3 Fc), and recombinant human CD137 Fc (rhCD137 Fc) as measured by Luminex assay. The graphs show the dependence of MFI values on antibody concentrations. Anti-TIM-3 antibodies tested in this study include pab2188w (IgG1 N297A) (FIG. 10B), AM-2 (IgG1 N297A) (FIG. 10C), and AM-6 (IgG1 N297A) (FIG. 10D).

In FIG. 11A and 11B are graphs showing the percent binding of recombinant human TIM-3 Fc (FIG. 11A) or recombinant cynomolgus TIM-3 Fc (FIG. 11B) to phosphatidylserine-expressing WR19L cells in the presence of a titrated dose of anti-TIM-3 antibodies or an isotype control. antibodies IgG ₁ N297A. Anti-TIM-3 antibodies tested in this study include pab2188w (IgG1 N297A), AM-2 (IgG1 N297A) and AM-6 (IgG1 N297A).

In FIG. 12A and 12B are bar graphs showing IFNy production induced by anti-TIM-3 antibodies or isotype control IgG1 antibody Ν297Α,πο alone or in combination with anti-PD-1 antibody pembrolizumab in human PBMC from two different donors upon SEA stimulation. TIM-3 antibodies tested include pab2188w ( _IgG1 N297A), AM-1 (IgG1 N297A), AM-2 (IgG1 N297A), AM-3 (IgG1 N297A), AM-4 (IgG1 N297A), AM-5 ( IgG1 N297A), AM-6 (IgG1 N297A), AM-7 (IgG1 N297A) and AM-8 (IgG1 N297A).

In FIG. 13A, 13B, 13C, 13D, 13E and 13F are graphs showing the production of IFN-a or TNF-α by primary tumor-infiltrating lymphocytes (IOLs) induced by anti-TIM-3 antibodies or isotype control antibody IgG1 N297A, alone or in combination with anti-PD-1 antibody pembrolizumab. These IOLs were isolated from non-small cell lung cancer (NSCLC) tumors (FIGS. 13A and 13B), gallbladder adenocarcinoma (FIGS. 13C and 13D), or breast cancer (FIGS. 13E and 13F), and activated with microbeads containing antibodies against

CD3/CD28. Anti-TIM-3 antibodies tested in this study include pab2188w (IgG ₁ N297A), AM-2 (IgG ₁ N297A) and AM-6 (IgG ₁ N297A).

In FIG. 14A, 14B and 14C are graphs showing the percentage of cell survival relative to the untreated control group after incubation with anti-TIM-3 antibody or IgG1 N297A isotype control antibody. In FIG. 14A and 14B show treatment with this antibody in combination with a secondary antibody-drug conjugate aHFc-NC-DM1. Cells tested were either Jurkat cells engineered to overexpress TIM-3 (FIG. 14A) or Kasumi-3 cells, an acute myeloid leukemia cell line endogenously expressing TIM-3 (FIG. 14B). In FIG. 14C shows treatment with the indicated antibody as a monomethylauristatin E (MMAE) conjugate. Anti-TIM-3 antibodies tested in this study include pab2188w (IgG ₁ N297A), AM-2 (IgG ₁ N297A), AM-6 (IgG ₁ N297A); as well as reference antibodies Hum11 (IgG ₄ S228P) and pab1944w (IgG ₁ N297A).

In FIG. 15 is a series of graphs depicting the internalization of TIM-3 in Jurkat cells expressing the HaloTag-TIM-3 fusion protein when incubated with 10 μg/ml of either anti-TIM-3 AM 2 antibody or isotype control antibody, as assessed using confocal fluorescent microscopy on live cells, at different time points (i.e. after 0-3.5 hours). Black dots mark the average fluorescence levels for each state at a certain point in time.

6. Detailed description

The invention provides antibodies that specifically bind to TIM-3 (eg human TIM-3) and antagonize TIM-3 function, eg TIM-3 mediated immune suppression. Also provided are pharmaceutical compositions containing said antibodies, nucleic acids encoding said antibodies, expression vectors and host cells for producing said antibodies, and methods of treating an individual with said antibodies. Antibodies as described herein are particularly suitable for increasing T cell activation in response to an antigen (e.g., tumor antigen or infectious disease antigen) and, accordingly, for treating cancer in an individual, or for treating or preventing an infectious disease in an individual. . In all cases, isolated antibodies are herein also understood to mean antibodies, which may be, but need not be, isolated. In all cases, isolated polynucleotides are herein also understood to mean polynucleotides, which may be, but need not be, isolated. In all cases, antibodies as used herein also include antibodies, which may be, but need not be, isolated. In all cases, polynucleotides in this document also refers to polynucleotides, which may be, but are not necessarily selected.

6.1. Definitions.

In this document, the terms approximately and approximately, when used to modify a numerical value or numerical range, indicate that for deviations of 5-10% from the upper limit of the values (for example, up to values 5-10% higher) and 5-10% relative to the lower limit of values (for example, to values 5-10% lower), the specified value or range continues to correspond to the specified specified value or range.

As used herein, the term TIM-3 refers to T-cell immunoglobulin and mucin domain 3 (also known as T-cell immunoglobulin and mucin domain 3 protein, or hepatitis A virus cell receptor 2 (HAVCR2)), which in humans encodes the HAVCR2 gene. . Swiss-Prot accession number Q8TDQ0-1 provides an example amino acid sequence for human TIM-3. The amino acid sequence of immature human TIM-3 is represented by SEQ ID NO:78. ID NO: 79.

As used herein, the terms antibody and antibodies include full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules containing CDRs, VH regions, or VL regions of antibodies. Examples of antibodies include monoclonal antibodies, recombinant antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies containing two heavy chain molecules and two light chain molecules. antibody light chain monomer, antibody heavy chain monomer, antibody light chain dimer, antibody heavy chain dimer, antibody light chain-antibody heavy chain pair, intrabodies, heteroconjugate antibodies, antibody drug conjugates, single domain antibodies, monovalent antibodies, single chain antibodies or single-chain Fv (scFv), camelized antibodies, affitel, Fab fragments, F(ab') ₂ fragments disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, antibodies against anti- -Id), and

- 15 043100 antigen-binding fragments of any of the listed antibodies. In some embodiments, the antibodies described herein refer to populations of polyclonal antibodies. Antibodies can be of any type (eg IgG, IgE, IgM, IgD, IgA or IgY), any class (eg IgG1, IgG2, IgG ₃ , IgG ₄ , IgA1 or IgA2) or any subclass (eg IgG _2a or IgG _2b ) immunoglobulin molecules. In some embodiments, the antibodies described herein are IgG antibodies, or antibodies of the IgG class (eg, human IgG1 or IgG ₄ ) or IgG subclass. In a specific embodiment, said antibody is a humanized monoclonal antibody. In another specific embodiment, said antibody is a human monoclonal antibody.

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

As used herein, the term CDR, or complementarity determining region, refers to non-contiguous antigen-binding sites found within the variable region of both heavy and light chain polypeptides. These specific areas have been described in the sources: Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest (1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987), as well as MacCallum et al., J. Mol. Biol. 262:732-745 (1996), each of which is incorporated herein by reference in its entirety, wherein said definitions include overlapping amino acid residues or subgroups of amino acid residues when compared to one another. In some embodiments, the term CDR is a CDR as defined in MacCallum et al., J. Mol. Biol. 262:732-745 (1996) and Martin A. Protein Sequence and Structure Analysis of Antibody Variable Domains, in Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In some embodiments, the term CDR is a CDR as defined in Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991). In some embodiments, heavy chain CDRs and light chain CDRs of antibodies are determined using different conventional systems. For example, in some embodiments, heavy chain CDRs are defined according to MacCallum (above) and light chain CDRs are defined according to Kabat (above). CDRH1, CDRH2 and CDRH3 are heavy chain CDRs and CDRL1, CDRL2 and CDRL3 are light chain CDRs.

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

In this document, the terms variable region and variable domain are used interchangeably and are commonly used in the art. By variable region is generally meant a portion of an antibody, usually a portion of a light or heavy chain, typically about 110-120 amino-terminal amino acids, or 110-125 amino-terminal amino acids in a mature heavy chain and approximately 90-115 amino-terminal amino acids in a mature light chain, which differ significantly in the sequences of different antibodies and are involved in the binding and specificity of a particular antibody for a particular antigen. Sequence variability is concentrated in these regions, called complementarity determining regions (CDRs), while the more highly conserved regions of the variable domain are called framework regions (FRs). Regardless of the specific mechanism or theory, it is believed that the light and heavy chain CDR regions are primarily responsible for the interaction of the specified antibody with the antigen and its specificity. In some embodiments, said variable region is a human variable region. In some embodiments, said variable region comprises rodent or mouse CDRs and human framework regions (FRs). In specific embodiments, the variable region is a primate (eg, non-human primate) variable region. In some embodiments, said variable region comprises rodent or mouse CDR regions and primate (eg, non-human primate) framework regions (FRs).

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

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

In this document, the terms constant region and constant domain are used interchangeably and are commonly used in the art. A constant region is a portion of an antibody, such as the carboxy-terminal portion of a light and/or heavy chain, that is not directly involved in binding the antibody to an antigen, but may exhibit various effector functions, such as interaction with an Fc receptor (for example, Fc gamma receptor). The constant region of an immunoglobulin molecule usually contains a more conservative amino acid sequence than the variable domain of an immunoglobulin.

As used herein, the term heavy chain in relation to an antibody may refer to any of the distinct types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant domain. from which the IgA, IgD, IgE, IgG and IgM antibody classes, respectively, are derived, including the IgG subclasses, eg IgG1, IgG2, IgG ₃ and IgG.

As used herein, the term light chain in relation to an antibody may refer to any of the distinct types, eg, kappa (k) or lambda (λ), based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. In specific embodiments, said light chain is a human light chain.

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

Binding affinity generally refers to the cumulative strength of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise indicated, herein, binding affinity refers to true binding affinity, which reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for partner Y can generally be described by a dissociation constant (K _D ). Affinity can be measured and/or expressed using several methods known in the art, including, but not limited to, equilibrium dissociation constant (K _D ) and equilibrium binding constant (K _A ). K _D is calculated based on the quotient koff/kon and K _A is calculated based on the quotient ko _n /k _ff . k _on refers to the rate constant of binding, eg, antibody to antigen, and ko _ff refers to the dissociation rate constant, eg, antibody to antigen. kon and kof can be determined using techniques known to those skilled in the art such as BIAcore® or KinExA. As used herein, lower affinity refers to higher K _D .

As used herein, the terms specifically binds, in particular recognizes, immunospecifically binds, and immunospecifically recognizes are analogous terms in the context of antibodies, and refer to molecules that bind to an antigen (e.g., epitope or immune complex) as such binding is understood. specialist in this field of technology. For example, a molecule that specifically binds to an antigen may bind to other peptides or polypeptides, typically with lower affinity as assessed using, for example, immunoassays, BIAcore®, KinExA 3000 instrument (Sapidyne Instruments, Boise, Idaho), or others. assays known in the art. In a specific embodiment, molecules that specifically bind to an antigen bind to said antigen with a K _A greater than the K _A for non-specific binding of molecules to another antigen by at least 2, 2.5, 3, 4 or more on a logarithmic scale (e.g. , with a factor of 10).

In another specific embodiment, molecules that specifically bind to an antigen do not cross-react with other proteins under similar binding conditions. In another specific embodiment, molecules that specifically bind to TIM3 do not cross-react with other proteins that are not TIM-3 proteins. In a specific embodiment, the present invention provides an antibody that binds to TIM-3 (eg, human TIM-3) with greater affinity compared to binding to another, unrelated antigen. In some embodiments, the present invention provides an antibody that binds to TIM-3 (e.g., human TIM-3) with an affinity that is 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 higher, 70%, 75%, 80%, 85%, 90%, 95% or more than the affinity for another, unrelated antigen, as assessed using, for example, radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay. In a specific embodiment, the degree of binding of an anti-TIM-3 antibody described herein to an unrelated protein that is not a TIM-3 protein is less than 10%, 15%, or 20% of the binding of said antibody to a TIM-3 protein, as assessed using , for example, radioimmunoassay.

As used herein, the term afucosylation or afucosylation in the context of Fc refers to the absence of substantially fucose covalently attached, directly or indirectly, to residue 297 of the human IgG1 Fc region according to the EU numbering system, or the corresponding residue in non-IgG1 or non-IgG1 immunoglobulins. being human IgG1. Accordingly, in

- 17 043100 composition containing a set of afucosylated antibodies, at least 70% of these antibodies are not directly or indirectly fucosylated (for example, through intermediate

Sugars) at residue 297 of the Fc region of these antibodies; in some embodiments, at least 80%, 85%, 90%, 95%, or 99% are not directly or indirectly fucosylated on the remainder

297 Fc regions.

As used herein, an epitope is a term known in the art and refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be represented, for example, by a contiguous amino acid sequence of a polypeptide (linear or continuous epitope); or an epitope may, for example, be composed of two or more non-contiguous regions of a polypeptide or polypeptides (conformational, non-linear, discontinuous or non-contiguous epitope). In some embodiments, said epitope to which an antibody binds can be determined, for example, by NMR spectroscopy, X-ray diffraction crystallography, ELISA, hydrogen/deuterium exchange combined with mass spectrometry (e.g., liquid chromatography/electrospray mass spectrometry). ), chip-based oligopeptide scanning (e.g., limiting peptide conformation using the CLIPS (Chemical Linkage of Peptides onto Scaffolds) method to map discontinuous or conformational epitopes), and/or mutagenesis-based mapping (e.g., mapping with using site-directed mutagenesis). Crystallization for X-ray crystallography can be carried out using any methods known in the art (for example, see sources: 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, each of which incorporated herein in its entirety by reference). Antibody:antigen crystals can be examined using well-known X-ray diffraction techniques and processed using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, for example, sources: Meth Enzymol ( 1985), vol. 114 & 115, eds Wyckoff H.W. et al.; US. 2004/0014194) and BUSTER (see sources: Bricogne G. (1993) Acta Crystallogr D Biol. Crystallogr. 49(Pt 1): 37- 60; Bricogne G. (1997) Meth. Enzymol. 276A: 361-423, ed Carter C. W.; Roversi P. et al., (2000) Acta Crystallogr D Biol. Crystallogr. 56 (Pt 10): 1316-1323, each of which are incorporated herein in their entirety by reference). Studies using mapping based mutagenesis can be carried out using any method known to a person skilled in the art. See, for example, descriptions of mutagenesis techniques, including alanine scan mutagenesis techniques, in Champe M. et al., (1995) J. Biol. Chem. 270: 1388-1394 and Cunningham W.S. & Wells J.A. (1989) Science 244: 1081-1085, each of which is incorporated herein by reference in its entirety. CLIPS (Chemical Linkage of Peptides onto Scaffolds - chemical linkage of peptides on scaffolds) is a technology for presenting one or more peptides in a structurally limited configuration, behaving as functional mimetics of complex protein domains. See, for example, US publications US 2008/0139407 A1 and US 2007/099240 A1, as well as US patent No. 7972993, each of which is incorporated herein in its entirety by reference. In a specific embodiment, an antibody epitope is determined by alanine scan mutagenesis assay. In a specific embodiment, an antibody epitope is determined using hydrogen-deuterium exchange in combination with mass spectrometry. In a specific embodiment, an antibody epitope is determined using CLIPS epitope mapping technology from Pepscan Therapeutics.

As used herein, the term epitope located within a region of human TIM-3, consisting of a specific amino acid sequence or set of amino acid residues, refers to an epitope containing one or more amino acid residues of a given region, where said defined region includes the first given amino acid residue and the last given amino acid residue of the indicated human TIM-3 region. In some embodiments, said epitope contains each of the amino acid residues located within a given region. In some embodiments, one or more additional human TIM-3 amino acid residues outside of a given region bind to an antibody in conjunction with an epitope located within a given region.

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 containing CDRs or TCR variable regions. Examples of TCRs include, but are not limited to, full-length TCRs, antigen-binding fragments of full-length TCRs, soluble TCRs without transmembrane and cytoplasmic regions, single-chain TCRs containing TCR variable regions attached by a flexible linker, TCR chains connected by an engineered disulfide bond, monospecific TCRs, multispecific TCRs ( including bispecific TCRs), fused TCRs, human TCRs, humanized TCRs, chimeric

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TCR, recombinant TCR and synthetic TCR. The term encompasses wild-type TCRs and genetically engineered TCRs (eg, a chimeric TCR containing a chimeric TCR chain containing a first portion from a first species TCR and a second portion from a second species TCR).

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

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

As used herein, the terms treat (treat) and treat (treat) refer to the therapeutic or prophylactic measures described herein. Methods of treatment or treatment include administering an antibody to an individual suffering from, or predisposed to, a disease or disorder, to prevent, treat, delay, reduce the severity or alleviate one or more symptoms of said disease or disorder, or recurrence of the disease or disorder, or to extending the duration of an individual's survival beyond that expected in the absence of such treatment.

As used herein, the term effective amount in the context of administering therapy to an individual refers to the amount of therapy that provides the desired prophylactic or therapeutic effect.

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

Determining the percent identity of two sequences (eg, amino acid sequences or nucleic acid sequences) can be performed using a mathematical algorithm. A specific non-limiting example of a mathematical algorithm used to compare two sequences is provided by the algorithm described in the source: Karlin S & Altschul S.F. (1990) PNAS 87: 22 64-22 68 and modified according to Karlin S. & Altschul S.F. (1993) PNAS 90: 5873-5877; each of these references is incorporated herein in its entirety by reference. Such an algorithm is included in the NBLAST and XBLAST programs (source: Altschul S.F. et al., (1990) J. Mol. Biol. 215: 403, incorporated herein by reference in its entirety). Nucleotide searches in BLAST can be performed using a set of NBLAST nucleotide programming parameters, eg, alignment score=100, wordlength=12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. Searches for BLAST proteins can be performed using a set of XBLAST program parameters, eg, alignment score 50, wordlength=3, to obtain amino acid sequences homologous to the protein molecule described herein. To obtain gapped alignments for comparison, the Gapped BLAST program can be used as described in the source: Altschul S.F. et al., (1997) Nuc. Acids. Res. 25: 3389-3402, which is hereby incorporated by reference in its entirety. Alternatively, the PSI BLAST program can be used to perform an iterative search to detect distant relationships between molecules (ibid.). When using the BLAST, Gapped BLAST, and PSI Blast programs, the default settings of the respective programs (for example, for XBLAST and NBLAST) may apply (see, for example, the National Center for Biotechnology Information (NCBI) website on the Internet available at www.ncbi.nlm.nih.gov). Another specific, non-limiting example of a mathematical algorithm used for sequence comparison is the algorithm described in Myers and Miller, 1988, CABIOS 4:11-17, which is incorporated herein by reference in its entirety. Such an algorithm is included in the ALIGN program (version 2.0), which is included in the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, the following parameters can be used: PAM120 residue substitution weight table, gap extension penalty=12, and sequence gap penalty=4.

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

As used herein, the term internalized or internalized refers to the entry of an antibody into the intracellular compartment of a cell upon binding of the antibody to an antigen expressed on the surface of said cell.

6.2. Antibodies against TIM-3.

According to one aspect of the present invention, antibodies are provided that specifically bind to TIM-3 (eg, human TIM-3) and antagonize TIM-3 function. The amino acid sequences of examples of antibodies are given in table. 1-4 in this document.

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Table 1. Amino acid sequences of examples of anti-TIM-3 antibodies

SEQ ID NO: Description* Amino acid sequence 1 BADD456-2919 CDRH1 SSYAMS 2 BADD456-2919 CDRH2 WVSAISGSGGSTY 3 BADD456-2919 CDRH3 AKGGDYGGNYFD 4 AM-1 CDRH1 KAGQSS 5 AM-2 CDRH1 RQNAWS 6 AM-3 CDRH1 MSGQTS 7 AM-4 CDRH1 GAGQSS 8 AM-5 CDRH1 SAQQAS 9 AM-6 CDRH1 ARNAWS 10 AM-7 CDRH1 RSQQAS eleven AM-8 CDRH1 TTQQAS 12 AM-9 CDRH1 GGQQAS 13 BADD197-1181 CDRL1 RASQSVSSSYLA 14 BADD412-2513 CDRL1 RASQSVSSYLA 15 BADD456-2928CDRL1 RASQGISNYLA 16 BADD466-3169 CDRL1 GASQSVSSSYLA 17 BADD197-1181 CDRL2 GASS RAT 18 BADD456-2928CDRL2 AASTLQS 19 BADD466-3165 CDRL2 GASTRAT 20 BADD466-3166CDRL2 DASS RAT 21 BADD466-3167 CDRL2 DASNRAT 22 BADD197-1181 CDRL3 QQYGSSPLT 23 BADD392-2234CDRL3 QQYGSSPIT EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAM SWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRF 24 BADD456-2919 VH TISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGDY GGNYFDYWGQGTLVTVSS EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAM SWVRRAPGKGLEWVSAISGSGGSTYYADSVKGRF 25 BADD466-3162 VH TISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGDY GGNYFDYWGQGTLVTVSS EVQLVESGGGLVQPRGSLRLSCAASGFTFSSYAM SWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRF 26 BADD466-3163 VH TISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGDY GGNYFDYWGQGTLVTVSS EVQLVESGGGLVQPGGSLRLSCAASGFTFKAGQS SWVRRAPGKGLEWVSAISGSGGSTYYADSVKGRF 27 AM-1-VH TISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGDY GGNYFDYWGQGTLVTVSS

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28 AM-2-VH EVQLVE S GGGLVQPGGS LRL S CAAS GET FRQNAW SWVRRAPGKGLEWVSAISGSGGSTYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGDY GGNYFDYWGQGTLVTVSS 29 AM-3-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFMSGQT SWVRRAPGKGLEWVSAISGSGGSTYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGDY GGNYFDYWGQGTLVTVSS thirty AM-4-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFGAGQS SWVRRAPGKGLEWVSAISGSGGSTYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGDY GGNYFDYWGQGTLVTVSS 31 AM-5-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSAQQA SWVRRAPGKGLEWVSAISGSGGSTYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGDY GGNYFDYWGQGTLVTVSS 32 AM-6-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFARNAW SWVRRAPGKGLEWVSAISGSGGSTYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGDY GGNYFDYWGQGTLVTVSS 33 AM-7-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFRSQQA SWVRRAPGKGLEWVSAISGSGGSTYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGDY GGNYFDYWGQGTLVTVSS 34 AM-8-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFTTQQA SWVRRAPGKGLEWVSAISGSGGSTYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGDY GGNYFDYWGQGTLVTVSS 35 AM-9-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFGGQQA SWVRRAPGKGLEWVSAISGSGGSTYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGDY GGNYFDYWGQGTLVTVSS 36 BADD197-1181 VL EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYL AWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGS GTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGG

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TKVEIK 37 BADD412-2513 VL EIVLTQSPGTLSLSPGERATLSCRASQSVSSYLA WYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSG TDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGT KVEIK 38 BADD456-2928 VL DIQMTQSPSSLSASVGDRVTITCRASQGISNYLA WYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSG TDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGT KVEIK 39 BADD466-3164 VL EIVLTQSPATLSLSPGERATLSCRASQSVSSYLA WYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSG TDFTLTISRLEPEDFAVYYCQQYGSSPLTTFGGGT KVKIK 40 BADD466-3165 VL EIVLTQSPATLSLSPGERATLSCRASQSVSSYLA WYQQKPGQAPRLLIYGASTRATGIPARFSGSGSG TDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGT KVEIK 41 BADD466-3166 VL EIVLTQSPGTLSLSPGERATLSCRASQSVSSYLA WYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSG TDFTLTISRLEPEDFAVYYCQQYGSSPLTTFGGGT KVEIK 42 BADD466-3167 VL KVEIK 43 BADD466-3168 VL EIVLTQSPGTLSLSPGERATLSCRASQSVSSYLA WYQQKPGQAPRLLIYDASNRATGIPARFSGSGSG TDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGT KVEIK 44 BADD466-3169 VL EIVLTQSPATLSLSPGERATLSCGASQSVSSSYL AWYQQKPGLAPRLLIYDASSRATGIPDRFSGSGS GTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGG TKVEIK 45 BADD466-3170 VL EIVLTQSPATLSLSPGERATLSCRASQSVSSYLA WYQQKPGQAPRLLIYDASNRATTGIPARFSGSGSG

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TDFTLTISSLEPEDFAVYYCQQYGSSPLTTFGGGT KVEIK 46 BADD466-3171 VL EIVLTQSPATLSLSPGERATLSCRASQSVSSYLA WYQQKPGQAPRLLIYDASNRATGIPASFSGSGSG TDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGT KVEIK 47 VL BADD466-3172 EIVLTQSPGTLSLSPGERATLSCRASQSVSSYLA WYQQKPGQAPRLLIYGASSRATGIPARFSGSGSG TDFTLTISRLEPEDFAVYYCQQYGSSPITFGGGT KVEIK 48 Consensus subsequence 1 CDRH1 X ₁ X ₂ X ₃ X ₄ X ₅ S, where: Xi is R, S, A, G, K, M or T; X ₂ is Q, S, A, G, R or T; X ₃ is N, Y, G or Q; X ₄ is A or Q; and X ₅ is W, M, A, S or T 49 Consensus subsequence 2 CDRH1 X ₂ X ₂ NAWS, where: X ₄ is R or A; and X ₂ is Q or R 50 Consensus subsequence 3 CDRH1 X ₂ X ₂ GQX ₃ S, where: X ₄ is K, M or G; X ₂ is A or S; and X ₃ is S or T 51 Consensus subsequence 4 CDRH1 X ₂ X ₂ QQAS, where: X ₄ represents S, R, T or G; and X ₂ is A, S, T or G 52 Consensus subsequence CDRL1 X ₄ ASQSVX ₂ SSYLA, where X ₄ is R or G; and X ₂ is absent or is S 53 Consensus subsequence CDRL2 X ₂ ASX ₂ RAT, where: X ₄ is D or G; and X ₂ is N, S or T 54 Consensus subsequence CDRL3 QQYGSSPX ₄ T, where X ₄ is L or I 55 Consensus EVQLVESGGGLVQPX ₁ GSLRLSCAASGFTF

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subsequence vh X ₂ X ₃ X ₄ X ₅ X ₆ SWVRX ₇ APGKGLEWVSAI SGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGDYGGNYF DYWGQGTLVTVSS where: Xx is G or R; X ₂ represents R, S, A, G, K, M or T; X ₃ is Q, S, A, G, R or T; X ₄ is N, Y, G or Q; X ₅ represents A or Q; X ₆ is W, M, A, S or T; and X ₇ is R or Q 56 Consensus subsequence VL EIVLTQSPX ₄ TLSLSPGERATLSCX ₂ ASQSVX ₃ SSYLAWYQQ KPGX ₄ APRLLIYX ₅ ASX ₆ RATGIPX ₇ X ₈ FSGSGSGTDFTLTIS XgLEPEDFAVYYCQQYGSSPXxoTFGGGTKVXnIK where: X ₄ is A or G; X ₂ represents R or G; X ₃ is absent or represents S; X ₄ is Q or L; X ₅ is D or G; X ₆ is N, S or T; X ₇ is A or D; X ₈ is S or R; X ₉ is R or S; X _1o is L or I; and X ₄₁ is E or K 57 Full-length IgGi heavy chain, pab2188 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRRAP GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSWTVPSS SLGTQTYICNV NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF R R K R K D T LMIS RT R E VT S VWD VS HEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSN KALPARIEECTISKAKGQPREPQVYT L R R S REEMT KNQ VS LT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 58 Full-length heavy chain IgGi N297A, pab2188 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRRAP GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSWTVPSS SLGTQTYICNV NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF

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R R K R K D T LMIS RT R E VT S VWD VS HEDPEVKFNWYVDGVEV HNAKTKPREEQYASTYRWSVLTVLHQDWLNGKEYKCKVSN KAL PARIECTISKAKGQ PREPQVYT L R R S REEMT KNQVS LT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 59 Full-length heavy chain IgG ₄ S228P, pab2188 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRRAP GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSSAST KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSWTVPSSSL GTKTYTCNV DHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPK PKDTLMISRTP EVT CVWDVS QED P EVQ FNWYVDGVEVHNA KTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLV KGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSRL TVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 60 Full-length IgGi heavy chain N297A, AM-1 EVQLVE S GGGLVQ R GGS LRL S CAAS GFT FKAGQ S SWVRRAP GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSL SSWTVPSSSLGTQTYICNV NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVT CVWDVS HEDPEVKFNWYVDGVEV HNAKTKPREEQYASTYRWSVLTVLHQDWLNGKEYKCKVSN KAL PAPIEKTISKAKGQ PREPQVYT L P P S REEMT KNQVS LT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 61 Full-length IgGi heavy chain N297A, AM-2 EVQLVESGGGLVQPGGSLRLSCAASGFTFRQNAWSWVRRAP GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSWTVPSS SLGTQTYICNV NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVT CVWDVS HEDPEVKFNWYVDGVEV HNAKTKPREEQYASTYRWSVLTVLHQDWLNGKEYKCKVSN KAL PAPIEKTISKAKGQ PREPQVYT L P P S REEMT KNQVS LT CLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 62 Full-length IgGi heavy chain N297A, AM-3 EVQLVE S GGGLVQ P GGS LRL S CAAS GFT FMS GQT SWVRRAP GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG

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ALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNV NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF R R K R K D T LMIS RT R E VT VWD VS HEDPEVKFNWYVDGVEV HNAKTKPREEQYASTYRWSVLTVLHQDWLNGKEYKCKVSN KALPARIEECTISK AKGQPREPQVYT L Р Р S REEMT KNQVS LT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 63 Full-length IgGi heavy chain N297A, AM-4 EVQLVE S GGGLVQ R GGS LRL S CAAS GFT FGAGQ S SWVRRAP GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSL SSWTVPSSSLGTQTYICNV NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVT WITH VWD VS HEDPEVKFNWYVDGVEV HNAKTKPREEQYASTYRWSVLTVLHQDWLNGKEYKCKVSN KAL PAPIECTIS KAKGQ PREPQVYT L P P S REEMT KNQ VS LT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 64 Full-length IgGi heavy chain N297A, AM-5 EVQLVESGGGLVQPGGSLRLSCAASGFTFSAQASWVRRAP GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSWTVPSS C LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 65 Full-length IgGi heavy chain N297A, AM-6 EVQLVESGGGLVQPGGSLRLSCAASGFTFARNAWSWVRRAP GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSWTVPSSSL GTQTYICNV NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVT C VWD VS HEDPEVKFNWYVDGVEV HNAKTKPREEQYASTYRWSVLTVLHQDWLNGKEYKCKVSN KAL PAPIEKTISKAKGQ PREPQVYT L P P S REEMT KNQVS LT CLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 66 Full-length IgGi heavy chain EVQLVESGGGLVQPGGSLRLSCAASGFTFRSQQASWVRRAP GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQ

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N297A, AM-7 MNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNV NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF R R K R K D T LM IS RT R E VT S VWD VS HEDPEVKFNWYVDGVEV HNAKTKPREEQYASTYRWSVLTVLHQDWLNGKEYKCKVSN KALRAPARIEKTISKAKGQPREPQVYT L R R S REEMT KNQVS LT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG 67 Full-length IgGi heavy chain N297A, AM-8 EVQLVESGGGLVQPGGSLRLSCAASGFTFTTQQASWVRRAP GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYYCAKGGDYGGNYFDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSWTVPSS SLGTQTYICNV NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVT WITH VWD VS HEDPEVKFNWYVDGVEV HNAKTKPREEQYASTYRWSVLTVLHQDWLNGKEYKCKVSN KAL PAPIECTISKAKGQ PREPQVYT L P P S REEMT KNQVS LT CLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 68 Full length heavy chain IgGi N297A, AM-9 EVQLVE S GGGLVQ P GGS LRL S CAAS GFT FGGQQASWVRRAP GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLY SLSSWTVPSSSLGTQTYICNV NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVT C VWD VS HEDPEVKFNWYVDGVEV HNAKTKPREEQYASTYRWSVLTVLHQDWLNGKEYKCKVSN KAL PAPIEKTISKAKGQ PREPQVYT L P P S REEMT KN QVS LT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 69 Full Length Light Chain Sequence BADD466-3171 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPG QAPRLLIYDASNRATGIPASFSGSGSGTDFTLTISRLEPED FAVYYCQQYGSSPLTFGGGTKVEIKRTVAAPSVFIFPPSDE Q L K S GT AS WC L LNN F Y P REAKVQWKVDNALQ S GN S Q E S VT EQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPV TKSFNRGEC 70 Human IgGi Glm3 allotype (without C-terminal lysine) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVT CVWD VS HEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRWSVL TVLHQDWLNGKEYKCK

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VS N KAL PAPIEKTIS KAKGQ PREPQVYT L P P S REEMT KNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G 71 Human IgGi Glm3 allotype ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVT C VWD TVLHQDWLNGKEYKCK VS N KAL PAPIEKTIS KAKGQ PREPQVYT L P P S REEMT KNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQGNVFSCSVMHEALHNHYTQKSLSLSP GK 72 IgGi N297A (no C-terminal lysine) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVT C VWD TVLHQDWLNGKEYKCK VS N KAL PAPIEKTIS KAKGQ PREPQVYT L P P S REEMT KNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G 73 IgGi N297A ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVT C VWD TVLHQDWLNGKEYKCK VS N KAL PAPIEKTIS KAKGQ PREPQVYT L P P S REEMT KNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQGNVFSCSVMHEALHNHYTQKSLSLSP GK 74 IgG ₄ S228P (no C-terminal lysine) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYT CNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLF PPKPKDTLMISRTPEVT C VWD VS QEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRWSVLTVL HQDWLNGKEYKCKVSN KGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 75 _IgG4 S228P ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYT CNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLF

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P P K P K D T LMIS RT P E VT C VWD VS Q E D P EVQ FNWYVD GVEV HNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSN KGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGK 76 Light chain constant region of the human kappa allotype IGKC*01 Kt3 RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLS S PVTKS FNRGEC 77 Light chain constant region of the human kappa allotype IGKC*01 Kt3 (with mutation T109S) RSVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLS S PVTKS FNRGEC 84 IGHV3-23*04 QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAK 85 IGKV1-27*O1 DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPG KVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPED VATYYCQKYNSAP 86 IGKV3-ll*01 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPG QAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPED FAVYYCQQRSNWP 87 IGKV3-20*01 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKP GQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPE DFAVYYCQQYGSSP 88 IGKV3D-20*01 EIVLTQSPATLSLSPGERATLSCGASQSVSSSYLAWYQQKP GLAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPE DFAVYYCQQYGSSP

*Heavy chain CDR regions are defined according to the MacCallum numbering system and light chain CDR regions are defined according to the Kabat numbering system.

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Table 2. Amino acid sequences of heavy chain CDRs of anti-TIM-3 antibody examples

vh CDRH1* SEQ ID NO: CDRH2* SEQ ID NO: CDRH3* SEQ ID NO: BADD456- SSYAMS 1 WVSAISGSGGSTY 2 AKGGDYGGNYFD 3 2919 BADD466- SSYAMS 1 WVSAISGSGGSTY 2 AKGGDYGGNYFD 3 3162 BADD466- SSYAMS 1 WVSAISGSGGSTY 2 AKGGDYGGNYFD 3 3163 AM-1 KAGQSS 4 WVSAISGSGGSTY 2 AKGGDYGGNYFD 3 AM-2 RQNAWS 5 WVSAISGSGGSTY 2 AKGGDYGGNYFD 3 AM-3 MSGQTS 6 WVSAISGSGGSTY 2 AKGGDYGGNYFD 3 AM-4 GAGQSS 7 WVSAISGSGGSTY 2 AKGGDYGGNYFD 3 AM-5 SAQQAS 8 WVSAISGSGGSTY 2 AKGGDYGGNYFD 3 AM-6 ARNAWS 9 WVSAISGSGGSTY 2 AKGGDYGGNYFD 3 AM-7 RSQQAS 10 WVSAISGSGGSTY 2 AKGGDYGGNYFD 3 AM-8 TTQQAS eleven WVSAISGSGGSTY 2 AKGGDYGGNYFD 3 AM-9 GGQQAS 12 WVSAISGSGGSTY 2 AKGGDYGGNYFD 3

*Defined according to the MacCallum numbering system.

Table 3. Amino acid sequences of light chain CDRs of exemplary anti-TIM-3 antibodies

VL CDRL1* SEQ ID NO: CDRL2* SEQ ID NO: CDRL3* SEQ ID NO: BADD197-1181 RASQSVSSSYLA 13 GASSRAT 17 QQYGSSPLT 22 BADD412-2513 RASQSVSSYLA 14 GASSRAT 17 QQYGSSPLT 22 BADD456-2928 RASQGISNYLA 15 AASTLQS 18 QQYGSSPLT 22 BADD466-3164 RASQSVSSYLA 14 GASSRAT 17 QQYGSSPLT 22 BADD466-3165 RASQSVSSYLA 14 GASTRAT 19 QQYGSSPLT 22 BADD466-3166 RASQSVSSYLA 14 DASSRAT 20 QQYGSSPLT 22 BADD466-3167 RASQSVSSYLA 14 DASNRAT 21 QQYGSSPLT 22 BADD466-3168 RASQSVSSYLA 14 DASNRAT 21 QQYGSSPLT 22 BADD466-3169 GASQSVSSSYLA 16 DASSRAT 20 QQYGSSPLT 22 BADD466-3170 RASQSVSSYLA 14 DASNRAT 21 QQYGSSPLT 22 BADD466-3171 RASQSVSSYLA 14 DASNRAT 21 QQYGSSPLT 22 BADD466-3172 RASQSVSSYLA 14 GASSRAT 17 QQYGSSPIT 23

*Determined according to the Kabat numbering system.

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Table 4 Examples of Anti-TIM-3 Antibodies

Antibody Variable region of severe SEQID NO: Variable region of the lung SEQ ID NO: chains chains pab2085 BADD456-2919 24 BADD197-1181 36 pab2088 BADD456-2919 24 BADD456-2928 38 pab2173 BADD466-3163 26 BADD466-3167 42 pab2174 BADD456-2919 24 BADD466-3167 42 pab2175 BADD456-2919 24 BADD466-3171 46 pab2176 BADD456-2919 24 BADD466-3168 43 pab2177 BADD466-3163 26 BADD466-3168 43 pab2178 BADD466-3163 26 BADD466-3171 46 pab2179 BADD466-3163 26 BADD466-3166 41 pab2180 BADD456-2919 24 BADD466-3166 41 pab2181 BADD466-3162 25 BADD466-3164 39 pab2182 BADD456-2919 24 BADD466-3172 47 pab2183 BADD466-3162 25 BADD466-3165 40 pab2184 BADD466-3163 26 BADD466-3172 47 pab2185 BADD466-3162 25 BADD412-2513 37 pab2186 BADD466-3162 25 BADD466-3170 45 pab2187 BADD466-3162 25 BADD466-3169 44 pab2188 BADD466-3162 25 BADD466-3171 46 pab2189 BADD466-3162 25 BADD466-3167 42 pab2190 BADD466-3162 25 BADD466-3166 41 pab2191 BADD466-3162 25 BADD466-3168 43 pab2192 BADD466-3162 25 BADD466-3172 47 AM-1 AM-1-VH 27 BADD466-3171 46 AM-2 AM-2-VH 28 BADD466-3171 46 AM-3 AM-3-VH 29 BADD466-3171 46 AM-4 AM-4-VH thirty BADD466-3171 46 AM-5 AM-5-VH 31 BADD466-3171 46 AM-6 AM-6-VH 32 BADD466-3171 46 AM-7 AM-7-VH 33 BADD466-3171 46 AM-8 AM-8-VH 34 BADD466-3171 46 AM-9 AM-9-VH 35 BADD466-3171 46

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Table 5. Proximate germline genes.

Variable SEQ ID NO of the gene severe area closest gene germinal chain or light germ line lines: chains BADD456-2919 VH IGHV3-23*04 84 BADD466-3162 VH IGHV3-23*04 84 BADD466-3163 VH IGHV3-23*04 84 AM-1-VH IGHV3-23*04 84 AM-2-VH IGHV3-23*04 84 AM-3-VH IGHV3-23*04 84 AM-4-VH IGHV3-23*04 84 AM-5-VH IGHV3-23*04 84 AM-6-VH IGHV3-23*04 84 AM-7-VH IGHV3-23*04 84 AM-8-VH IGHV3-23*04 84 AM-9-VH IGHV3-23*04 84 VL BADD197-1181 IGKV3-20*01 87 VL BADD412-2513 IGKV3-20*01 87 VL BADD456-2928 IGKV1-27*O1 85 VL BADD466-3164 IGKV3-20*01 87 VL BADD466-3165 IGKV3-20*01 87 VL BADD466-3166 IGKV3-20*01 87 VL BADD466-3167 IGKV3-ll*01 86 VL BADD466-3168 IGKV3-20*01 87 VL BADD466-3169 IGKV3D-20*01 88 VL BADD466-3170 IGKV3-ll*01 86 VL BADD466-3171 IGKV3-ll*01 86 VL BADD466-3172 IGKV3-20*01 87

Table 6. Example TIM-3 sequences

SEQ ID NO: Description* Amino acid sequence 78 immature protein Human TIM-3 (Q8TDQ0-1) FRKGDVS L LRDS RLANDLRDSGATIRIGIYIGAGICAGLALA

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LIFGALIFKWYSHSKEKIQNLSLISLANLPPSGLAN AVAEGIRSEENIYTIEENVYEVEEPNEYYCYVSSRQ QPSQPLGCRFAMP 79 Mature human TIM3 protein SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGA CPVFECGNWLRTDERDVNYWTSRYWLNGDFRKGDV SLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVI KPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTL GSLPDINLTQISTLANELRDSRLANDLRDSGATIRI GIYIGAGICAGLAALIFGALIFK WYS HS KEKIQNL SLISLANLPPSGLANAVAEGIRSEENIYTIEENVYE VEEPNEYYCYVSSRQQPSQPLGCRFAMP 10 1 TIM-3 human, F40A SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGA CPVAECGNWLRTDERDVNYWTSRYWLNGDFRKGDV SLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVI KPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTL GSLPDINLTQISTLANELRDSRLANDLRDSGATIRI GIYIGAGICAGLALALIFGALIFK WYS HS KEKIQNL SLISLANLPPSGLANAVAEGIRSEENIYTIEENVYE VEEPNEYYCYVSSRQQPSQPLGCRFAMP 10 2 Human TIM-3 fragment SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGA CPVFECGNWLRTDERDVNYWTSRYWLNGDFRKGDV SLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVI KPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTL GSLPDINLTQISTLANELRDSRLANDLRDSGATIR 93 TIM-3 epitope PVFECGN 94 Epitope TIM-3 VCWGKGACPVFECCGNWL 95 TIM-3 epitope RIQIPGIMND 96 Epitope TIM-3 RIQIPGIMNDEKFNLKL 97 Epitope TIM-3 EKFNLKL 98 TIM-3 epitope PAAPGNLVP 99 Epitope TIM-3 GKGACPVFE 10 0 TIM-3 epitope DFTAAFPR

In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) comprising a VH domain comprising one, two, or all three of the VH domain CDRs shown in Table 1. 1 in this document. In some embodiments, said antibody contains a CDRH1 of one of the VH domains shown in Table 1. 1. According to some variants of implementation of the specified antibody contains CDRH2 of one of the VH domains presented in table. 1. In some embodiments, said antibody contains a CDRH3 of one of the VH domains shown in Table 1. 1.

According to some embodiments of the present invention, an isolated antibody is provided that specifically binds to TIM-3 (e.g., human TIM-3) containing a VL domain containing one, two, or all three CDR regions of the VL domain shown in Table 1. 1 in this document. In some embodiments, said antibody contains a CDRL1 of one of the VL domains shown in Table 1. 1. According to some variants of implementation of the specified antibody contains CDRL2 of one of the VL domains presented in table. 1. According to some variants of implementation of the specified antibody contains CDRL3 of one of the VL domains presented in table. 1.

In some embodiments, CDR antibodies can be defined according to the source: MacCallum RM et al., (1996) J. Mol. Biol. 262: 732-745, incorporated herein in its entirety by reference. See also, for example, source: Martin A. Protein Sequence and Structure

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Analysis of Antibody Variable Domains, in Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001), incorporated herein in its entirety by reference. In some embodiments, the antibody heavy chain CDRs are determined by MacCallum and the antibody light chain CDRs are determined according to another method.

In some embodiments, CDR antibodies may be defined according to Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest (1991), each of which is incorporated herein by reference in its entirety. In some embodiments, antibody light chain CDRs are determined by Kabat and antibody heavy chain CDRs are determined by MacCallum (above).

In some embodiments of the CDR, antibodies can be defined according to the Chothia numbering scheme, which refers to the location of structural loops of the immunoglobulin (see, for example, sources: Chothia C. & Lesk A.M., (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): 17 5-82 and US Pat. Typically, when using the Kabat numbering system, the Chothia CDRH1 loop is located at heavy chain amino acid positions 26-32, 33, or 34, the Chothia CDRH2 loop is located at heavy chain amino acid positions 52-56, and the Chothia CDRH3 loop is located at heavy chain amino acid positions chains 95-102, while the Chothia CDRL1 loop is located at light chain amino acid positions 24-34, the Chothia CDRL2 loop is located at light chain amino acid positions 50-56, and the Chothia CDRL3 loop is located at light chain amino acid positions 89-97. The location of the end of the CDRH1 loop according to Chothia when numbering using the Kabat numbering system varies from H32 to H34 depending on the length of the loop (this is due to the fact that in the Kabat numbering scheme, insertions are located at positions H35A and H35B; in the absence of both 35A and 35B loop ends at position 32; with only 35A, the loop ends at position 33; with both 35A and 35B, the loop ends at position 34).

In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (eg, human TIM-3) comprising VH CDR regions of the Chothia VH domain, Table. 1 in this document. In some embodiments, the invention provides an isolated antibody that specifically binds to TIM-3 (eg, human TIM-3) comprising VL CDR regions of the Chothia VL domain, Table 1. 1 in this document. In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (eg, human TIM-3) comprising Chothia VH CDR regions and Chothia VL CDR regions of the antibody, Table 1. 1 in this document. In some embodiments, antibodies that specifically bind to TIM-3 (eg, human TIM-3) contain one or more CDRs, with the Chothia and Kabat CDR regions having the same amino acid sequence. In some embodiments, the invention provides an isolated antibody that specifically binds to TIM-3 (eg, human TIM-3) and contains combinations of Kabat CDR regions and Chothia CDR regions.

In some embodiments, CDR antibodies may be defined according to the IMGT numbering system as described in Lefranc M-P, (1999) The Immunologist 7: 132-136 and Lefranc M.-P. et al., (1999) Nucleic Acids Res 27: 209-212, each of which is incorporated herein by reference in its entirety. According to the IMGT numbering scheme, CDRH1 is 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 5052; and CDRL3 is located at positions 89-97.

According to some embodiments of the present invention, antibodies are provided that specifically bind to TIM-3 (eg, human TIM-3) and contain the CDR regions of the antibody shown in Table 1. 1 in this document, as assessed using the IMGT numbering system, for example, as described in the sources: Lefranc M.-R. (1999), supra, and Lefranc M.-R. et al., (1999), supra.

In some embodiments, antibody CDR regions can be defined according to the AbM numbering scheme, which refers to AbM hypervariable regions representing a compromise between Kabat CDR regions and Chothia structural loops, and is used in Oxford's AbM antibody modeling software. Molecular (Oxford Molecular Group, Inc.), incorporated herein in its entirety by reference. According to a specific embodiment of the present invention, antibodies are provided that specifically bind to TIM-3 (eg, human TIM-3) and contain the CDR regions of the antibody shown in Table 1. 1 in this document as evaluated using the AbM numbering scheme.

According to some embodiments of the present invention, an isolated antibody is provided that specifically binds to TIM-3 (e.g., human TIM-3), characterized in that said antibody comprises a heavy chain variable region comprising the amino acid sequences of the VH domain CDRH1, CDRH2, and CDRH3 regions, given in SEQ sequences

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ID NOs: 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35, and a light chain variable region containing the amino acid sequences of the VL domain CDRL1, CDRL2, and CDRL3 regions shown in the SEQ ID sequences NO: 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47, each CDR defined according to MacCallum's CDR definition, Kabat definition, Chothia definition, Kabat definition combination and definitions by Chothia, the IMGT numbering system, or the AbM definition.

In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) comprising:

(a) CDRH1 contains the amino acid sequence X ₁ X ₂ X ₃ X ₄ X ₅ S (SEQ ID NO: 48), where

X ₁ represents R, S, A, G, R, M or T,

X ₂ is Q, S, A, G, R or T,

X ₃ is N, Y, G or Q,

X4 is A or Q, and

X ₅ represents W, M, A, S or T; and/or (b) CDRH2 contains the amino acid sequence WVSAISGSGGSTY (SEQ ID NO: 2); and/or (c) CDRH3 contains the amino acid sequence AKGGDYGGNYFD (SEQ ID NO: 3); and/or (d) CDRL1 contains the amino acid sequence X1ASQSVX2SSYLA (SEQ ID NO: 52), where X ₁ is R or G, and

X ₂ is absent or represents S; and/or (e) CDRL2 contains the amino acid sequence X1ASX2RAT (SEQ ID NO: 53), where

X1 is D or G, and

X2 is N, S or T; and/or (f) CDRL3 contains the amino acid sequence QQYGSSPX1T (SEQ ID NO: 54), where X ₁ is L or I.

In some embodiments, CDRH1 comprises the amino acid sequence X ₁ X ₂ NAWS (SEQ ID NO: 49), where X ₁ is R or A; and X ₂ is Q or R. In some embodiments, CDRH1 comprises the amino acid sequence X ₁ X ₂ GQX ₃ S (SEQ ID NO: 50), where: X ₁ is K, M, or G; X ₂ is A or S; and X ₃ is S or T. In some embodiments, CDRH1 comprises the amino acid sequence X1X2QQAS (SEQ ID NO: 51), where X ₁ is S, R, T, or G; and X ₂ is A, S, T, or G. In some embodiments, CDRH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 4-12. In some embodiments, the CDRL1 contains an amino acid sequence selected from the group consisting of the sequences of SEQ ID NOs: 13-16. In some embodiments, the CDRL2 contains an amino acid sequence selected from the group consisting of the sequences of SEQ ID NOs: 17-21. In some embodiments, the CDRL3 contains an amino acid sequence selected from the group consisting of SEQ ID NOS: 22 and 23.

According to some embodiments of the present invention, an isolated antibody is provided that specifically binds to TIM-3 (e.g., human TIM-3), characterized in that said antibody contains a VH domain containing the amino acid sequences CDRH1, CDRH2, and CDRH3, the sequences of SEQ ID NO: 1, 2 and 3; 4, 2 and 3; 5, 2 and 3; 6, 2 and 3; 7, 2 and 3; 8, 2 and 3; 9, 2 and 3; 10, 2 and 3; 11, 2 and 3; or 12, 2 and 3, respectively. According to some embodiments of the present invention, an isolated antibody is provided that specifically binds to TIM-3 (e.g., human TIM-3), characterized in that said antibody contains a VH domain containing the amino acid sequences CDRH1, CDRH2, and CDRH3, the sequences of SEQ ID NO: 1, 2 and 3, respectively. According to some embodiments of the present invention, an isolated antibody is provided that specifically binds to TIM-3 (e.g., human TIM-3), characterized in that said antibody contains a VH domain containing the amino acid sequences CDRH1, CDRH2, and CDRH3, the sequences of SEQ ID NO: 5, 2 and 3, respectively. According to some embodiments of the present invention, an isolated antibody is provided that specifically binds to TIM-3 (e.g., human TIM-3), characterized in that said antibody contains a VH domain containing the amino acid sequences CDRH1, CDRH2, and CDRH3, the sequences of SEQ ID NO: 9, 2 and 3, respectively.

According to some embodiments of the present invention, an isolated antibody is provided that specifically binds to TIM-3 (e.g., human TIM-3), characterized in that said antibody contains a VL domain containing the amino acid sequences CDRL1, CDRL2, and CDRL3, sequences of SEQ ID NO: 13, 17 and 22; 14, 17 and 22; 15, 18 and 22; 14, 19 and 22; 14, 20 and 22; 14, 21 and 22; 16, 20 and 22; or 14, 17 and 23, respectively. According to some embodiments of the present invention, an isolated antibody is provided that specifically binds to TIM-3 (e.g., human TIM-3), characterized in that said antibody contains a VL domain containing the amino acid sequences CDRL1, CDRL2, and CDRL3, sequences of SEQ ID NO: 14, 21 and 22,

- 35 043100 respectively.

In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), characterized in that said antibody comprises a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3 regions, and a light chain variable region. a chain containing CDRL1, CDRL2 and CDRL3 regions, wherein said CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 regions contain amino acid sequences, sequences of SEQ ID NOs: 1, 2, 3, 14, 21 and 22; 4, 2, 3, 14, 21 and 22; 5, 2, 3, 14, 21 and 22; 6, 2, 3, 14, 21 and 22; 7, 2, 3, 14, 21 and 22; 8, 2, 3, 14, 21 and 22; 9, 2, 3, 14, 21 and 22; 10, 2, 3, 14, 21 and 22; 11, 2, 3, 14, 21 and 22; or 12, 2, 3, 14, 21 and 22, respectively. In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), characterized in that said antibody comprises a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3 regions, and a light chain variable region. a chain containing CDRL1, CDRL2 and CDRL3 regions, wherein said CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 regions contain the amino acid sequences of SEQ ID NOs: 1, 2, 3, 14, 21 and 22, respectively. In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), characterized in that said antibody comprises a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3 regions, and a light chain variable region. a chain containing CDRL1, CDRL2 and CDRL3 regions, wherein said CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 regions contain amino acid sequences, sequences of SEQ ID NOs: 5, 2, 3, 14, 21 and 22, respectively. In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), characterized in that said antibody comprises a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3 regions, and a light chain variable region. a chain containing CDRL1, CDRL2 and CDRL3 regions, while said CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 regions contain amino acid sequences, sequences of SEQ ID NOs: 9, 2, 3, 14, 21 and 22.

In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 55. In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) containing a heavy chain variable region containing an amino acid sequence of at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) identical to the amino acid sequence shown in the sequence of SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In some embodiments, said antibody comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NOs: 24, 25, 26, 27, 28, 29, 30 , 31, 32, 33, 34, or 35. In some embodiments, said antibody comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 24. In some embodiments, said antibody comprises a heavy chain variable region comprising an amino acid sequence, SEQ ID NO: 25. In some embodiments, said antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, said antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: : 27. In some embodiments, said antibody comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 28. In some embodiments, said antibody comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 29. According to In some embodiments, said antibody comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 30. In some embodiments, said antibody comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 31. In some embodiments, said the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 32. In some embodiments, said antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 33. In some embodiments, said antibody comprises the variable region heavy chain containing the amino acid sequence of SEQ ID NO: 34. In some embodiments, said antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 35. In some embodiments, the N-terminal glutamate (E) residue of the variable heavy chain regions of an antibody as described herein are replaced with a pyroglutamate (pE) residue.

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In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 56. In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) containing a light chain variable region containing an amino acid sequence 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 or 99%) identical to the amino acid sequence shown in the sequence of SEQ ID NOs: 36, 37, 38, 39 , 40, 41, 42, 43, 44, 45, 46, or 47. In some embodiments, said antibody comprises a light chain variable region comprising an amino acid sequence of SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47. In some embodiments, said antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, said antibody comprises a light chain variable region comprising the amino acid sequence , sequence SEQ ID NO: 37. In some embodiments, said antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 38. In some embodiments, said antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 39. In some embodiments, said antibody comprises a light chain variable region comprising an amino acid sequence of SEQ ID NO: 40. In some embodiments, said antibody comprises a light chain variable region comprising an amino acid sequence of SEQ ID NO: 41. In some embodiments, said antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, said antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 43. In some embodiments, said antibody comprises a light chain variable region comprising an amino acid sequence of SEQ ID NO: 44. In some embodiments, said antibody comprises a light chain variable region comprising an amino acid sequence of SEQ ID NO: 45. In some embodiments, said antibody comprises a variable a light chain region comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, said antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 47. In some embodiments, the N-terminal glutamate residue (E) The light chain variable region of an antibody as described herein is replaced by a pyroglutamate (pE) residue.

In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 55, and a light chain variable region comprising amino acid sequence, sequences of SEQ ID NO: 56. In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) comprising a heavy chain variable region comprising an amino acid sequence 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, or 99%) sequence identical amino acids represented in the sequence of SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35, and a light chain variable region containing an amino acid sequence 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, or 99%) identical to the amino acid sequence represented by in the sequence of SEQ ID NO: 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47. In some embodiments, said antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO : 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35, and a light chain variable region containing the amino acid sequence of SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47. In some embodiments, said antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 24 and 36; 24 and 38; 26 and 42; 24 and 42; 24 and 46; 24 and 43; 26 and 43; 26 and 46; 26 and 41; 24 and 41; 25 and 39; 24 and 47; 25 and 40; 26 and 47; 25 and 37; 25 and 45; 25 and 44; 25 and 46; 25 and 42; 25 and 41; 25 and 43; 25 and 47; 27 and 46; 28 and 46; 29 and 46; 30 and 46; 31 and 46; 32 and 46; 33 and 46; 34 and 46; or 35 and 46, respectively. In some embodiments, said antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 24 and 36, respectively. In some embodiments, said antibody comprises a heavy chain variable region and a light chain variable region, comprising

- 37 043100 amino acid sequences, sequences of SEQ ID NOs: 24 and 38, respectively. In some embodiments, said antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 26 and 42, respectively. In some embodiments, said antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 24 and 42, respectively. In some embodiments, said antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 24 and 46, respectively. In some embodiments, said antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 24 and 43, respectively. In some embodiments, said antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 26 and 43, respectively. In some embodiments, said antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 26 and 46, respectively. In some embodiments, said antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 26 and 41, respectively. In some embodiments, said antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 24 and 41, respectively. In some embodiments, said antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 25 and 39, respectively. In some embodiments, said antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 24 and 47, respectively. In some embodiments, said antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 25 and 40, respectively. In some embodiments, said antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 26 and 47, respectively. In some embodiments, said antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 25 and 37, respectively. In some embodiments, said antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 25 and 45, respectively. In some embodiments, said antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 25 and 44, respectively. In some embodiments, said antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 25 and 46, respectively. In some embodiments, said antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 25 and 42, respectively. In some embodiments, said antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 25 and 41, respectively. In some embodiments, said antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 25 and 43, respectively. In some embodiments, said antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 25 and 47, respectively. In some embodiments, said antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 27 and 46, respectively. In some embodiments, said antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 28 and 46, respectively. In some embodiments, said antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 29 and 46, respectively. In some embodiments, said antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 30 and 46, respectively. In some embodiments, said antibody comprises a severe variable region

- 38 043100 chain and variable region of the light chain, containing the amino acid sequences, the sequence of SEQ ID NO: 31 and 46, respectively. In some embodiments, said antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 32 and 46, respectively. In some embodiments, said antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 33 and 46, respectively. In some embodiments, said antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 34 and 46, respectively. In some embodiments, said antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 35 and 46, respectively. In some embodiments, the N-terminal glutamate (E) residue of the heavy chain variable region of an antibody as described herein is replaced with a pyroglutamate (pE) residue; and/or the N-terminal glutamate residue (E) of the light chain variable region of said antibody is replaced by a pyroglutamate residue (pE).

In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) comprising a heavy chain variable region comprising an amino acid sequence derived from the human IGHV3-23 germline sequence (e.g., IGHV3- 23*04, for example, containing the amino acid sequence of SEQ ID NO: 84). One or more regions selected from framework 1, framework 2, framework 3, CDRH1, and CDRH2 (e.g., two, three, four, or five of these regions) can be derived from the human IGHV3-23 germline sequence (e.g., IGHV323*04, for example, containing the amino acid sequence of SEQ ID NO: 84). In one embodiment, framework 1, framework 2, framework 3, CDRH1, and CDRH2 are derived from a human IGHV3-23 germline sequence (eg, IGHV3-23*04, eg, containing the amino acid sequence of SEQ ID NO: 84).

In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) comprising a light chain variable region comprising an amino acid sequence derived from a human germline sequence selected from the group consisting of IGKV1 -27 (for example, IGKV1-27*01, for example, containing the amino acid sequence of SEQ ID NO: 85), IGKV3-11 (for example, IGKV3-11*01, for example, containing the amino acid sequence of SEQ ID NO: 86), IGKV320 (for example, , IGKV3-20*01, for example, containing the amino acid sequence of SEQ ID NO: 87) and IGKV3D-20 (for example, IGKV3D-20*01, for example, containing the amino acid sequence of SEQ ID NO: 88). One or more regions selected from framework 1, framework 2, framework 3, CDRL1, and CDRL2 (e.g., two, three, four, or five of these regions) may be derived from a human germline sequence selected from the group consisting of IGKV1-27 (for example, IGKV1-27*01, for example, containing the amino acid sequence of SEQ ID NO: 85), IGKV3-11 (for example, IGKV311*01, for example, containing the amino acid sequence of SEQ ID NO: 8 6), IGKV3-2 0 (for example , IGKV3-20*01, for example, containing the amino acid sequence of SEQ ID NO: 87) and IGKV3D-20 (for example, IGKV3D-20*01, for example, containing the amino acid sequence of SEQ ID NO: 88). In one embodiment, the sequences from framework 1, framework 2, framework 3, CDRL1, and CDRL2 are all derived from a human germline sequence selected from the group consisting of IGKV1-27 (e.g., IGKV1-27*01, e.g., containing the amino acid sequence SEQ ID NO: 85), IGKV3-11 (for example, IGKV3-11*01, for example, containing the amino acid sequence of SEQ ID NO: 8 6), IGKV3-2 0 (for example, IGKV3-2 0*01, for example, containing the amino acid sequence SEQ ID NO: 87) and IGKV3D-20 (eg IGKV3D-20*01, eg containing the amino acid sequence of SEQ ID NO: 88).

In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) comprising a heavy chain variable region comprising an amino acid sequence derived from the human IGHV3-23 germline sequence (e.g., IGHV3- 23*04, for example, containing the amino acid sequence of SEQ ID NO: 84), and a light chain variable region containing an amino acid sequence derived from a human germline sequence selected from the group consisting of IGKV1-27 (for example, IGKV1-27*01 , for example, containing the amino acid sequence of SEQ ID NO: 85), IGKV3-11 (for example, IGKV3-11*01, for example, containing the amino acid sequence of SEQ ID NO: 86), IGKV3-20 (for example, IGKV3-20*01, for example , containing the amino acid sequence of SEQ ID NO: 87) and IGKV3D-20 (for example, IGKV3D-20*01, for example, containing the amino acid sequence of SEQ ID NO: 88).

In some embodiments, the present invention provides an isolated antibody that cross-competes for binding to TIM-3 (e.g., human TIM-3) with an antibody comprising the amino acid sequences of the heavy and light chain variable regions, the sequences of SEQ ID NO: 24 and 36; 24 and 38; 26 and 42; 24 and 42; 24 and 46; 24 and 43; 26 and 43; 26 and 46; 26 and 41; 24 and

41; 25 and 39; 24 and 47; 25 and 40; 26 and 47; 25 and 37; 25 and 45; 25 and 44; 25 and 46; 25 and 42; 25 and 41; 25 and 43; 25 and 47; 27 and 46; 28 and 46; 29 and 46; 30 and 46; 31 and 46; 32 and 46; 33 and 46; 34 and 46; or 35 and 46, respectively.

In some embodiments, the present invention provides an isolated antibody that binds to the same or overlapping TIM-3 epitope (e.g., a human TIM-3 epitope) as the antibody described herein, e.g., an antibody comprising the amino acid sequences of the heavy variable regions and light chains, the sequences of SEQ ID NOs: 24 and 36; 24 and 38; 26 and 42; 24 and 42; 24 and 46; 24 and 43; 26 and 43; 26 and 46; 26 and 41; 24 and 41; 25 and 39; 24 and 47; 25 and 40; 26 and 47; 25 and 37; 25 and 45; 25 and 44; 25 and 46; 25 and 42; 25 and 41; 25 and 43; 25 and 47; 27 and 46; 28 and 46; 29 and 46; 30 and 46; 31 and 46; 32 and 46; 33 and 46; 34 and 46; or 35 and 46, respectively. In some embodiments, said antibody epitope can be determined, for example, using NMR spectroscopy, surface plasmon resonance (BIAcore®), X-ray diffraction crystallography, ELISA ELISA, hydrogen-deuterium exchange combined with mass spectrometry (for example, liquid chromatography/ electrospray mass spectrometry), chip-based oligopeptide scanning, and/or mutagenesis-based mapping (eg, mapping using site-directed mutagenesis). Crystallization for X-ray crystallography can be carried out using any methods known in the art (for example, from sources: 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 N E (1997) Structure 5: 1269-1274 McPherson A (1976) J Biol Chem 251: 6300-6303, each of which is included herein in its entirety by reference). Antibody:antigen crystals can be examined using well-known X-ray diffraction techniques and processed using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, for example, sources: Meth Enzymol ( 1985) volumes 114 & 115, eds Wyckoff H. W. et al., and US 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 C. W.; Roversi P. et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10): 1316-1323, each of which is incorporated herein by reference in its entirety). Studies using mapping based mutagenesis can be carried out using any method known to a person skilled in the art. See, for example, descriptions of mutagenesis techniques, including alanine scanning mutagenesis techniques, in Champe M. et al., (1995), supra, and Cunningham, B.C. & Wells J.A. (1989), supra. In a specific embodiment, said antibody epitope is determined by performing an alanine scan mutagenesis assay. In addition, antibodies that recognize and bind to the same or overlapping TIM-3 epitopes (e.g., human TIM-3) can be identified using routine techniques such as immunoassays, for example, by demonstrating the ability of one antibody to block the binding of another. antibodies with the target antigen, ie. competitive binding analysis. Competitive binding assays can also be used to determine if two antibodies have similar binding specificity for an epitope. Competitive binding can be determined in an assay in which the immunoglobulin being tested inhibits the specific binding of a reference antibody to a common antigen such as TIM-3 (eg, human TIM-3). Many types of competitive binding assays are known, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (FIA), competitive sandwich assay (see Stahli C. et al., (1983) Methods Enzymol. 9: 242-253); solid phase direct biotin-avidin FIA (see Kirkland T. N. et al., (1986) J. Immunol. 137: 3614-9); labeled direct solid phase assay, labeled direct sandwich assay (see Harlow E. & Lane D., (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press); solid phase direct RIA using the I-125 label (see Morel G. A. et al., (1988) Mol. Immunol. 25(1): 7-15); solid phase direct biotin-avidin FIA (see Cheung R. C. et al., (1990) Virology 176: 546-52); and direct labeled RIA (see Moldenhauer G. et al., (1990) Scand J. Immunol. 32: 77-82); each of these sources is incorporated herein in its entirety by reference. Typically, such an assay involves the use of a purified antigen (eg, TIM-3, such as human TIM-3) bound to a solid surface, or cells bearing either an unlabeled test immunoglobulin or a labeled reference immunoglobulin. Competitive inhibition can be measured by determining the amount of label bound to a solid surface or cells in the presence of the immunoglobulin being tested. Typically, the immunoglobulin being tested is present in excess. Typically, when a competing antibody is present in excess, it inhibits the specific binding of the reference antibody to the common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more. Competitive binding assays can be performed in a variety of different formats using either labeled antigen or labeled antibody. In a general variant of this assay, the antigen is immobilized on a 96-well plate. Then, using radioactive or enzyme labels, the ability of unlabeled antibodies to block the binding of labeled antibodies to the antigen is measured. Additional information can be found, for example, in Wagener C. et al. (1983) J. Immunol. 130:

23O8-2315; 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, each of which is incorporated herein in its entirety by reference.

In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) comprising a heavy chain comprising the amino acid sequence of SEQ ID NOs: 57, 58, 59, 60, 61, 62 , 63, 64, 65, 66, 67, or 68. In some embodiments, said antibody comprises a heavy chain comprising the amino acid sequence, SEQ ID NO: 57. In some embodiments, said antibody comprises a heavy chain comprising the amino acid sequence, SEQ ID NO: 58. In some embodiments, said antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 59. In some embodiments, said antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 60. In some embodiments, implementation of the specified antibody contains a heavy chain containing the amino acid sequence, the sequence of SEQ ID NO: 61. According to some embodiments, the specified antibody contains a heavy chain containing the amino acid sequence, the sequence of SEQ ID NO: 62. According to some embodiments, the specified antibody contains a heavy chain containing amino acid sequence, sequence SEQ ID NO: 63. In some embodiments, said antibody comprises a heavy chain comprising an amino acid sequence, sequence SEQ ID NO: 64. In some embodiments, said antibody comprises a heavy chain comprising an amino acid sequence, sequence SEQ ID NO: 65. In some embodiments, said antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 66. In some embodiments, said antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 67. In some embodiments, said antibody contains a heavy chain containing the amino acid sequence of SEQ ID NO: 68. In some embodiments, the N-terminal glutamate (E) residue of the heavy chain of an antibody as described herein is replaced by a pyroglutamate (pE) residue.

In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) comprising a light chain containing the amino acid sequence of SEQ ID NO: 69. In some embodiments, an N-terminal glutamate residue (E) The light chain of an antibody as described herein is replaced with a pyroglutamate residue (pE).

In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (eg, human TIM-3) comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 57; and a light chain comprising the amino acid sequence of SEQ ID NO: 69. In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) comprising a heavy chain containing the amino acid sequence of sequence SEQ ID NO: 58; and a light chain comprising the amino acid sequence of SEQ ID NO: 69. In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) comprising a heavy chain containing the amino acid sequence of sequence SEQ ID NO: 59; and a light chain comprising the amino acid sequence of SEQ ID NO: 69. In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) comprising a heavy chain containing the amino acid sequence of sequence SEQ ID NO: 60; and a light chain comprising the amino acid sequence of SEQ ID NO: 69. In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) comprising a heavy chain containing the amino acid sequence of sequence SEQ ID NO: 61; and a light chain comprising the amino acid sequence of SEQ ID NO: 69. In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) comprising a heavy chain containing the amino acid sequence of sequence SEQ ID NO: 62; and a light chain comprising the amino acid sequence of SEQ ID NO: 69. In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) comprising a heavy chain containing the amino acid sequence of sequence SEQ ID NO: 63; and a light chain containing the amino acid sequence of SEQ ID NO: 69. In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) comprising a heavy chain containing amino acid sequence, sequence SEQ ID NO: 64; and a light chain comprising the amino acid sequence of SEQ ID NO: 69. In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) comprising a heavy chain containing the amino acid sequence of sequence SEQ ID NO: 65; and a light chain comprising the amino acid sequence of SEQ ID NO: 69. In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) comprising a heavy chain containing the amino acid sequence of sequence SEQ ID NO: 66; and a light chain comprising the amino acid sequence of SEQ ID NO: 69. In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) comprising a heavy chain containing the amino acid sequence of sequence SEQ ID NO: 67; and a light chain comprising the amino acid sequence of SEQ ID NO: 69. In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) comprising a heavy chain containing the amino acid sequence of sequence SEQ ID NO: 68; and a light chain containing the amino acid sequence of SEQ ID NO: 69. In some embodiments, the N-terminal glutamate (E) residue of the heavy chain of an antibody as described herein is replaced with a pyroglutamate (pE) residue; and/or the N-terminal glutamate residue (E) of the light chain of said antibody is replaced by a pyroglutamate residue (pE).

Any Ig constant region can be used in antibodies as described herein. In some embodiments, said Ig region is a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any immunoglobulin molecule of any class (e.g., IgG1, IgG2, IgG ₃ , IgG ₄ , IgA1 and IgA2), or any subclass (e.g. , _IgG2a and _IgG2b ).

In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) comprising a heavy chain constant region comprising an amino acid sequence of SEQ ID NOs: 70, 71, 72, 73, 74 or 75. In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) comprising a light chain constant region comprising an amino acid sequence of SEQ ID NO: 76 or 77.

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

In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) such that the number of cysteine residues in the hinge region changes (e.g., increases or decreases) as described, e.g. , in US Pat. No. 5,677,425, incorporated herein in its entirety by reference. The number of cysteine residues in the hinge region of the CH1 domain can be changed, for example, to facilitate the assembly of light and heavy chains, or to change (eg, increase or decrease) the stability of the antibody.

In a specific embodiment, one, two, or more amino acid mutations (e.g., substitutions, insertions, or deletions) are introduced into an IgG constant domain or FcRn-binding fragment (preferably an Fc domain or hinge-Fc fragment) to change (e.g., increase or decrease ) the in vivo half-life of said antibody. See, for example, sources: international publications WO 02/060919; No. WO 98/23289; and No. WO 97/34631; and US Pat. No. 5,869,046, No. 6,121,022, No. 6,277,375, and No. 6,165,745, each of which is incorporated herein in its entirety by reference, which provides examples of mutations that alter (eg, decrease or increase) the half-life of an antibody in vivo. In some embodiments, one, two, or more amino acid mutations (e.g., substitutions, insertions, or deletions) are introduced into the IgG constant domain or FcRn-binding fragment (preferably an Fc domain or hinge-Fc fragment) to reduce the half-life of said antibody in vivo. In other embodiments, one, two, or more amino acid mutations (e.g., substitutions, insertions, or deletions) are introduced into the IgG constant domain or FcRn-binding fragment (preferably an Fc domain or hinge-Fc fragment) to increase the in vivo half-life of said antibody. . According to a specific implementation variant, these antibodies may contain one or more amino acid mutations (for example, for

- 42 043100 men) in the second constant domain (CH2) (human IgG1 residues 231-340) and/or the third constant domain (CH3) (human IgG1 residues 341-447) according to the EU numbering system. In a specific embodiment, the constant region of an IgG1 antibody described herein comprises a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254; and replacing threonine (T) with glutamic acid (E) at position 256 according to the EU numbering system. See US Pat. No. 7,658,921, which is incorporated herein by reference in its entirety. This type of IgG mutant, called the YTE mutant, has been shown to exhibit a 4-fold increased half-life compared to wild-type variants of the same antibody (see source: Dall'Acqua W.F. et al., (2006) J. Biol. Chem 281:23514-24, which is incorporated herein by reference in its entirety). In some embodiments, the antibody comprises an IgG constant domain containing one, two, three or more amino acid residue substitutions at positions 251-257, 285-290, 308-314, 385-389 and 428-436 according to the EU numbering system.

In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of an antibody described herein (e.g., into the CH2 domain (human IgG1 residues 231-340) and/or the CH3 domain (residues 341 -447 human IgG1), and/or a hinge region according to the EU numbering system, to increase or decrease the affinity of said antibody for an Fc receptor (e.g., an activated Fc receptor) on the surface of an effector cell. Mutations in the Fc region of an antibody, which reduce or increase the affinity of an antibody for the Fc receptor, and methods for introducing such mutations into the Fc receptor or fragment thereof are known to those skilled in the art. Fc receptors are described, for example, in Smith P et al., (2012) PNAS 109: 6181-6186, US Pat. No. 6,737,056 and International Publications WO 02/060919; and WO 97/34631, each of which is incorporated herein in its entirety by reference.

In a further embodiment, one, two, or more amino acid substitutions are introduced into the Fc region of an IgG constant domain to alter the effector function or functions of said antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 according to the EU numbering system may be substituted for other amino acid residues such that the affinity of said antibody for effector ligand has changed, while maintaining the antigen-binding ability of the original antibody. The effector ligand for which the affinity is changed may be, for example, the Fc receptor or the C1 component of complement. This approach is described in more detail in US patent No. 5624821 and No. 5648260, each of which is incorporated herein in its entirety by reference. In some embodiments, deletion or inactivation (through the use of point mutations or other means) of the constant region domain may reduce binding to the Fc receptor of a circulating antibody, thereby promoting tumor localization. See, for example, US Pat. Nos. 5,585,097 and 8,591,886, each incorporated herein by reference in its entirety, for descriptions of mutations that provide for the deletion or inactivation of the constant domain and thus promote tumor localization. In some embodiments, one or more amino acid substitutions can be introduced into the Fc region of an antibody described herein to remove potential glycosylation sites on the Fc region that may reduce binding to the Fc receptor (see, e.g., Shields R.L. et al., (2001) J Biol Chem 276: 6591-604, which is incorporated herein by reference in its entirety). In various embodiments, one or more of the following mutations can be made in the constant region of an antibody described herein: N297A substitution; N297Q replacement; L235A replacement and L237A replacement; L234A replacement and L235A replacement; replacement for E233P; replacement L234V; replacement L235A; deletion C236; replacement R238A; D265A replacement; A327Q replacement; or replacement of P329A in accordance with the EU numbering system. In some embodiments, a mutation selected from the group consisting of D265A, P329A, and combinations thereof, according to the EU numbering system, can be made in the constant region of an antibody described herein.

In a specific embodiment, an antibody described herein comprises an IgG1 constant domain with the amino acid substitution N297Q or N297A according to the EU numbering system. In one embodiment, an antibody described herein comprises an IgG1 constant domain with a mutation selected from the group consisting of D265A, P329A, and combinations thereof, according to the EU numbering system. In another embodiment, an antibody described herein comprises an IgG1 constant domain with a mutation selected from the group consisting of L234A, L235A, and combinations thereof, according to the EU numbering system. In some embodiments, amino acid residues in the constant region of an antibody described herein at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain according to the EU numbering system are not L, L, and D, respectively. This method is described in detail in international publication WO 14/108483, which is incorporated herein by reference in its entirety. In a specific embodiment, the amino acids corresponding to positions L234, L235, and D265 in the human IgGi heavy chain are F, E, and A; or A, A and A, respectively, according to the EU numbering system.

In some embodiments, one or more amino acids selected from amino acid residues 329, 331, and 322 in the constant region of an antibody described herein, in accordance with the EU numbering system, can be replaced with a different amino acid residue so as to change and/or reduce C1q binding by said antibody, or eliminate complement-dependent cytotoxicity (CDC). This method is described in more detail in US patent No. 6194551 (Idusogie et al.), which is incorporated herein in its entirety by reference. In some embodiments, one or more amino acid residues at amino acid positions 231 to 238 in the N-terminal region of the CH2 domain of an antibody described herein are altered to thereby modify the ability of said antibody to fix complement, according to the EU numbering system. This method is described in more detail in international publication WO 94/29351, which is incorporated herein in its entirety by reference. In some embodiments, the Fc region of an antibody described herein is modified to increase the ability of said antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of said antibody for the Fcy receptor by mutating one or more amino acids (e.g., introducing amino acid substitutions) at the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289 ,290,292,293,294,295,296,298,301,303,305,307,309,312,315,320,322,324,326,327,328,329,330,331,333, 334 , 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439 according to the EU numbering system. This method is described in more detail in international publication WO 00/42072, which is incorporated herein in its entirety by reference.

In some embodiments, an antibody described herein contains an IgG ₄ antibody constant region and serine is replaced with proline at amino acid residue position 228 of the heavy chain, according to the EU numbering system. In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) comprising a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) containing a heavy chain constant region containing the amino acid sequence of SEQ ID NO: 75.

In some embodiments, any of the constant region mutations or modifications described herein may be introduced into one or more heavy chain constant regions of an antibody described herein containing two heavy chain constant regions.

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

In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) and reduces TIM-3 (e.g., human TIM-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 using the methods described herein and/or known one of skill in the art regarding the activity of TIM-3 (e.g., human TIM-3) without the use of any antibody, or with the use of an unrelated antibody (e.g., an antibody that does not bind to TIM-3 (e.g., human TIM-3) specifically). In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) and reduces the activity of TIM-3 (e.g., human TIM-3) by at least about 1.2-fold, 1 ,3x, 1.4x, 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 6 -x, 7x, 8x, 9x, 10x, 15x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x fold or 100 fold as assessed using the methods described herein and/or known to one of skill in the art for TIM-3 (e.g., human TIM-3) activity without any antibody or with an unrelated antibody (eg, an antibody that does not bind to TIM-3 (eg, human TIM-3) specifically). Non-limiting examples of TIM-3 (eg, human TIM-3) activity may include TIM-3 signaling (eg, human TIM-3), TIM-3 (eg, human TIM-3) binding to a TIM-3 ligand (eg, TIM -3 human) (eg, phosphatidylserine) and inhibition of cytokine production (eg, IFN-γ and/or TNF-α). In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (eg, human TIM-3) and deactivates, reduces, or inhibits the activity of TIM-3 (eg, human TIM-3).

- 44 043100

In specific embodiments, the decrease in TIM-3 (eg, human TIM-3) activity is assessed as described in the examples section below.

Specific embodiments of the present invention provide an isolated antibody that specifically binds to TIM-3 (eg, human TIM-3) and reduces the binding of TIM-3 (eg, human TIM-3) to its ligand (eg, phosphatidylserine) by at least approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, as assessed using methods described herein (see examples section below) or known to one of skill in the art regarding the binding of TIM-3 (e.g., human TIM-3) to its ligand (e.g., phosphatidylserine) without the use of any antibody or with the use of an unrelated antibody (eg, an antibody that does not bind to TIM-3 (eg, human TIM-3) specifically). Specific embodiments of the present invention provide an isolated antibody that specifically binds to TIM-3 (eg, human TIM-3) and reduces the binding of TIM-3 (eg, human TIM-3) to its ligand (eg, phosphatidylserine) by at least approximately 1.2x, 1.3x, 1.4x, 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5 -x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20x, 30x, 40x, 50x, 60x, 70x -fold, 80-fold, 90-fold, or 100-fold, as assessed using the methods described herein (see the Examples section, below) or known to one of skill in the art for TIM-3 binding (e.g., TIM- 3 human) with its ligand (eg, phosphatidylserine) without the use of any antibody or with the use of an unrelated antibody (eg, an antibody that does not bind to TIM-3 (eg, human TIM-3) specifically).

Specific embodiments of the present invention provide an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) and increases cytokine (e.g., IFN-γ and/or TNF-α) production by at least about 5, 10 , 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98, or 99% as assessed using the methods described herein (see examples section below) or those known to one of skill in the art regarding cytokine production without the use of any antibody, or with the use of an unrelated antibody (e.g., an antibody that does not specifically bind to TIM-3 (e.g., human TIM-3 )). Specific embodiments of the present invention provide an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) and increases cytokine (e.g., IFN-γ and/or TNF-α) production by at least about 1.2- multiple, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20x, 30x, 40x, 50x, 60x, 70x fold, 80-fold, 90-fold, or 100-fold, as assessed using the methods described herein (see examples section below) or known to one of skill in the art for cytokine production without the use of any antibody or when using an unrelated antibody (eg, an antibody that does not bind to TIM-3 (eg, human TIM-3) specifically).

Specific embodiments of the present invention provide an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) and either alone or in combination with an anti-PD-1 antibody (e.g., pembrolizumab or nivolumab) increases IFN-α production. γ in human peripheral blood mononuclear cells (PBMC) in response to stimulation with staphylococcus enterotoxin A (SEA) at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, or 100x as assessed using the methods described herein document (see examples section below) or known to one of skill in the art regarding IFN-γ production without the use of any antibody, or using an unrelated antibody (e.g., an antibody that does not bind to TIM-3 (e.g., TIM- 3 people) specifically).

In some embodiments, in human peripheral blood mononuclear cells (PBMCs) stimulated with staphylococcus enterotoxin A (SEA) in the presence of an antibody described herein that specifically binds to TIM-3 (e.g., human TIM-3), the production of IFN-γ increases 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 , 4.5x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20x, 30x, 40x, 50x, 60x -fold, 70-fold, 80-fold, 90-fold, or 100-fold relative to PBMC stimulated with SEA alone, without the use of any antibody, or with the use of an unrelated antibody (for example, an antibody that does not bind to TIM-3 ( eg, human TIM-3) specifically), as assessed using the methods described herein (see examples section, below) or known to one of skill in the art.

- 45 043100

Specific embodiments provide an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) and either alone or in combination with an anti-PD-1 antibody (e.g., pembrolizumab or nivolumab) increases IFN-γ production. and/or TNFa in tumor-infiltrating lymphocytes (IOLs) in response to stimulation with anti-CD3 antibody and anti-CD28 antibody at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2- multiple, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 6x, 7x, 8x, 9x, 10x fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold, as assessed using the methods described herein (see examples section below) or known to one of skill in the art regarding the production of IFN-γ and/or TNFa without the use of an antibody that specifically binds to TIM-3 (eg, human TIM-3). In one embodiment, said IOLs are derived from a non-small cell lung cancer (NSCLC) tumor. In another embodiment, said IOLs are derived from a gallbladder adenocarcinoma tumor. In another embodiment, said IOLs are derived from a breast cancer tumor.

In some embodiments, tumor infiltrating lymphocytes (TILs) stimulated with anti-CD3 and anti-CD28 antibodies in the presence of an antibody described herein that specifically binds to TIM-3 (e.g., human TIM-3) increased the production of IFN-γ and/ or TNFa 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- times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold relative to IOLs stimulated with only anti-CD3 and anti-CD28 antibodies without an antibody that specifically binds to TIM-3 (e.g., human TIM-3), by evaluation using the methods described herein (see the examples section below) or known to a person skilled in the art. In one embodiment, said IOLs are derived from a non-small cell lung cancer (NSCLC) tumor. In another embodiment, said IOL is derived from a gallbladder adenocarcinoma tumor. In another embodiment, said IOLs are derived from a breast cancer tumor.

In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (eg, human TIM-3) and internalizes when bound to cells expressing TIM-3 (eg, human TIM-3). In specific embodiments, 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% an antibody described herein is internalized when bound to cells expressing TIM-3 (e.g., human TIM-3), as assessed using the methods described herein (see examples section, below) or known to one of skill in the art technology. In some embodiments, in the presence of an antibody described herein, a lower percentage of cells expressing TIM-3 (e.g., human TIM-3) survive than in the presence of a reference anti-TIM-3 antibody (e.g., human TIM-3), in analysis, including the following steps:

(a) seeding cells expressing TIM-3 (eg, human TIM-3) at a density of 2x10 ⁴ cells per well in a tissue culture plate;

(b) adding aHFc-NC-DM1 and an antibody described herein or a reference anti-TIM-3 antibody (e.g., human TIM-3) at equal concentrations (e.g., 1.5, 4.6, 13.7, 41.2, 123.5, 370, 1111 or 3333 ng/ml) to a final volume of 100 µl/well;

(c) incubation at 37°C and 5% CO ₂ for 72 h;

(d) measuring the survival of cells expressing TIM-3 (eg, human TIM-3); and (e) calculating percent relative cell survival compared to untreated cells expressing TIM-3 (eg, human TIM-3).

In some embodiments, the percentage of cell survival in the presence of an antibody described herein is 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% lower than the percentage of cell survival in the presence of a reference anti-TIM-3 antibody (eg, human TIM-3). In some embodiments, the percent survival of cells in the presence of an antibody described herein is at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2, 5x, 3x, 3.5x, 4x, 4.5x, 5x, 6x, 7x, 8x, 9x, 10x, 15x fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold lower than the percentage of cell survival in the presence of a reference anti-TIM antibody -3 (for example, human TIM-3). In some embodiments, said anti-TIM-3 reference antibody (eg, human TIM-3) is pab1944w (IgG1 N297A). In some embodiments, said reference anti-TIM-3 antibody (eg, human TIM-3) is Hum11 (IgG4 S228P). In some embodiments, said cells expressing TIM-3 (eg, human TIM-3) are Kasumi-3 cells. In some embodiments, said TIM-3 expressing cells (eg, human TIM-3) are Kasumi-3 (ATCC® CRL-2725™) cells. In some embodiments, said cells expressing TIM-3 (eg, human TIM-3) are Jurkat cells engineered to express TIM-3 (eg, human TIM-3).

In some embodiments, a maximum of 50% of cells expressing TIM-3 (e.g., human TIM-3) survive in the presence of the antibody described herein relative to the number of untreated cells expressing TIM-3 (e.g., human TIM-3), in an analysis that includes the following steps:

(a) seeding cells expressing TIM-3 (eg, human TIM-3) at a density of 2x10 ⁴ cells per well in a tissue culture plate;

(b) adding aHFc-NC-DM1 and an antibody described herein at equal concentrations (e.g., 1.5, 4.6, 13.7, 41.2, 123.5, 370, 1111, or 3333 ng/mL) to a final volume of 100 µl/well;

(c) incubation at 37°C and 5% CO ₂ for 72 h;

(d) measuring the survival of cells expressing TIM-3 (eg, human TIM-3); and (e) calculating percent relative cell survival compared to untreated cells expressing TIM-3 (eg, human TIM-3).

In some embodiments, a maximum of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of cells expressing TIM-3 (e.g., human TIM-3) survive in the presence of an antibody described herein, relative to the number of untreated cells expressing TIM-3 (eg, human TIM-3). In some embodiments, aHFc-NC-DM1 and the antibody described herein are added at a concentration of 1111 ng/mL and a maximum of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of cells expressing TIM -3 (eg, human TIM-3) survive in the presence of the antibody described herein relative to the number of untreated cells expressing TIM-3 (eg, human TIM-3). In some embodiments, aHFcNC-DM1 and the antibody described herein are added at a concentration of 1111 ng/mL and a maximum of 50% of cells expressing TIM-3 (e.g., human TIM-3) survive in the presence of the antibody described herein. document, regarding the number of untreated cells expressing TIM-3 (for example, human TIM-3).

In some embodiments, an isolated antibody is provided that specifically binds to TIM-3 (e.g., human TIM-3), characterized in that at least about 5, 10, 15, 20, 25, 3θ, 35, 40, 45 , 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the indicated antibody is internalized when bound to cells expressing TIM-3 (e.g., human TIM-3), as assessed using the methods described herein (see the examples section below) or known to a person skilled in the art, and while the specified antibody contains a CDRH1 containing the amino acid sequence X1X ₂ X ₃ X ₄ X ₅ S (SEQ ID NO: 48), Where

X1 is R, S, A, G, K, M or T,

X ₂ is Q, S, A, G, R or T,

X ₃ is N, Y, G or Q,

X ₄ is A or Q, and

X ₅ is W, M, A, S or T.

In some embodiments, an isolated antibody is provided that specifically binds to TIM-3 (e.g., human TIM-3), characterized in that at least about 5, 10, 15, 20, 25, 3θ, 35, 40, 45 , 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the indicated antibody is internalized when bound to cells expressing TIM-3 (e.g., human TIM-3), as assessed using methods described herein (see the examples section below) or known to a person skilled in the art, and wherein the specified antibody contains a CDRH1 containing the amino acid sequence X1X2NAWS (SEQ ID NO: 49), where

X1 is R or A; And

X ₂ is Q or R.

In some embodiments, an isolated antibody is provided that specifically binds to TIM-3 (e.g., human TIM-3), characterized in that at least about 5, 10, 15, 20, 25, 3θ, 35, 40, 45 , 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the indicated antibody is internalized when bound to cells expressing TIM-3 (e.g., human TIM-3), as assessed using methods described herein (see the examples section below) or known to a person skilled in the art, and wherein the specified antibody contains a CDRH1 containing the amino acid sequence X1X2GQX3S (SEQ ID NO: 50), where

X1 is K, M or G;

X ₂ is A or S; And

X ₃ is S or T.

In some embodiments, an isolated antibody is provided that

- 47 043100 specifically binds to TIM-3 (for example, human TIM-3), characterized in that 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% of the indicated antibody is internalized when bound to cells expressing TIM-3 (e.g., human TIM-3) as assessed using the methods described herein ( see examples section, below) or known to a person skilled in the art, and said antibody contains a CDRH1 containing the amino acid sequence X1X ₂ QQAS (SEQ ID NO: 51), where

X1 is S, R, T or G; And

X ₂ is A, S, T or G.

In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), characterized in that 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% of the indicated antibody is internalized when bound to cells expressing TIM-3 (e.g., human TIM-3), as measured by using the methods described herein (see the examples section below) or known to a person skilled in the art, and while the specified antibody contains a CDRH2 containing the amino acid sequence WVSAISGSGGSTY (SEQ ID NO: 2).

In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), characterized in that 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% of the indicated antibody is internalized when bound to cells expressing TIM-3 (e.g., human TIM-3), as measured by using the methods described herein (see the examples section below) or known to a person skilled in the art, and while the specified antibody contains a CDRH3 containing the amino acid sequence AKGGDYGGNYFD (SEQ ID NO: 3).

In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), characterized in that 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% of the indicated antibody is internalized when bound to cells expressing TIM-3 (e.g., human TIM-3), as measured by using the methods described herein (see the examples section, below) or known to a person skilled in the art, and while the specified antibody contains a CDRL1 containing the amino acid sequence X1ASQSVX2SSYLA (SEQ ID NO: 52), where X ₁ represents R or G, and X ₂ is absent or is S.

In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), characterized in that 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% of the indicated antibody is internalized when bound to cells expressing TIM-3 (e.g., human TIM-3), as measured by using the methods described herein (see the examples section, below) or known to a person skilled in the art, and while the specified antibody contains a CDRL2 containing the amino acid sequence X1ASX2RAT (SEQ ID NO: 53), where

X1 is D or G and X ₂ is N, S or T.

In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), characterized in that 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% of the indicated antibody is internalized when bound to cells expressing TIM-3 (e.g., human TIM-3), as measured by using the methods described herein (see the examples section below) or known to a person skilled in the art, and while the specified antibody contains a CDRL3 containing the amino acid sequence QQYGSSPX1T (SEQ ID NO: 54), where X1 represents L or I .

In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), characterized in that 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% of the indicated antibody is internalized when bound to cells expressing TIM-3 (e.g., human TIM-3), as measured by using the methods described herein (see Examples section below) or known to those skilled in the art, wherein said antibody cross-competes for binding to TIM-3 (e.g., human TIM-3) with an antibody containing amino acid sequences heavy and light chain variable regions, sequence SEQ ID NOs: 24 and 36; 24 and 38; 26 and 42; 24 and 42; 24 and 46; 24 and 43; 26 and 43; 26 and 46; 26 and 41; 24 and 41; 25 and 39; 24 and 47; 25 and 40; 26 and 47; 25 and 37; 25 and 45; 25 and 44; 25 and 46; 25 and 42; 25 and 41; 25 and 43; 25 and 47; 27 and 46; 28 and 46; 29 and 46; 30 and 46; 31 and 46; 32 and 46; 33 and 46; 34 and 46; or 35 and 46, respectively.

In some embodiments, the present invention provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), characterized in that

- 48 043100 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% of the specified antibody is internalized when bound to cells expressing TIM-3 (e.g., human TIM-3), as assessed using the methods described herein (see Examples section, below) or known to one of skill in the art, and said antibody binds to the same or overlapping TIM-3 epitope (e.g., a human TIM-3 epitope) as an antibody described herein, e.g., antibodies containing heavy and light chain variable region amino acid sequences, SEQ ID NOs: 24 and 36; 24 and 38; 26 and 42; 24 and 42; 24 and 46; 24 and 43; 26 and 43; 26 and 46; 26 and 41; 24 and 41; 25 and 39; 24 and 47; 25 and 40; 26 and 47; 25 and 37; 25 and 45; 25 and 44; 25 and 46; 25 and 42; 25 and 41; 25 and 43; 25 and 47; 27 and 46; 28 and 46; 29 and 46; 30 and 46; 31 and 46; 32 and 46; 33 and 46; 34 and 46; or 35 and 46, respectively.

In some embodiments, an isolated antibody is provided that specifically binds to TIM-3 (e.g., human TIM-3), characterized in that at least about 5, 10, 15, 20, 25, 3θ, 35, 40, 45 , 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the indicated antibody is internalized when bound to cells expressing TIM-3 (e.g., human TIM-3), as assessed using methods described herein (see Examples section below) or known to one of skill in the art, wherein said antibody comprises a human IgG heavy chain constant region that is a variant of a wild-type human IgG heavy chain constant region, wherein said the human IgG heavy chain constant region variant binds to the human Fc-gamma receptor with an affinity lower than that of the wild-type human IgG heavy chain constant region to the human Fc-gamma receptor. In some embodiments, said human Fc-gamma receptor is selected from the group consisting of FcyRI, FcyRII, and FcyRIII. In some embodiments, said human IgG heavy chain constant region variant is an IgG1 constant region containing the N297A mutation according to the EU numbering system.

6.3. pharmaceutical compositions.

The present invention provides compositions comprising an anti-TIM-3 antibody (e.g., human TIM-3) as described herein, in the desired purity, in a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co. ., Easton, PA). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate buffer, citrate buffer, and other organic acid buffers; antioxidants, including ascorbic acid and methionine; preservatives (such as ammonium octadecyldimethylbenzyl 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; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; complexes with metals (eg Zn complexes with proteins); and/or non-ionic surfactants such as TWEEN™, Pluronics (™) or polyethylene glycol (PEG).

In a specific embodiment, the pharmaceutical compositions comprise an anti-TIM-3 antibody (eg, human TIM-3) described herein and, optionally, one or more additional prophylactic or therapeutic agents in a pharmaceutically acceptable carrier. In a specific embodiment, the pharmaceutical compositions comprise an effective amount of an antibody described herein and, optionally, one or more additional prophylactic or therapeutic agents in a pharmaceutically acceptable carrier. In some embodiments, said antibody is the only active ingredient included in said pharmaceutical composition. The pharmaceutical compositions described herein may be suitable for use in inhibiting TIM-3 (eg, human TIM-3) activity and treating a condition such as cancer or an infectious disease. In one embodiment, the present invention relates to a pharmaceutical composition of the present invention comprising an anti-TIM-3 antibody of the present invention, for use as a medicament. According to another implementation variant, the present invention relates to a pharmaceutical composition according to the present invention for use in a method of treating cancer or an infectious disease. According to another embodiment, the present invention relates to the use of a pharmaceutical composition according to the present invention for the manufacture of a medicament for the treatment of cancer or an infectious disease.

Pharmaceutically acceptable carriers for use in parenteral formulations include aqueous bases, non-aqueous bases, antimicrobial agents, isotonic agents, buffers, antioxidant

- 49 043100 you, local anesthetics, suspending and dispersing agents, emulsifying agents, complexing or chelating agents and other pharmaceutically acceptable substances. Examples of aqueous bases include sodium chloride injection, Ringer's solution for injection, isotonic dextrose solution for injection, sterile water for injection, Ringer's solution for injection with dextrose and lactate. Non-aqueous parenteral bases include fixed vegetable oils, cottonseed oil, corn oil, sesame oil and peanut oil. Bacteriostatic or fungistatic concentrations of antimicrobial agents may be added to parenteral formulations packaged in multi-dose containers, which include phenols or cresols, mercury-containing agents, benzyl alcohol, chlorobutanol, esters of methyl and propyl phydroxybenzoic acid, thimerosal, benzalkonium chloride, and benzethonium chloride. . Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate buffer. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspension and dispersing agents include sodium carboxymethylcellulose, hydroxypropylmethylcellulose, and polyvinylpyrrolidone. Emulsifying agents include Polysorbate-80 (TWEEN® 80). The metal ion complexing or chelating agent includes EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible bases; and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.

The pharmaceutical composition may be formulated for administration to an individual via any route. Specific examples of administration routes include intranasal, oral, pulmonary, transdermal, intradermal, and parenteral. Parenteral administration, characterized by the use of subcutaneous, intramuscular or intravenous injections, is also contemplated by the present invention. Injectable formulations can be obtained in the form of standard forms: liquid solutions or suspensions; solid forms suitable for dissolution or suspension in liquid prior to injection; or emulsions. Injectable formulations, solutions and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. In addition, if necessary, pharmaceutical compositions for administration may also contain minor amounts of non-toxic co-substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, for example, sodium acetate, sorbitan monolaurate, triethanolaminoleate, and cyclodextrins. .

Formulations for parenteral administration of the antibody include prepared sterile injectable solutions, sterile dry soluble products, such as prepared lyophilized powders for mixing with a solvent immediately before use, including tablets for subcutaneous administration, prepared sterile suspensions for administration, sterile dry insoluble prepared products for mixing with the base immediately before use; and sterile emulsions. Said solutions may be aqueous or non-aqueous.

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

Antibody-containing topical mixtures are prepared as described for topical and systemic administration. The resulting mixture may be a solution, suspension, emulsion, or the like, and may be incorporated into creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, irrigants, sprays, suppositories, dressings, dermal patches, or any other formulation suitable for topical administration.

An anti-TIM-3 antibody (e.g., human TIM-3) described herein can be formulated into a topical aerosol, for example, by inhalation (see, for example, US Pat. steroid delivery aerosols suitable for the treatment of inflammatory diseases, in particular asthma, which are incorporated herein by reference in their entirety). Said respiratory formulations may be in the form of an aerosol or nebulizer solution, or in the form of an ultrafine powder for insufflation, alone or in combination with an inert carrier such as lactose. In this case, the particles of the composition, according to one embodiment, have a diameter of less than 50 microns, according to one embodiment, less than 10 microns.

An anti-TIM-3 antibody (e.g., human TIM-3) described herein may be formulated for topical or topical application or application, e.g., topical application to the skin and mucous membranes, e.g., the eyes, in the form of gels, creams and lotions; for application to the eye, or for intracisternal or intraspinal use. Local administration is carried out by transdermal delivery, as well as by administration to the eyes or mucous membranes, or by inhalation therapy. Nasal solutions of the antibody may also be administered, alone or in combination with other pharmaceutically acceptable excipients.

Transdermal patches, including iontophoretic and electrophoretic devices,

- 50 043100 are well known to those skilled in the art and can be used to administer an antibody.

For example, such patches are described in US Pat. Nos. 6,267,983; 6,261,595;

6010715, 5985317, 5983134, 5948433 and 5860957, each of which is incorporated herein in its entirety by reference.

In some embodiments, a pharmaceutical composition comprising an antibody described herein is a lyophilized powder that can be reconstituted for administration as solutions, emulsions, and other mixtures. It may also be reconstituted and incorporated into a solid formulation or gel. Said lyophilized powder is prepared by dissolving the antibody described herein, or a pharmaceutically acceptable derivative thereof, in a suitable solvent. In some embodiments, said lyophilized powder is sterile. Said solvent may contain a stability aid or other pharmacological component of the powder or reconstituted solution obtained from said powder. Suitable adjuvants include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agent. The solvent may also contain a buffer, such as citrate, sodium or potassium phosphate, or other such buffer known to those skilled in the art, providing, in one embodiment, pH values near neutral. Subsequent sterilizing filtration of the solution and lyophilization under standard conditions known to those skilled in the art provide the desired formulation. In one embodiment, the final solution is dispensed into lyophilization vials. Each vial contains one dose or multiple doses of the compound. The lyophilized powder may be stored under suitable conditions, for example at a temperature of from about 4°C to room temperature. Reconstitution of said lyophilized powder with water for injection provides a formulation for parenteral administration. To reconstitute the lyophilized powder, it is added to sterile water or other suitable vehicle. The exact amount depends on the selected compound. Such an amount can be determined empirically.

The anti-TIM-3 antibodies (e.g., human TIM-3) described herein and other compositions of the present invention may also be formulated to target a particular tissue, receptor, or other area of the body of the individual to be treated. Many such targeting methods are well known to those skilled in the art. All such targeting methods are contemplated by the present invention for use in the proposed compositions. Non-limiting examples of targeting methods are, for example, US Pat. Nos. 6,316,652; 6,274,552; 6,271,359; 004534, 5985307, 5972366, 5900252, 5840674, 5759542 and 5709874 each of which is incorporated herein in its entirety by reference. In a specific embodiment, an antibody described herein is targeted to a tumor.

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

6.4. Methods of application and application.

According to another aspect of the present invention, there is provided a method of treating a subject with antibodies against TIM-3 (eg, human TIM-3) as described herein. Treatment of any disease or disorder in an individual that may benefit from inhibition of TIM-3 (eg, human TIM-3) function may be carried out using antibodies against TIM-3 (eg, human TIM-3) as described herein. Anti-TIM-3 antibodies (eg, human TIM-3) as described herein are particularly suitable for inhibiting immune system tolerance to tumors, and thus can be used as immunotherapy for individuals suffering from cancer. For example, according to some embodiments of the present invention, there is provided a method of increasing T cell activation in response to an antigen in an individual, comprising administering to said individual an effective amount of an anti-TIM-3 antibody (e.g., human TIM-3) or a pharmaceutical composition containing it, as described herein. document. In some embodiments, the present invention provides a method of treating cancer in an individual, comprising administering to said individual an effective amount of an antibody or pharmaceutical composition as described herein. In some embodiments, the present invention provides an antibody or pharmaceutical composition as described herein for use in a method of treating cancer or an infectious disease. In some embodiments, the present invention provides an antibody or pharmaceutical composition as described herein for use as a medicament. According to another embodiment of the present invention, the use of an antibody or a pharmaceutical composition as described herein is provided for the preparation of a medicament for the treatment of cancer or an infectious disease.

Cancers that can be treated with anti-TIM-3 antibodies (e.g., human TIM-3) or pharmaceutical compositions as described herein include, but are not limited to, solid tumor, hematologic cancer (e.g., leukemia, lymphoma , myeloma, for example, multiple myeloma) and metastatic focus. In one embodiment, said cancer is a solid tumor. Examples of solid tumors include malignancies, such as sarcomas and carcinomas, such as adenocarcinomas of various organ systems, such as those affecting the lungs, breast, ovaries, lymphoid tissue, gastrointestinal tract (eg, colon), anus, genitals, and urinary tract (eg, , kidney, urothelium, bladder cells, prostate), pharynx, CNS (eg, brain, nerve or glial cells), head and neck, skin (eg, melanoma), and pancreas, and adenocarcinomas, which include malignancies such as colon cancer, rectal cancer, renal cell carcinoma, liver cancer, lung cancer (eg, non-small cell lung cancer or small cell lung cancer), small intestine cancer, and cancer of the esophagus. Said cancer may be an early, intermediate, late stage, or metastatic cancer. In some embodiments, said cancer is associated with increased PD-1 activity (eg, increased PD-1 expression).

In one embodiment, said cancer is selected from lung cancer (e.g. adenocarcinoma cancer or non-small cell lung cancer (NSCLC) (e.g. NSCLC with squamous and/or non-squamous histology or NSCLC adenocarcinoma)), melanoma (e.g. advanced melanoma), kidney cancer (eg, renal cell carcinoma), liver cancer (eg, renal cell carcinoma), myeloma (eg, multiple myeloma), prostate cancer, breast cancer (eg, breast cancer not expressing one, two, or all of estrogen receptors, progesterone or Her2/neu, e.g., triple-negative breast cancer), ovarian cancer, colorectal cancer, pancreatic cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma (HCHC), anal cancer, gastroesophageal cancer (eg, esophageal squamous cell carcinoma), mesothelioma, nasopharyngeal cancer, thyroid cancer, cervical cancer, epithelial cancer, peritoneal cancer; or a lymphoproliferative disease (eg, post-transplant lymphoproliferative disease).

In one embodiment, said cancer is NSCLC. In one embodiment, said cancer is renal cell carcinoma. In one embodiment, said cancer is ovarian cancer. In a specific embodiment, said ovarian cancer is platinum-refractory ovarian cancer.

In one embodiment, said cancer is a hematological cancer, such as leukemia, lymphoma, or myeloma. In one embodiment, said cancer is a leukemia, e.g., acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), chronic lymphocytic leukemia (CLL), or hairy cell leukemia. In one embodiment, said cancer is a lymphoma, e.g., B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), diffuse activated B-cell (ABC-type) large B-cell lymphoma, diffuse B-cell B-large cell lymphoma germinal center (GCB-type), mantle cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, relapsed non-Hodgkin's lymphoma, refractory non-Hodgkin's lymphoma, recurrent follicular non-Hodgkin's lymphoma, Burkitt's lymphoma, small cell lymphoma, follicular lymphoma, lymphoplasmacytic lymphoma, or extranodal marginal zone lymphoma. In one embodiment, said cancer is a myeloma, such as multiple myeloma.

In another embodiment, said cancer is selected from carcinoma (eg, advanced or metastatic carcinoma), melanoma, or lung carcinoma, eg, non-small cell lung carcinoma.

In one embodiment, said cancer is lung cancer, such as lung adenocarcinoma, non-small cell lung cancer, or small cell lung cancer.

In one embodiment, said cancer is melanoma, such as advanced melanoma. In one embodiment, said cancer is advanced or inoperable melanoma not responding to other therapies. In other embodiments, said cancer is a BRAF-mutated melanoma (eg, BRAF V600 mutation). In other further embodiments, said anti-TIM-3 antibody (e.g., human TIM-3) or a pharmaceutical composition as described herein is administered after treatment with an anti-CTLA-4 antibody (e.g., ipilimumab) with or without the use of a BRAF inhibitor (e.g., vemurafenib or dabrafenib).

In another embodiment, said cancer is a hepatocarcinoma, eg, advanced hepatocarcinoma, with or without viral infection, eg, chronic viral hepatitis.

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In another embodiment, said cancer is prostate cancer, eg advanced prostate cancer.

In another embodiment, said cancer is a myeloma, such as multiple myeloma.

In another embodiment, said cancer is a renal cancer, eg, renal cell carcinoma (RCC) (eg, metastatic RCC, clear cell renal cell carcinoma (SPCC), or papillary renal cell carcinoma).

In yet another embodiment, said cancer is selected from lung cancer, melanoma, kidney cancer, breast cancer, colon cancer, leukemia, or metastatic cancer.

In some embodiments, the present invention provides a method for preventing or treating an infectious disease in an individual, comprising administering to said individual an effective amount of an anti-TIM-3 antibody (e.g., human TIM-3) or a pharmaceutical composition containing it, as described herein. In one embodiment, the present invention provides methods for preventing and/or treating an infection (eg, a viral infection, a bacterial infection, a mycotic infection, a protozoal infection, or a parasitic infection). An infection that is prevented and/or treated according to these methods may be caused by the infectious agent identified herein. In a specific embodiment, an anti-TIM-3 antibody (eg, human TIM-3) described herein, or a composition containing it, is the only active agent that is administered to an individual. In some embodiments, an anti-TIM-3 antibody (e.g., human TIM-3) described herein, or a composition comprising it, is used in combination with an anti-infective intervention (e.g., antiviral, antibacterial, antimycotic, or anthelmintic agents) to treat infectious diseases. Accordingly, according to one implementation variant, the present invention relates to an antibody and/or a pharmaceutical composition according to the present invention for use in a method for preventing and/or treating an infectious disease, while said antibody or said pharmaceutical composition is optionally a single active agent that is administered to an individual, or said antibody or said pharmaceutical composition is used in combination with an anti-infective intervention.

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

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

Protozoal diseases or protozoal infections that can be prevented and/or treated according to the methods described herein include, but are not limited to, leishmaniasis, coccidiosis, trypanosomiasis, schistosomiasis, or malaria. Parasitic diseases or parasitic infections caused by parasites, the prevention and/or treatment of which can be carried out in accordance with the methods described in

- 53 043100 of this document include, but are not limited to, those caused by chlamydia and rickettsia.

Mycotic diseases or mycotic infections, the prevention and/or treatment of which can be carried out in accordance with the methods described herein, include, but are not limited to, caused by Candida infections, zygomycosis, caused by Candida mastitis, progressive disseminated trichosporonosis with latent trichosporonemia, disseminated candidiasis, pulmonary paracoccidioidomycosis, pulmonary aspergillosis, pneumocystis pneumonia, cryptococcal meningitis, coccidioidal meningoencephalitis and cerebrospinal vasculitis, Aspergillus niger infection, Fusarium keratitis, paranasal mycoses, Aspergillus fumigatus endocarditis, tibial dyschondroplasia caused by Candida glabrata vaginitis, oropharyngeal candidiasis, chronic granulomatous disease with X-linked inheritance, tinea pedis, cutaneous candidiasis, mycotic placentitis, disseminated trichosporonosis, allergic bronchopulmonary aspergillosis, mycotic keratitis, Cryptococcus neoformans infection, mycotic peritonitis, Curvularia geniculata infection, staphylococcal endophthalmitis, sporotrichosis and dermatophytosis.

In some embodiments, said methods further comprise administering an additional therapeutic agent to said individual. In some embodiments, said additional therapeutic agent is a chemotherapeutic agent, a radiotherapeutic agent, or a checkpoint-targeting agent. In some embodiments, said chemotherapeutic agent is a hypomethylating agent (eg, azacitidine). In some embodiments, said checkpoint-targeting agent is selected from the group consisting of an anti-CTLA-4 antagonist antibody, an anti-PD-L1 antagonist antibody, an anti-PD-L2 antagonist antibody, an anti-PD-1 antagonist antibody, an anti-PD-1 antibody, an anti-TIM-3 antagonist antibody, an anti-LAG-3 antagonist antibody, an anti-CEACAM1 antagonist antibody, an anti-CD137 agonist antibody, an anti-TIGIT antagonist antibody, an anti-VISTA antagonist antibody, an anti-GITR agonist antibody, and an anti-0X40 agonist antibody.

According to one implementation variant, the present invention relates to an antibody and/or a pharmaceutical composition according to the present invention for use in a method according to the present invention, characterized in that said method further comprises administering an additional therapeutic agent to said individual. In one embodiment, the present invention relates to (a) an antibody and/or pharmaceutical composition of the present invention, and (b) an additional therapeutic agent for use as a medicament. According to one implementation variant, the present invention relates to (a) an antibody and/or pharmaceutical composition according to the present invention; and (b) an additional therapeutic agent for use in a method of treating cancer. According to an additional implementation variant of the present invention relates to a pharmaceutical composition, kit or composite kit containing (a) an antibody and/or pharmaceutical composition according to the present invention; and (b) an additional therapeutic agent. In one embodiment, said additional therapeutic agent is a chemotherapeutic agent, a radiotherapeutic agent, or a checkpoint targeting agent.

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

Additional non-limiting examples of antibodies against PD-1 that can be used in methods of treatment as described herein are described in the following patents and patent applications, all of which are incorporated herein by reference in their entirety for any purpose: US patent No. 6808710; US patent No. 7332582; US patent No. 7488802; US patent No. 8008449; US patent No. 8114845; US patent No. 8168757; US patent No. 8354509; US patent No. 8686119; US patent No. 8735553; US patent No. 8747847; US patent No. 8779105; US Patent No.

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8927697; US patent No. 8993731; US patent No. 9102727; US patent No. 9205148; US Publication No. US 2013/020262 3 A1; US Publication No. US 2013/0291136 A1; US Publication No. US 2014/0044738 A1; US Publication No. US 2014/0356363 A1; US Publication No. US 2016/0075783 A1; and PCT Publication WO 2013/033091 A1; PCT Publication WO 2015/036394 A1; PCT Publication WO 2014/179664 A2; PCT Publication WO 2014/209804 A1; PCT Publication WO 2014/206107 A1; PCT Publication WO 2015/058573 A1; PCT Publication WO 2015/085847 A1; PCT Publication WO 2015/200119 A1; PCT Publication WO 2016/015685 A1; and PCT Publication WO 2016/020856 A1.

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

Non-limiting examples of antibodies against PD-L1 that can be used in methods of treatment as described herein are described in the following patents and patent applications, all of which are incorporated herein by reference in their entirety for any purpose: US patent No. 7943743; US patent No. 8168179; US patent No. 8217149; US patent No. 8552154; US patent No. 8779108; US patent No. 8981063; US patent No. 9175082; US Publication No. US 2010/02 03 05 6 A1; US Publication No. US 2003/0232323 A1; US Publication No. US 2013/0323249 A1; US Publication No. US 2014/0341917 A1; US Publication No. US 2014/0044738 A1; US Publication No. US 2015/0203580 A1; US Publication No. US 2015/0225483 A1; US Publication No. US 2015/0346208 A1; US Publication No. US 2015/0355184 A1; and PCT Publication WO 2014/100079 A1; PCT Publication WO 2014/022758 A1; PCT Publication WO 2014/055897 A2; PCT Publication WO 2015/061668 A1; PCT Publication WO 2015/109124 A1; PCT Publication WO 2015/195163 A1; PCT Publication WO 2016/000619 A1; and PCT Publication WO 2016/030350 A1.

In some embodiments, an anti-TIM-3 antibody (e.g., human TIM-3) as described herein is administered to an individual in combination with a compound that targets an immunomodulatory enzyme or enzymes such as IDO (indolamine-(2,3)-dioxygenase) and/or TDO (tryptophan 2,3-dioxygenase). Accordingly, in one embodiment, said additional therapeutic agent is a compound that targets an immunomodulatory enzyme or enzymes, such as an indolamine-(2,3)-dioxygenase (IDO) inhibitor. In some embodiments, such a compound is selected from the group consisting of epacadostat (Incyte Corp; see, for example, WO 2010/005958, incorporated herein by reference in its entirety), F001287 (Flexus Biosciences/Bristol-Myers Squibb), indoxymod ( NewLink Genetics) and NLG919 (NewLink Genetics). In one embodiment, said compound is epacadostat. In another embodiment, said compound is F001287. In another embodiment, said compound is indoxymod. In another embodiment, said compound is NLG919. In a specific embodiment, an anti-TIM-3 antibody (eg, human TIM-3) as described herein is administered to an individual in combination with an IDO inhibitor for the treatment of cancer. An IDO inhibitor as described herein for use in cancer treatment is presented as a solid dosage form of a pharmaceutical composition such as a tablet, pill or capsule, said pharmaceutical composition comprising an IDO inhibitor and a pharmaceutically acceptable excipient.

Accordingly, an antibody as described herein and an IDO inhibitor as described herein may be administered separately, sequentially, or simultaneously as separate dosage forms. In one embodiment, said antibody is administered parenterally and said IDO inhibitor is administered orally. In specific embodiments, said inhibitor is selected from the group consisting of epacadostat (Incyte Corporation), F001287 (Flexus Biosciences/Bristol-Myers Squibb), indoxymod (NewLink Genetics), and NLG919 (NewLink Genetics). Epacadostat has been described in PCT Publication WO 2010/005958, which is incorporated herein by reference in its entirety for all purposes. In one embodiment, said inhibitor is epacadostat. In another embodiment, said inhibitor is F001287. In another embodiment, said inhibitor is indoxymod. In another embodiment, said inhibitor is NLG919.

In some embodiments, an anti-TIM-3 antibody (eg, human TIM-3) as described herein is administered to an individual in combination with a vaccine. Said vaccine may be, for example, a peptide vaccine, a DNA vaccine or an RNA vaccine. According to

- 55 043100 in some embodiments, said vaccine is a heat shock protein-based antitumor vaccine or a heat shock protein-based pathogen vaccine. In some embodiments, an anti-TIM-3 antibody (e.g., human TIM-3) as described herein is administered to an individual in combination with a vaccine as described in WO 2016/183486 (e.g., a vaccine containing at least one synthetic peptide containing a specific for cancer, a mutation present in a cancer in said individual), incorporated herein in its entirety by reference. In a specific embodiment, an anti-TIM-3 antibody (eg, human TIM-3) as described herein is administered to an individual in combination with a heat shock protein based antitumor vaccine. Heat shock proteins (HSPs) are a family of highly conserved proteins that are widely distributed in all species. As a result of heat shock or other forms of stress, including exposure to toxins, oxidative stress or glucose deprivation, marked induction of significantly higher levels of their expression can occur. Based on molecular weight, five families have been classified: HSP-110, -90, -70, -60 and -28. HSPs deliver immunogenic peptides via a 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 peptides that form complexes capable of inducing tumor-specific immunity. When released from dying tumor cells, the HSP-antigen complexes are taken up by antigen-presenting cells (APCs), while these antigens are processed into peptides that bind MHC class I and class II molecules, which leads to the activation of antitumor CD8+ and CD4+ T cells. The immunity elicited by HSP complexes derived from tumor compounds is directed specifically against the unique repertoire of antigenic peptides expressed by the cancer in each individual. Accordingly, in one embodiment, the present invention relates to (a) an antibody and/or pharmaceutical composition of the present invention, and (b) a vaccine for use as a medicament, for example, for use in a method of treating cancer. According to one implementation variant, the present invention relates to a pharmaceutical composition, kit or composite kit containing (a) an antibody and/or pharmaceutical composition according to the present invention; and (b) a vaccine. In one embodiment, said vaccine is a heat shock protein-based cancer vaccine. In one embodiment, said vaccine is a heat shock protein vaccine against a pathogen.

The peptide-heat shock protein complex (HSPPC) is a protein-peptide complex consisting of heat shock protein in a non-covalent complex with antigenic peptides. HSPPCs elicit both innate and adaptive immune responses. In a specific embodiment, said antigenic peptide or peptides exhibit antigenicity for the cancer being treated. HSPPCs are efficiently taken up by APCs via membrane receptors (primarily CD91) or by binding to Toll-like receptors. Internalization of HSPPC leads to the functional maturation of APC with the production of chemokines and cytokines, leading to the activation of natural killer (NK) cells, monocytes, and a Th1 and Th-2 mediated immune response. In some embodiments, the HSPPCs used in the methods described herein comprise one or more heat shock proteins from the hsp60, hsp70, or hsp90 stress protein families in complex with antigenic peptides. In some embodiments, the HSPPCs comprise hsc70, hsp70, hsp90, hsp110, grp170, gp96, calreticulin; or combinations of two or more of the listed proteins.

In a specific embodiment, said peptide-heat shock protein complex (HSPPC) comprises recombinant heat shock proteins (eg, hsp70 or hsc70) or their peptide-binding domain in complex with recombinant antigenic peptides. Recombinant heat shock proteins can be generated using recombinant DNA technology, for example using the human hsc70 sequence from Dworniczak and Mirault, Nucleic Acids Res. 15:5181-5197 (1987) and GenBank Accession No. P11142 and/or Y00371, each of which is incorporated herein by reference in its entirety. In some embodiments, the Hsp70 sequences are as described in Hunt and Morimoto Proc. Natl. Acad. sci. U.S.A. 82 (19), 6455-6459 (1985), and GenBank Accession No. P0DMV8 and/or M11717, each of which is incorporated herein by reference in its entirety. Antigenic peptides may also be prepared using recombinant DNA techniques known in the art.

In some embodiments, said antigenic peptides comprise a modified amino acid. In some embodiments, said modified amino acid contains a post-translational modification. In some embodiments, said modified amino acid contains a post-translational modification mimetic. In some embodiments, said modified amino acid is Tyr, Ser, Thr, Arg, Lys, or His that has been phosphorylated at the hydroxyl or amino group of the side chain. In some embodiments, said modified amino acid is a mimetic of the amino acid Tyr, Ser, Thr, Arg, Lys, or His that has been phosphorylated at the hydroxyl or amino group of the side chain.

In a specific embodiment, an anti-TIM-3 antibody (e.g., human TIM-3) as described herein is administered to an individual in combination with a heat shock peptide-protein complex (HSPPC), e.g. 96), for the treatment of cancer. HSPPC-96 contains the 96 kDa heat shock protein (Hsp), gp96, complexed with antigenic peptides. HSPPC-96 is a cancer immunotherapy derived from an individual's tumor and contains the antigenic fingerprint of said cancer. In some embodiments, said fingerprint contains unique antigens that are present only on specific cancer cells of said particular individual, and injection of said vaccine is intended to stimulate said individual's immune system to recognize and attack any cells bearing the specific cancer fingerprint. Accordingly, in one embodiment, the present invention relates to an antibody and/or pharmaceutical composition of the present invention in combination with a peptide-heat shock protein complex (HSPPC) for use as a medicament and/or for use in a method of treating cancer.

In some embodiments, said HSPPC, such as HSPPC-96, is derived from the tumor tissue of an individual. In a specific embodiment, said HSPPC (eg, HSPPC-96) is derived from a tumor of the cancer type or metastasis being treated. In another specific embodiment, said HSPPC (eg, HSPPC-96) is autologous to the individual being treated. In some embodiments, said tumor tissue is non-necrotic tumor tissue. In some embodiments, the vaccine regimen uses 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. In some embodiments, following surgical resection, non-necrotic tumor tissue is frozen prior to use in vaccine preparation. In some embodiments, said HSPPC, eg, HSPPC-96, is isolated from tumor tissue using purification techniques, filtered, and an injectable vaccine is obtained. In some embodiments, the individual is administered 6-12 doses of said HSPPC, eg HSPCC-96. In such embodiments, doses of said HSPPC, eg, HSPPC-96, may be administered weekly for the first 4 doses, and then biweekly for 2-8 additional doses.

Additional examples of HSPPCs that can be used in accordance with the methods described herein are presented in the following patents and patent applications, each of which is incorporated herein by reference in its entirety: US Pat. , 6447781 and 6610659.

In some embodiments, an anti-TIM-3 antibody as described herein is administered to an individual in combination with an adjuvant. Depending on the context of treatment, various adjuvants may be used. Non-limiting examples of suitable adjuvants include, but are not limited to, Complete Freund's Adjuvant (CFA), Incomplete Freund's Adjuvant (IFA), Montanide (Montanide ISA, Incomplete Seppic Adjuvant), Ribi's Adjuvant System (RAS), Titer Max, Muramyl Peptides, Adjuvant Formulation SAF (Syntex Adjuvant Formulation), alum (aluminum hydroxide and/or aluminum phosphate), aluminum salt adjuvants, Gerbu® adjuvants, nitrocellulose adsorbed antigen, encapsulated or captured antigen, 3 de-O-acylated monophosphoryl lipid A (3 D-MPL ), immunostimulatory oligonucleotides, Toll-like receptor (TLR) ligands, mannan-binding lectin (MBL) ligands, STING agonists, immunostimulatory complexes such as saponins, Quil A, QS-21, QS-7, ISCOMATRIX and others. Other adjuvants include CpG oligonucleotides and double stranded RNA molecules such as poly(A) and poly(C). Combinations of the above adjuvants may also be used. See, for example, US Pat. Nos. 6,645,495; No. 7029678; and No. 7858589, each of which is incorporated herein in its entirety by reference. In one embodiment, the adjuvant used according to the present invention is QS-21 STIMULON.

In some embodiments, an anti-TIM-3 antibody as described herein is administered to an individual in combination with an additional TCR-containing therapeutic agent. In some embodiments, said additional therapeutic agent is a soluble TCR. In some embodiments, said additional therapeutic agent is a TCR expressing cell. Accordingly, in one embodiment, the present invention provides an antibody and/or a pharmaceutical composition of the present invention in combination with an additional TCR-containing therapeutic agent for use as a medicament and/or for use in a method of treating cancer.

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In some embodiments, an anti-TIM-3 antibody as described herein is administered to an individual in combination with a cell expressing a chimeric antigen receptor (CAR). In some embodiments, said cell is a T cell.

In some embodiments, an anti-TIM-3 antibody as described herein is administered to an individual in combination with a TCR mimic antibody. In some embodiments, said TCR mimic antibody is an antibody that specifically binds to a peptide-MHC complex. Non-limiting examples of TCR-mimicking antibodies are given, for example, in US Pat.

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

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

The amount of an antibody or composition effective in treating and/or preventing a condition depends on the nature of the disease and can be determined using standard clinical techniques.

The exact dosage to be used in the composition also depends on the route of administration, and the severity of the infection or disease caused by it, and should be selected based on the opinion of the attending physician, taking into account the circumstances for each individual. For example, effective doses may also vary depending on the route of administration, the target site, the physiological state of the patient (including age, body weight, and health status), whether the patient is a human or an animal, other medications administered, or whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, but non-human mammals, including transgenic mammals, may also be treated. Dosages for treatment are optimally titrated to optimize safety and efficacy.

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

- 58 043100 vitro, optionally characterized in that said anti-TIM-3 antibody is conjugated to a radionuclide or a detectable label and/or carries the label described herein, and/or characterized by the use of an immunohistological method.

It is understood that the analysis of the level of expression of the TIM-3 protein (for example, human TIM-3) includes a qualitative or quantitative measurement or calculation of the level of TIM-3 protein (for example, human TIM-3) in the first biological sample, either directly (for example, by determining or calculating an absolute protein level) or relative (eg, by comparison with a disease-associated protein level in a second biological sample). The expression level of a TIM-3 polypeptide (eg, human TIM-3) in the first biological sample can be measured or calculated and compared to a standard TIM-3 protein (eg, human TIM-3) level, with the standard derived from the second biological sample. derived from an individual not suffering from the specified disorder, or determined based on average levels in a population of individuals not suffering from the specified disorder. As is known in the art, once a standard level of a TIM-3 polypeptide (eg, human TIM-3) has been determined, it can be repeatedly used as a reference standard. Accordingly, in a further embodiment, the present invention relates to an in vitro method for assaying and/or detecting TIM-3 protein levels, e.g., human TIM3 protein levels, in a biological sample, comprising qualitatively or quantitatively measuring or assessing a TIM-3 protein level, e.g., human TIM-3 protein, in a biological sample using an immunohistological method.

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

An anti-TIM-3 antibody (e.g., human TIM-3) described herein can be used for prognostic, diagnostic, monitoring, and screening applications, including in vitro and in vivo applications, well known and standard in the art based on the present description. Predictive, diagnostic, monitoring, and screening assays and kits for in vitro assessment and investigation of immune system status and/or immune response can be used to predict, diagnose, and monitor patient specimens, including known or suspected immune system dysfunction , or to investigate a suspected or desired immune system response, antigen response, or vaccine response. These assessments and investigations of immune system status and/or immune response are also suitable for use in determining a patient's eligibility for a clinical drug trial or for the administration of a particular chemotherapeutic agent, radiotherapeutic agent, or antibody, including combinations thereof, versus another agent or antibody. . This type of prognostic and diagnostic monitoring and evaluation is already practiced for the use of antibodies against the HER2 protein in breast cancer (HercepTest™, Dako), when this assay is also used to evaluate therapy with Herceptin® antibody in patients. In vivo applications include targeted cell therapy and modulation of the immune system, and radioimaging of the immune response. Accordingly, in one embodiment, the present invention provides an anti-TIM-3 antibody and/or a pharmaceutical composition of the present invention for use as a diagnostic agent. In one embodiment, the present invention provides an anti-TIM-3 antibody and/or a pharmaceutical composition of the present invention for use in a method for predicting, diagnosing and/or monitoring an individual suffering from or suspected of having an immune system dysfunction and/or for investigating suspected or the desired response of the immune system, response to an antigen, or response to a vaccine. According to another embodiment, the present invention relates to the use of an anti-TIM-3 antibody of the present invention for predicting, diagnosing and/or monitoring in an individual suffering from or suspected of suffering from immune system dysfunction, and/or for studying a putative or desired immune system response, a response to antigen or vaccine response by analyzing and/or detecting human TIM-3 protein levels in an in vitro biological sample obtained from said individual.

In one embodiment, an anti-TIM-3 antibody (eg, human TIM-3) can be used in the immunohistochemistry of biopsy specimens. According to one implementation variant of the specified method is a method for use in vitro. In another embodiment, an anti-TIM-3 antibody (e.g., human TIM-3) can be used to detect levels of TIM-3 (e.g., human TIM-3) or levels of cells that contain TIM-3 (e.g., human TIM-3) on the surface of the membranes; these levels can then be associated with certain symptoms of the disease. The anti-TIM-3 antibodies (e.g., human TIM-3) described herein may carry a detectable or functional label, and/or may be conjugated to

- 59 043100 radionuclide or detectable label. When using fluorescent labels, currently available fluorescence-activated microscopic analysis and cell sorting (FACS) methods, or a combination of both methods known in the art, can be used to identify and quantify specific binding participants. The anti-TIM-3 antibodies (eg, human TIM-3) described herein may be fluorescently labeled or conjugated to a fluorescent label. Examples of fluorescent labels include, for example, reactive and conjugated probes such as aminocoumarin, fluorescein, and Texas red, Alexa Fluor dyes, Cy dyes, and DyLight dyes. An anti-TIM- ³ antibody (eg, human TIM-3) may be radioactively labeled or radionuclide, or may be conjugated to a radioactive label or radionuclide, such as the isotopes ³ H, ¹⁴ C, 32 P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr , ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁶⁷ Cu, ⁹⁰ Y, ⁹⁹ Tc, ⁿ¹ In, ¹¹⁷ Lu, ¹²¹ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ 1, ¹⁹⁸ Au, ²¹¹ At, ²¹³ Bi, ²²⁵ Ac and ¹⁸⁶ Re. When using radioactive labels to identify and quantify the specific binding of an anti-TIM-3 antibody (eg, human TIM-3) to TIM-3 (eg, human TIM-3), currently available scoring procedures known in the art can be used. . When the label is an enzyme, detection can be performed using any of the modern colorimetric, spectrophotometric, fluorospectrophotometric, amperometric, or gasometric techniques known in the art. This can be achieved by contacting the sample or control sample with an anti-TIM-3 antibody (eg, human TIM-3) under conditions that allow the formation of a complex between the antibody and TIM-3 (eg, human TIM-3). Any complexes formed by the indicated antibody and TIM-3 (eg, human TIM-3) are detected and compared in the sample and in the control. Given the specific binding of the antibodies described herein to TIM-3 (eg, human TIM-3), these antibodies can be used to specifically detect expression of TIM-3 (eg, human TIM-3) on the surface of cells. The antibodies described herein can also be used for immunoaffinity purification of TIM-3 (eg, human TIM-3). Also provided by the present invention is an assay system that may be in the form of a test kit, kit, or composite kit to quantify the amount of presence of, for example, TIM-3 (e.g., human TIM-3) or TIM-3 complexes (e.g., TIM-3 human)/ TIM-3 ligand (e.g. human TIM-3). Said system, test kit, kit, or composite kit may contain a labeled component, such as a labeled antibody, and one or more additional immunochemical reagents.

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

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

As used herein, an isolated polynucleotide or nucleic acid molecule is a polynucleotide or nucleic acid molecule that has been separated from other nucleic acid molecules present in the natural source (eg, mouse or human) of said nucleic acid molecule. In addition, an isolated nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. For example, the term substantially free includes polynucleotide or nucleic acid molecule formulations containing less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (in particular, less than about 10%) of other material. , for example, cell material, culture medium, other nucleic acid molecules, chemical precursors, and/or other chemicals. In a specific embodiment, the nucleic acid molecule(s) encoding the antibody described herein is isolated or purified.

According to specific aspects of the present invention, polynucleotides are provided that contain nucleotide sequences encoding antibodies that specifically bind to a TIM-3 polypeptide (for example, human TIM-3) and contain an amino acid sequence as described herein, as well as antibodies that compete with such antibodies. for binding to a TIM-3 polypeptide (eg, human TIM-3) (eg, in a dose-dependent manner) or bind to the same epitope as such antibodies.

In certain aspects of the present invention, polynucleotides are provided comprising a nucleotide sequence encoding a light chain or heavy chain of an antibody described herein. These polynucleotides may contain nucleotide sequences encoding a light chain containing the FR and CDR VL of the antibodies described herein (see, for example, Table 1), or nucleotide sequences encoding a heavy chain containing the FR and CDR VH of the antibodies described in this document (see, for example, Table 1).

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

In some embodiments, an optimized polynucleotide sequence encoding an anti-TIM-3 antibody (e.g., human TIM-3) described herein, or a fragment thereof (e.g., VL domain and/or VH domain) can hybridize to an antisense (e.g., complementary) polynucleotide of a non-optimized polynucleotide sequence encoding an anti-TIM-3 antibody (e.g., human TIM-3) described herein, or a fragment thereof (e.g., VL domain and/or VH domain). In specific embodiments, an optimized nucleotide sequence encoding an anti-TIM-3 antibody (e.g., human TIM-3) described herein, or a fragment thereof, hybridizes under very high stringency conditions to an antisense polynucleotide with a non-optimized polynucleotide sequence encoding an anti-TIM antibody. -3 (eg, human TIM-3) described herein, or a fragment thereof. In a specific embodiment, an optimized nucleotide sequence encoding an anti-TIM-3 antibody (e.g., human TIM-3) described herein, or a fragment thereof, hybridizes under very high stringency, medium stringency, or less stringency hybridization conditions with a non-optimized sequence antisense polynucleotide. nucleotides encoding an anti-TIM-3 antibody (eg, human TIM-3) described herein, or a fragment thereof. Information regarding hybridization conditions is provided, for example, in US Published Application US 2005/0048549 (eg, paragraphs 72-73), which is incorporated herein by reference in its entirety.

Said polynucleotides can be obtained and the nucleotide sequence of said polynucleotides determined using any method known in the art. The nucleotide sequences encoding the antibodies described herein, for example, the antibodies described in table. 1 and modified variants of said antibodies can be determined using methods well known in the art, i.e. nucleotide codons known to encode specific amino acids are assembled in such a way as to produce a nucleic acid that encodes said antibody. Such an antibody-encoding polynucleotide can be assembled from chemically synthesized oligonucleotides (for example, as described in Kutmeier G. et al., (1994), BioTechniques 17: 242-6, incorporated herein in its entirety by reference), wherein Briefly, it involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding said antibody, annealing and ligation of said oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody described herein can be obtained from a nucleic acid from a suitable source (eg, hybridoma) using methods well known in the art (eg, PCR and other molecular cloning methods). For example, PCR amplification can be performed using synthetic primers hybridizing to the 3' and 5' ends of a known sequence using

- 61 043100 genomic DNA obtained from hybridoma cells producing the antibody of interest. Such PCR amplification methods can be used to produce nucleic acids containing a sequence encoding the light chain and/or heavy chain of an antibody. Such PCR amplification methods can be used to produce nucleic acids containing a sequence encoding a light chain variable region and/or an antibody heavy chain variable region. The amplified nucleic acids can be cloned into vectors for expression in host cells and for further cloning, for example to produce chimeric and humanized antibodies.

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

DNA encoding anti-TIM-3 antibodies (eg, human TIM-3) described herein can be easily isolated and sequenced using standard procedures (eg, using oligonucleotide probes that are capable of specifically binding to genes encoding heavy and light chains of said anti-TIM-3 antibodies (eg, human TIM-3)). Hybridoma cells can serve as a source of such DNA. Once isolated, said DNA can be placed into expression vectors which are then transfected into host cells such as E. coli cells, COS monkey cells, Chinese Hamster Ovary (CHO) cells (e.g. CHO cells from CHO GS System™ (Lonza)) , or myeloma cells, otherwise not producing immunoglobulin protein, allowing the synthesis of anti-TIM-3 antibodies (eg, human TIM-3) in said recombinant host cells.

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

Said DNA can also be modified, for example, by replacing the coding sequence with human heavy and light chain constant domain sequences instead of mouse sequences, or by covalently attaching all or part of the non-immunoglobulin polypeptide coding sequence to an immunoglobulin coding sequence.

Also provided are polynucleotides that hybridize to polynucleotides that encode an antibody described herein under high, medium, or less stringency hybridization conditions. In specific embodiments, the polynucleotides described herein hybridize under high, medium, or less stringency hybridization conditions with polynucleotides that encode a VH domain and/or a VL domain of the present invention.

Hybridization conditions have been described in the art and known to the person skilled in the art. For example, stringent hybridization may include hybridization to filter-bound DNA in bx sodium chloride/sodium citrate (SSC) at about 45°C followed by one or more washes in 0.2x SSC/0.1% SDS at about 50- 65°C; hybridization

- 62 043100 very stringent conditions may include hybridization to the filter-bound nucleic acid in 6x SSC at about 45°C followed by one or more washes in 0.1 x SSC/0.2% SDS at about 68°C. Hybridization under other stringent conditions is known to those skilled in the art and has been described, for example, in Ausubel F.M. et al., eds., (1989) Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, pp 6.3.1-6.3.6, 2.10.3, which is hereby incorporated by reference in its entirety.

In certain aspects, the present invention provides cells (eg, host cells) expressing (eg, recombinantly) the antibodies described herein that specifically bind to TIM-3 (eg, human TIM-3), and related polynucleotides and expression vectors. The present invention provides vectors (e.g., expression vectors) containing polynucleotides containing nucleotide sequences encoding anti-TIM-3 antibodies (e.g., human TIM-3) or fragments thereof, for recombinant expression in host cells, preferably mammalian cells. . The present invention also provides host cells containing such vectors for recombinant expression of anti-TIM-3 antibodies (eg, human TIM-3) described herein (eg, human or humanized antibodies). According to a specific aspect of the present invention, methods for producing an antibody described herein are provided, comprising expressing such an antibody in a host cell.

Recombinant expression of an antibody described herein (e.g., full length antibody, heavy and/or light chain antibody, or single chain antibody described herein) that specifically binds to TIM-3 (e.g., human TIM-3) involves constructing an expression vector containing a polynucleotide that encodes the specified antibody. Once a polynucleotide encoding an antibody molecule, heavy and/or light chain of an antibody, or fragment thereof (e.g., heavy and/or light chain variable regions) described herein has been obtained, a vector can be obtained to obtain an antibody molecule using recombinant DNA technology, using techniques well known in the art. Accordingly, methods are described herein for producing a protein by expressing a polynucleotide containing the nucleotide coding sequence of an antibody or antibody fragment (eg, light chain or heavy chain). Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequences for an antibody or antibody fragment (eg, light chain or heavy chain) and suitable transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, in vivo synthesis and genetic recombination techniques. Also provided are replicable vectors comprising a nucleotide sequence encoding an antibody molecule as described herein, an antibody heavy or light chain, an antibody heavy or light chain variable region or fragment thereof, or a heavy or light chain CDR operably linked to a promoter. Such vectors may, for example, include a nucleotide sequence encoding the constant region of the antibody molecule (see, for example, international publications WO 86/05807 and WO 89/01036; and US patent No. 5122464, which are incorporated herein by reference in their entirety), and antibody variable regions can be cloned into such a vector to express the entire heavy chain, the entire light chain, or both the entire heavy and the entire light chain.

The expression vector can be transferred into a cell (eg, a host cell) using standard techniques, and the resulting cells can then be cultured using standard techniques to produce an antibody described herein, or a fragment thereof. Accordingly, the present invention provides host cells comprising a polynucleotide encoding an antibody as described herein, or fragments thereof, or a heavy or light chain or fragment thereof, or a single chain antibody as described herein, operably linked to a promoter for expression of such sequences in said host cell. In some embodiments for the expression of double chain antibodies, vectors encoding both heavy and light chains individually can be co-expressed in a host cell to express an entire immunoglobulin molecule, as detailed below. In some embodiments, the host cell comprises a vector containing a polynucleotide encoding both the heavy chain and the light chain of an antibody described herein, or a fragment thereof. In specific embodiments, the host cell contains two different vectors; the first vector contains a polynucleotide encoding the heavy chain or variable region of the heavy chain of the antibody described herein, or a fragment thereof, and the second vector contains a polynucleotide encoding the light chain or variable region of the light chain of the antibody described herein, or a fragment thereof. In other embodiments, the first host cell comprises a first vector containing a polynucleotide encoding the heavy chain or variable region of the heavy chain of an anti

- 63 043100 of the body described herein, or a fragment thereof, and the second host cell contains a second vector containing a polynucleotide encoding the light chain or variable region of the light chain of the antibody described herein. In specific embodiments, a heavy chain/heavy chain variable region expressed by a first cell is linked to a light chain/light chain variable region of a second cell to form an anti-TIM-3 antibody (e.g., human TIM-3) described herein. In some embodiments, the present invention provides a population of host cells comprising such a first host cell and such a second host cell.

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

Various host and expression vector systems can be used to express the antibody molecules described herein (see, for example, US Pat. No. 5,807,715, which is incorporated herein by reference in its entirety). Such host expression systems provide a framework from which coding sequences of interest can be generated and then purified, but are also cells that can, when transformed or transfected with suitable nucleotide coding sequences, express the antibody molecule described herein in situ. . These systems include, but are not limited to, microorganisms such as bacteria (eg, E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences; yeast (eg Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (eg, baculovirus) containing antibody coding sequences; plant cell systems (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 antibody coding sequences; or mammalian cell systems (e.g. COS cells (e.g. COS1 or COS), CHO, BHK, MDCK, HEK 293, NSO, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa and NIH 3T3, HEK-293T, HepG2, SP210 , R1.1, B-W, L-M, BSC1, BSC40, YB/20, and BMT10) carrying recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or mammalian viruses (e.g., adenoviral late promoter; promoter vaccinia virus 7.5K). In a specific embodiment, the cells for expressing the antibodies described herein are CHO cells, eg CHO cells from CHO GS System™ (Lonza). In a specific embodiment, the cells for expressing the antibodies described herein are human cells, eg, human cell lines. In a specific embodiment, the mammalian expression vector is pOptiVEC™ or pcDNA3.3. In a specific embodiment, bacterial cells, such as Escherichia coli, or eukaryotic cells (eg, mammalian cells) are used to express the recombinant antibody molecule, in particular to express the whole recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary (CHO) cells, in combination with a vector such as the core promoter element of human cytomegalovirus immediate-early genes, provide an efficient expression system for antibodies (sources: Foecking M.K. & Hofstetter H. (1986 ) Gene 45: 101-5; and Cockett M. I. et al., (1990) Biotechnology 8(7): 662-7, each of which is incorporated herein by reference in its entirety). In some embodiments, the antibodies described herein are produced by CHO cells or NS0 cells. In a specific embodiment, expression of nucleotide sequences encoding antibodies described herein that specifically bind to TIM-3 (eg, human TIM-3) regulates a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

For bacterial systems, a number of expression vectors can be selected to provide benefits depending on the intended use of the antibody molecule to be expressed. For example, if it is intended to produce large amounts of such an antibody to prepare pharmaceutical compositions of the antibody molecule, it may be desirable to use vectors that direct the expression of high levels of fusion protein products that are easy to purify. Such vectors include, but are not limited to, the E. coli pUR278 expression vector (Ruether U. & Mueller-Hill B. (1983) EMBO J. 2: 1791-1794) wherein the antibody coding sequence

- 64 043100 can be ligated into the vector individually within reading frame with the lac Z coding region such that a fusion protein is produced; pIN vectors (Inouye S. & Inouye M. (1985) Nuc. Acids Res. 13: 3101-3109; Van Heeke G & Schuster SM (1989) J. Biol. Chem. 24: 5503-5509); etc., each of the listed sources is incorporated herein in its entirety by reference. For example, pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione-5-transferase (GST). In general, such fusion proteins are soluble and can be readily purified from lysed cells by adsorption and binding to glutathione-agarose support beads followed by elution in the presence of free glutathione. The pGEX vectors are constructed to include protease cleavage sites for thrombin or factor Xa so that the cloned target gene product can be released from the GST fragment.

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

A number of virus-based expression systems can be used in mammalian host cells. Where an adenovirus is used as the expression vector, the antibody coding sequence of interest can be ligated into an adenoviral transcription/translation control complex, such as a late promoter and triple leader sequence. Said chimeric gene may then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., E1 or E3 region) results in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (e.g., see source: Logan J. & Shenk T. (1984) PNAS 81(12 ): 3655-9, which is hereby incorporated by reference in its entirety). For efficient translation of the inserted antibody coding sequences, specific initiation signals may also be required. These signals include the start codon ATG and adjacent sequences. In addition, the start codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translation control signals and initiation codons can be of various origins, both natural and synthetic. Expression efficiency can be enhanced by the inclusion of suitable transcriptional enhancer elements, transcription terminators, and the like. (See, for example, Bitter G. et al., (1987) Methods Enzymol. 153: 516-544, which is incorporated herein by reference in its entirety).

In addition, a host cell strain can be selected to modulate the expression of the inserted sequences, or to modify and process the gene product in the specific manner desired. Such modifications (eg, glycosylation) and processing (eg, cleavage) of protein products may be important for protein function. Different host cells have characteristic specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure correct modification and processing of the expressed foreign protein. For this purpose, eukaryotic host cells with cellular mechanisms 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, or immunoglobulin chains), CRL7O3O, COS (eg COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB/20, BMT10 and HsS78Bst. In some embodiments, anti-TIM-3 antibodies (eg, human TIM-3) described herein are produced in mammalian cells, such as CHO cells.

In a specific embodiment, the antibodies described herein are characterized by reduced fucose or no fucose. Such antibodies can be obtained using techniques known to the person skilled in the art. For example, these antibodies can be expressed in cells deficient in fucosylation or incapable of fucosylation. According to a specific example, cell lines with a knockout of both alleles of α1,6-fucosyltransferase can be used to generate antibodies with a reduced content of fucose. The Potelligent® system (Lonza) is an example of such a system suitable for use in the preparation of fucose-reduced antibodies.

For long-term, highly productive production of recombinant proteins, cells with stable expression can be obtained. For example, cell lines can be engineered to stably express an anti-TIM-3 antibody (eg, human TIM-3) described herein. In specific embodiments, the cell of the present invention stably expresses a light chain/light chain variable region and a heavy chain/heavy chain variable region that bind to form the antibody described herein.

In certain aspects, instead of using expression vectors that contain viral origins of replication, host cells can be transformed with DNA under the control of suitable expression control elements (e.g., promoter, enhancer sequences, transcription terminators, polyadenylation sites, etc.), and selectable marker. After the introduction of foreign DNA/polynucleotide, the engineered cells can grow for 1-2 days in an enriched medium, after which they are transferred to a selective medium. The selectable marker in the recombinant plasmid confers resistance to selection, allows cells to stably integrate the plasmid into chromosomes and grow to form foci, which in turn can be cloned and propagated to form cell lines. This method may be advantageous when used to construct cell lines that express an anti-TIM-3 antibody (eg, human TIM-3) described herein, or a fragment thereof. Such engineered cell lines may be particularly suitable for use in screening and evaluating compositions that interact directly or indirectly with an antibody molecule.

A number of selection systems can be used, including, but not limited to, herpes simplex virus thymidine kinase genes (Wigler M. et al., (1977) Cell 11(1): 223-32), hypoxanthineguanine phosphoribosyltransferase (Szybalska E.H. & Szybalski W. (1962) PNAS 48(12): 2026-2034) and adenine phosphoribosyl transferase (Lowy I. et al., (1980) Cell 22(3): 817-23), in tk-, hgprt- or aprt- cells, respectively; each of these references is incorporated herein by reference in its entirety. Antimetabolite resistance due to the following genes can also be used as a basis for selection: 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 R.C. & Berg P. (1981) PNAS 78(4): 2072-6); neo which confers resistance to the aminoglycoside G-418 (Wu G.Y. & Wu C.H. (1991) Biotherapy 3: 87-95; Tolstoshev P. (1993) Ann. Rev. Pharmacol. Toxicol. 32: 573-596; Mulligan R. C. (1993 ) Science 260: 926-932 and Morgan R. A. & Anderson W. F. (1993) Ann. Rev. Biochem. 62: 191-217; Nabel G. J. & Feigner P. L. (1993) Trends Biotechnol. 11(5): 2115); and hygro, which confers resistance to hygromycin (Santerre R.F. et al., (1984) Gene 30(1-3): 14756); each of these references is incorporated herein by reference in its entirety. Methods well known in the art of recombinant DNA technology can be routinely used to select the desired recombinant clone; such methods are described, for example, in the sources: Ausubel F.M. et al., (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler M., Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); in chapters 12 and 13 of the manual: Dracopoli N.C. et al., (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); and in Colbere-Garapin F. et al., (1981) J. Mol. Biol. 150: 1-14; each of these references is incorporated herein by reference in its entirety.

Expression levels of an antibody molecule can be increased by vector amplification (for an overview, see Bebbington C.R. & 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), incorporated herein in its entirety by reference). When an amplifiable marker is used in a vector system expressing an antibody, an increase in the level of inhibitor present in the culture of the host cell provides an increase in the copy number of said marker gene. Since the amplified region is associated with the gene of the antibody, the production of said antibody is also increased (source: Crouse G.F. et al., (1983) Mol. Cell Biol. 3: 257-66, incorporated herein in its entirety by reference).

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

Alternatively, a single vector can be used that encodes and is capable of expressing both heavy and light chain polypeptides. In such situations, the light chain must be placed before the heavy chain to avoid excess toxic free heavy chain (sources: Proudfoot N.J. (1986) Nature 322: 562-565; and Kohler G. (1980) PNAS 77: 2197-2199, each of which are incorporated herein in their entirety by reference). The heavy and light chain coding sequences may comprise cDNA or genomic DNA. The expression vector may be monocistronic or multicistronic. A multicistronic nucleic acid construct can

- 66 043100 encode 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, or an amount in the range of 2-5, 5-10, or 10-20 gene/nucleotide sequences. For example, a bicistronic nucleic acid construct may comprise, in that order: a promoter, a first gene (eg, the heavy chain of an antibody described herein), and a second gene (eg, a light chain of an antibody described herein). In such an expression vector, the transcription of both genes can be controlled by a promoter, while translation of mRNA from the first gene can be carried out by a cap-dependent scanning mechanism, and translation of mRNA from the second gene can be carried out by a cap-independent mechanism, for example, IRES.

Once an antibody molecule described herein has been produced by recombinant expression, it can be purified by any method known in the art for purifying an immunoglobulin molecule, e.g., by chromatography (e.g., ion exchange, affinity, in particular based on against a specific antigen after protein A, and size exclusion column chromatography), centrifugation, differential solubility, or using any other standard protein purification technique. In addition, the antibodies described herein may be fused to heterologous polypeptide sequences as described herein or others known in the art to facilitate purification.

In specific embodiments, an antibody described herein is isolated or purified. Typically, an isolated antibody is an antibody substantially free of other antibodies with different antigenic specificity from said isolated antibody. For example, in a specific embodiment, an antibody formulation described herein is substantially free of cellular material and/or chemical precursors. Expression essentially free of cellular material includes antibody formulations wherein said antibody is separated from the cellular components of the cells from which it is isolated or recombinantly produced. Accordingly, an antibody substantially free of cellular material includes antibody formulations containing less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of a heterologous protein (also referred to as contaminant protein herein) and/or antibody variants, for example, containing other post-translational modifications of antibody forms or other distinct antibody variants (eg, antibody fragments). When an antibody is obtained by a recombinant method, it also generally contains essentially no culture medium, i. the culture medium is less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% by volume of the protein formulation. When an antibody is produced by chemical synthesis, it generally contains substantially no chemical precursors or other chemicals, i.e. separated from chemical precursors or other chemicals that are involved in the synthesis of said protein. Accordingly, such antibody formulations contain less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest. In a specific embodiment, the antibodies described herein are isolated or purified.

Antibodies or fragments thereof that specifically bind to TIM-3 (eg, human TIM-3) can be made by any method known in the art for antibody synthesis, eg, chemical synthesis or using recombinant expression techniques. The methods described herein involve, unless otherwise indicated, standard techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization and related fields within this field of technology. These techniques are described, for example, in references herein and are explained in detail in the literature. See, for example, Maniatis T. et al., (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook J. et al., (1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook J. et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel F.M. et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 edition and annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 edition 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, each of which is incorporated herein by reference in its entirety.

In a specific embodiment, an antibody described herein is an antibody (e.g., a recombinant antibody) made, expressed, generated, or isolated by any method, including generating, for example, through synthesis, genetic engineering of DNA sequences. In some embodiments, such an antibody contains sequences (eg, DNA sequences or amino acid sequences) not naturally found in the in vivo animal or mammalian (eg, human) germline antibody repertoire.

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According to one aspect of the present invention, there is provided a method for producing an antibody that specifically binds to TIM-3 (eg, human TIM-3), comprising culturing a cell or host cell as described herein. In one embodiment, said method is carried out in vitro. According to a specific aspect of the present invention, a method is provided for producing an antibody that specifically binds to TIM-3 (e.g., human TIM-3), comprising expression (e.g., recombinant expression) of said antibody using a cell or host cell described herein ( for example, a cell or host cell containing polynucleotides encoding an antibody described herein). In a particular embodiment, said cell is an isolated cell. In a specific embodiment, exogenous polynucleotides have been introduced into said cell. In a specific embodiment, said method further comprises the step of purifying an antibody derived from said cell or host cell.

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

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

In specific embodiments, a monoclonal antibody herein is an antibody produced by a single cell (e.g., a hybridoma or a host cell producing a recombinant antibody), wherein said antibody specifically binds to TIM-3 (e.g., human TIM-3) as assessed by , for example, the results of an ELISA or other antigen binding assay or competitive binding assay known in the art or described in the Examples section of this document. In specific embodiments, the monoclonal antibody may be a chimeric antibody or a humanized antibody. In some embodiments, the monoclonal antibody is a monovalent antibody or a multivalent (eg, bivalent) antibody. In specific embodiments, the monoclonal antibody is a monospecific or multispecific antibody (eg, a bispecific antibody). The monoclonal antibodies described herein may, for example, be prepared by the hybridoma method as described in Kohler G. & Milstein C. (1975) Nature 256:495, incorporated herein in its entirety by reference, or may, for example, be isolated from phage libraries, for example, using the techniques described herein. Other methods for obtaining clonal cell lines and thus expressed monoclonal antibodies are well known in the art (see, for example, chapter 11 in the manual: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel F. M. et al., supra) .

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

In some embodiments, mice (or other animals such as rats, monkeys, donkeys, pigs, sheep, hamsters, or dogs) can be immunized with an antigen (e.g., TIM-3 (e.g., human TIM-3)), and after an immune response is detected, for example, detected

- 68 043100 antibodies specific for the specified antigen in the serum of mice, spleens of mice are collected and splenocytes are isolated. The splenocytes are then fused using well known techniques with any suitable myeloma cells, for example, SP20 cells available from the American Type Culture Collection (ATCC®) (Manassas, Virginia) to form hybridomas. Hybridomas are selected and cloned by limited dilutions. In some embodiments, lymph nodes from immunized mice are harvested and fused with NS0 myeloma cells.

The hybridoma cells thus obtained are seeded and cultured in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of unfused original myeloma cells. For example, if the parent myeloma cells lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HHPT or HPT), the culture medium for hybridomas typically includes hypoxanthine, aminopterine, and thymidine (HAT medium), substances that prevent the growth of HHPT-deficient cells.

Specific embodiments involve myeloma cells that fuse efficiently, maintain stable production of high antibody levels by selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Such myeloma cell lines include mouse myeloma lines, such as the NS0 cell line, or cells derived from MOPC-21 and MPC-11 mouse tumors, available from the Salk Institute Cell Distribution Center, San Diego, Calif. 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 (sources: 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).

Analyze the production of monoclonal antibodies directed against TIM-3 (for example, TIM-3 person), in a culture medium where hybridoma cells grow. The binding specificity of monoclonal antibodies produced by hybridoma cells is determined using methods known in the art, for example, immunoprecipitation, or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

After identifying hybridoma cells producing antibodies with the desired specificity, affinity and/or activity, clones can be subcloned using limited dilution procedures and cultured by standard methods (Goding J.W. (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 can be cultured in vivo as ascitic tumors in an animal body.

Monoclonal antibodies secreted by said subclones are suitably isolated from the culture medium, ascitic fluid or serum using standard immunoglobulin purification procedures such as, for example, protein A Sepharose purification, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

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

In addition, the antibodies described herein can also be obtained using various phage display methods known in the art. In phage display methods, functional domains of an antibody are displayed on the surface of phage particles that carry their encoding polynucleotide sequences. In particular, DNA sequences encoding the VH and VL domains are amplified from animal cDNA libraries (eg cDNA libraries of diseased human or mouse tissues). The DNA encoding the VH and VL domains is recombined with the scFv linker by PCR and cloned into a phagemid vector. This vector is introduced into E. coli by electroporation and infected with E. coli helper phage. The phage for use in these methods is typically filamentous phage, including fd and M13, and the VH and VL domains are typically fused to gene III or gene VIII of said phage in a recombinant manner. A phage expressing an antigen binding domain that binds to a particular antigen can be selected or identified using the antigen, for example, a labeled antigen or an antigen bound or entrapped on a solid surface or bead. Examples of phage display methods that can be used to generate the antibodies described herein include

- 69 043100 cited in: Brinkman U. et al., (1995) J. Immunol. Methods 182: 41-50; Ames R.S. et al., (1995) J. Immunol. Methods 184: 177-186; Kettleborough C.A. et al., (1994) Eur J. Immunol. 24:952-958; Persic L. et al., (1997) Gene 187: 9-18; Burton D.R. & Barbas C.F. (1994) Advan. Immunol. 57:191-280; PCT application PCT/GB91/001134; international publications WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/1 1236, WO 95/15982, WO 95/20401 and WO 97/13844; and US Pat. 5969108, each of which is incorporated herein in its entirety by reference.

As described in the references listed above, once a phage has been selected, antibody coding regions from said 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 cells. mammalian, insect cells, plant cells, yeast and bacteria, for example, as described below. Techniques for recombinant production of antibody fragments such as Fab-, Fab'- and F(ab') ₂ fragments can also be used using methods known in the art, such as those described in: PCT Publication WO 92/22324 ; Mullinax RL et al., (1992) BioTechniques 12(6): 864-9; Sawai H. et al., (1995) Am. J. Reprod. Immunol. 34:26-34; and Better M. et al., (1988) Science 240: 1041-1043, each of which is incorporated herein by reference in its entirety.

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

A chimeric antibody is an antibody molecule whose different parts are derived from different immunoglobulin molecules. For example, a chimeric antibody may comprise the variable region of a mouse or rat monoclonal antibody fused to a constant region of a human antibody. Methods for producing chimeric antibodies are known in the art. See, for example, sources: Morrison S.L. (1985) Science 229: 1202-7; Oi VT & Morrison S.L. (1986) BioTechniques 4: 214-221; Gillies S.D. et al., (1989) J. Immunol. Methods 125: 191-202; and US Pat. Nos. 5,807,715; 4,816,567;

The humanized antibody is capable of binding to a predetermined antigen and comprises a framework region essentially having the amino acid sequence of a human immunoglobulin and CDR regions essentially having the amino acid sequence of a non-human immunoglobulin (eg, mouse immunoglobulin). In specific embodiments, the humanized antibody also comprises at least a portion of an immunoglobulin (Fc) constant region, typically a human immunoglobulin. Said antibody may also include the heavy chain CH1 region, hinge, CH2, CH3 and CH4 regions. The humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG1, IgG ₂ , IgG ₃ and IgG4. Humanized antibodies can be obtained using various techniques known in the art, including, but not limited to, CDR grafting (European patent EP 239400; international publication WO 91/09967; and US patents 5225539, 5530101 and 5585089), veneer or surface modification (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), strand permutations (US Pat. No. 5,565,332), and techniques described, for example, in US Pat. No. 6,407,213, US Pat. No. 5,766,886, International Publication WO 93/17105; sources: Tan P. et al., (2002) J. Immunol. 169:1119-25; Caldas C. et al., (2000) Protein Eng. 13(5): 353-60; Morea V. et al., (2000) Methods 20(3): 267-79; Baca M. et al., (1997) J. Biol. Chem. 272(16): 10678-84; Roguska MA et al., (1996) Protein Eng. 9(10): 895 904; Couto JR et al., (1995) Cancer Res. 55 (23 Supp): 5973s-5977s; Couto JR et al., (1995) Cancer Res. 55(8): 1717-22; Sandhu JS (1994) Gene 150(2): 409-10 and Pedersen JT et al., (1994) J. Mol. Biol. 235(3): 95973, each of which is incorporated herein in its entirety by reference. See also US Published Application US 2005/0042664 Al (February 24, 2005), which is incorporated herein by reference in its entirety.

Methods for producing multispecific (eg, bispecific) antibodies have been described, see, for example, US Pat. Nos. 7,951,917; 7183076; 8227577; 5837242; 5989830; 5869620; 6132992 and 8586713, each of which is incorporated herein in its entirety by reference.

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Single domain antibodies, for example, antibodies lacking light chains, can be prepared using methods well known in the art. See sources: Riechmann L. & Muyldermans S. (1999) J. Immunol. 231:25-38; Nuttall S.D. et al., (2000) Curr. Pharm. Biotechnol. 1(3): 253-263; Muyldermans S., (2001) J. Biotechnol. 74(4): 277-302; US patent No. 6005079; and international publications WO 94/04678, WO 94/25591 and WO 01/44301, each of which is incorporated herein in its entirety by reference.

In addition, antibodies that specifically bind to the TIM-3 antigen (eg, human TIM-3) can in turn be used to generate anti-idiotypic antibodies that mimic the antigen using techniques well known to those skilled in the art. See, for example, sources: Greenspan N.S. & Bona S.A. (1989) FASEB J. 7(5): 437-444; and Nissinoff A. (1991) J. Immunol. 147(8): 2429-2438, each of which is incorporated herein in its entirety by reference.

In specific embodiments, an antibody described herein that binds to the same epitope of TIM-3 (e.g., human TIM-3) as an anti-TIM-3 antibody (e.g., human TIM-3) described herein is is a human antibody. In specific embodiments, an antibody described herein that competitively blocks (eg, in a dose-dependent manner) the binding of any of the antibodies described herein to TIM-3 (eg, human TIM-3) is a human antibody. Human antibodies can be obtained using any method known in the art. For example, transgenic mice incapable of expressing functional endogenous immunoglobulins but capable of expressing human immunoglobulin genes can be used. In particular, human immunoglobulin heavy and light chain gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and D region can be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The functionality of mouse immunoglobulin heavy and light chain genes can be eliminated separately or simultaneously with the introduction of human immunoglobulin loci through homologous recombination. In particular, homozygous deletion of the JH region prevents the production of endogenous antibodies. Modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring expressing human antibodies. Transgenic mice are routinely immunized with a selected antigen, eg, the entire antigen or a portion of the antigen (eg, TIM-3 (eg, human TIM-3)). Monoclonal antibodies directed against the specified antigen can be obtained from immunized transgenic mice using standard hybridoma technology. Human immunoglobulin transgenes carried by transgenic mice undergo rearrangement during B cell differentiation and then undergo class switching and somatic mutations. Accordingly, the use of such a technique allows the production of therapeutically useful IgG, IgA, IgM and IgE antibodies. An overview of this technology for the production of human antibodies is given in the source: Lonberg N. & Huszar D. (1995) Int. Rev. Immunol. 13:65-93, incorporated herein by reference in its entirety. A detailed description of this technology for obtaining human antibodies and human monoclonal antibodies, as well as protocols for obtaining such antibodies are given, for example, in international publications WO 98/24893, WO 96/34096 and WO 96/33735; and US Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; Examples of mice capable of producing human antibodies, each of which is incorporated herein by reference in its entirety, include Xenomouse™ (Abgenix, Inc.; US Pat. Nos. 6,075,181 and 6,150,184), HuAb-Mouse™ (Mederex, Inc./Gen Pharm; US Nos. 5545806 and 5569825), Trans Chromo Mouse™ (Kirin), and KM Mouse™ (Medarex/Kirin).

Human antibodies that specifically bind to TIM-3 (e.g., human TIM-3) can be generated by various methods known in the art, including the phage display methods described above, using antibody libraries derived from immunoglobulin sequences. person. See also US Pat. Nos. 4,444,887, 4,716,111 and 5,885,793; and international publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741, each of which is included (a) in this document in its entirety by reference.

In some embodiments, human antibodies can be generated using mouse-human hybridomas. For example, Epstein-Barr virus (EBV)-transformed human peripheral blood lymphocytes 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 for hybridomas that secrete human monoclonal antibodies that specifically bind to the target antigen (for example, TIM-3 (for example,

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TIM-3 human)). Such methods are known and described in the art, see, for example, references: 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 in its entirety by reference.

6.6. Sets.

Also provided are kits containing one or more of the antibodies described herein, or a pharmaceutical composition containing them, or conjugates thereof. In a specific embodiment, the present invention provides a pharmaceutical kit or kit containing one or more containers filled with one or more of the ingredients of the pharmaceutical compositions described herein, for example, one or more antibodies of the present invention. In some embodiments, said kits contain a pharmaceutical composition as described herein and any prophylactic or therapeutic agent such as those described herein. In some embodiments, said kits may contain a T cell mitogen such as, for example, phytohemagglutinin (PHA) and/or phorbol myristate acetate (PMA); or an antibody that stimulates the TCR complex, such as an anti-CD3 antibody and an anti-CD28 antibody. Such container or containers may optionally be associated with a notice in the form prescribed by a government agency that regulates the manufacture, use, or sale of pharmaceuticals or biological products, reflecting that authority's approval of manufacture, use, or sale for human administration.

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

7. Examples

The examples given in this section (ie section 7) are for illustration and not limitation.

7.1. Example 1 Preparation and characterization of novel anti-human TIM-3 antibodies.

This example describes the production and characterization of antibodies that bind to human T-cell immunoglobulin and mucin domain 3 (TIM-3). In particular, this example describes the production of human antibodies that specifically bind to human TIM-3 and inhibit the function of human TIM-3.

In some of the studies described below, the activity of the anti-TIM-3 antibodies of the present invention was compared with the activity of the reference anti-TIM-3 antibody pab1944w or Hum11. Received the antibody pab1944w based on the variable regions of the antibody 8213 HV0LV0 described in US patent No. 8552156 (included herein in its entirety by reference). The pab1944w sequences are shown in Table 1. 7. The pab1944w antibody was expressed as an IgG1 antibody containing the N297A mutation in the Fc region according to the EU numbering system. The Hum11 antibody was derived from the variable regions of the ABTIM3-hum11 antibody described in US Patent Publication US 2015/0218274 (incorporated herein by reference in its entirety). Hum11 sequences are shown in Table 1. 7. The Hum11 antibody was expressed as an IgG ₄ antibody containing the S228P mutation in the Fc region according to the EU numbering system.

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Table 7. Anti-TIM-3 Reference Antibody Sequences

SEQ ID Description Amino acid sequence NO: 80 VH pabl944w QVQLVQS GAEVKKPGASVKVS CKAS GYT FT S YWMHWVRQA PGQGLEWMGEINPSNGRTNYNEKFKTRVTITADT S T S TAY MELSSLRSEDTAVYYCARGYYLYFDYWGQGTLVTVSS 81 VL pabl944w DIQMTQSPSSLSASVGDRVTITCHASQGIRINIGWYQQKP GKAPKLLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCVQYGQFPWTFGQGTKLEIK 89 Full size heavy chain pabl944w (IgGi N297A) QVQLVQS GAEVKKPGASVKVS CKAS GYT FT S YWMHWVRQA PGQGLEWMGEINPSNGRTNYNEKFKTRVTITADT S T S TAY MELSSLRSEDTAVYYCARGYYLYFDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSW TVPSSSLGTQTYIC NVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYASTYRWSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG 90 Full size I light chain pabl944w (IgGi N297A) DIQMTQSPSSLSASVGDRVTITCHASQGIRINIGWYQQKP GKAPKLLIYHGTNLEDGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCVQYGQFPWTFGQGTKLEIKRTVAAPSVFIFPP SDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQG LSSPVTKSFNRGEC 82 VH Humll QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYNMHWVRQA PGQGLEWMGDIYPGNGDTSYNQKFKGRVTITADKSTSTVY MELSSLRSEDTAVYYCARVGGAFPMDYWGQGTTVTVSS 83 VL Humll AIQLTQSPSSLSASVGDRVTITCRASESVEYYGTSLMQWY QQKPGKAPKLLIYAASNVESGVPSRFSGSGSGTDFTLTIS SLQPEDFATYFCQQSRKDPSFGGGTKVEIK 91 Full length Humll heavy chain (IgG ₄ S228P) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYNMHWVRQA PGQGLEWMGDIYPGNGDTSYNQKFKGRVTITADKSTSTVY MELSSLRSEDTAVYYCARVGGAFPMDYWGQGTTVTVSSAS TKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN

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SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYT CNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGV EVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCK VSNKGLPSSIEKTISKAKGQPREPQV YTLPPSQEEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLS LSLG 92 Full length Humll I light chain (IgG ₄ S228P) AIQLTQSPSSLSASVGDRVTITCRASESVEYYGTSLMQWY QQKPGKAPKLLIYAASNVESGVPSRFSGSGSGTDFTLTIS SLQPEDFATYFCQQSRKDPSFGGGTKVEIKRTVAAPSVF IFPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEV THQGLSSPVTKSFNRGEC

7.1.1. Obtaining antibodies against TIM-3 using Retrocyte Display™ technology.

The preparation of the Retrocyte Display™ library is described in this document. To obtain inserts for the library, total RNA was extracted with phenol-chloroform from FACS-sorted CD19-positive human B-lymphocytes. This total RNA was used for first strand cDNA synthesis using the Fermentas RevertAid First Strand cDNA Synthesis Kit (Cat. No. K1621 and K1622). The variable regions of the antibodies were amplified from cDNA using PCR and cloned into retroviral expression vectors (pCMA). These constructs were then used to transduce mouse B cell progenitors for surface antibody expression using Retrocyte Display™ technology.

The Retrocyte Display™ library prepared as described above with recombinant human TIM-3 and recombinant cynomolgus TIM-3 was screened to identify two antibodies named pab2085 and pab2088. The pab2085 and pab2088 variable region sequence information is summarized in Table 1. 4. The pab2085 and pab2088 antibodies were expressed as IgG ₁ antibodies and analyzed as described below.

7.1.2. Binding of anti-TIM-3 antibodies to TIM-3 expressing cells.

The pab2085 and pab2088 antibodies were tested for binding to TIM-3 expressing cells using flow cytometry. Briefly, wild type mouse 1624-5 cells or 1624-5 cells engineered to express human TIM-3 were incubated with a mouse Fc receptor blocker (BD Pharmingen cat#553142) to reduce non-specific binding. After washing, cells were stained with anti-TIM-3 antibody or isotype control antibody and analyzed on FACSCalibur (BD Biosciences). Both pab2085 and pab2088 showed binding to 1624-5 cells expressing human TIM-3, but not to wild-type 1624-5 cells (FIG. 1).

7.1.3. Analysis of the selectivity of antibodies against TIM-3.

The selectivity of pab2085 and pab2088 for TIM-3 was evaluated on members of the TIM1 and TIM-4 families using suspension microarray technology. Luminex® microspheres were coupled with recombinant human TIM-3 Fc (R&D Systems cat. no. 2365-TM), recombinant human TIM-3 His (Sino Biological cat. no. 10390-H08H), recombinant cynomolgus monkey TIM-3 Fc (R&D Systems cat. no. 7914-TM), recombinant human TIM-1 His (R&D Systems cat. no. 1750-TM) or recombinant human TIM-4 His (R&D cat. no. 2929-TM) via amine coupling with COOH on the surface of the granules. Purified pab2085, pab2088, and IgG1 isotype control antibody were diluted in assay buffer (Roche, cat. no. 11112589001) to concentrations of 10 ng/mL, 100 ng/mL, and 1000 ng/mL. Each dilution (25 µl) was incubated in the dark (20°C, 650 rpm) with 1500 Luminex® microspheres in 5 µl assay buffer for 1 hour in 96-well half-well filter plates (Millipore, cat. no. MABVN1250 ). Built standard curves using 25 μl of the standard IgG ₁ -kappa human (Sigma, cat. No. 15154) in duplicate in a series of dilutions 1:3 (0.08540 ng/ml). Detection was performed using 60 μl of R-phycoerythrin-labeled IgG F(ab) ₂ goat anti-human antibody (2.5 μg/ml; Jackson ImmunoResearch cat. no. 109-116-097) and incubated for an additional hour (20 °С, 650 rpm). The plates were analyzed using the Luminex® 200 system (Millipore). A total of 100 pellets per well were counted in a sample volume of 48 µl. PE MFI values were used to determine specific or non-specific binding to the recombinant proteins mentioned above.

Both pab2085 (FIG. 2A) and pab2088 (FIG. 2B) showed specific binding to human and cynomolgus TIM-3; no significant binding of TIM-1 or TIM-4 was observed at the concentrations tested.

7.1.4. Optimization of anti-TIM-3 antibodies using Retrocyte Display™ technology.

The pab2085 and pab2088 antibodies contain the same heavy chain. To generate additional anti-TIM-3 antibodies, a Retrocyte Display™ heavy chain sub-library based on pab2085 and pab2088 heavy chains was generated and combined with a more diverse light chain library. This novel Retrocyte Display™ library was additionally screened on recombinant human TIM-3 and recombinant cynomolgus TIM-3 to identify light chain optimized variants: pab2173, pab2174, pab2175, pab2176, pab2177, pab2178, pab2179, pab2180, pab2181, pab2182, pab2183, pab2184, pab2185, pab2186, pab2187, pab2188, pab2189, pab2190, pab2191 and pab2192. Information about the sequences of the variable regions of these options with an optimized light chain is given in table. 4. These light chain optimized variants were expressed as antibodies containing a wild-type IgG ₁ Fc region or a variant IgG ₁ Fc region. In this variant of the IgG ₁ Fc region, the effector functions of the Fc region are not affected.

The light chain-optimized pab2188 antibody is an antibody containing a T109S substitution (i.e. a threonine to serine substitution at position 109 relative to the wild-type sequence) according to Kabat numbering in the light chain constant domain, which facilitates in-frame cloning of the variable region readouts in the constant region. The specified mutation is a conservative modification that does not affect the binding or function of the antibody. A wild-type equivalent named pab2188w was also prepared, which contains a threonine at position 109 of the light chain according to Kabat numbering. The pab2188w antibody was expressed as an antibody containing the Fc region of Igd N297A.

7.1.5. Binding of anti-TIM-3 antibodies to TIM-3 expressing cells.

The binding of these optimized light chain variants to cells expressing human or cynomolgus TIM-3 was evaluated in a flow cytometric assay similar to that described above. All variants exhibited binding to mouse 1624-5 cells engineered to express human TIM-3 (FIGS. 3A and 3B) or cynomolgus TIM-3 (FIGS. 3C and 3D), but not wild-type mouse 1624-5 cells. (data not shown).

The binding of optimized light chain variants to primary human T cells was compared to that of the parent antibody pab2085. Briefly, peripheral blood mononuclear cells (PBMCs) isolated in a ficoll gradient from the buffy coat from healthy donors (Research Blood Components, LLC) were enriched for raw pan-T cells using magnetic field separation (Miltenyi Biotec). The enriched T lymphocyte populations were then activated with plate-bound anti-CD3 antibody (SP34, 3 μg/mL) and soluble anti-CD28 antibody (CD28.1, 2 μg/mL) for 3 days in RPMI supplemented with 10% heat-inactivated PBS, at 37°C and 5% CO ₂ . After activation, cells were incubated with a human Fc receptor blocker for 15 min at room temperature to reduce non-specific binding (FcR blocker, Biolegend). Anti-TIM-3 antibodies or IgG isotype control antibodies (12-point dose titration, 10,000 ng/ml to 0.06 ng/ml) were added to individual samples and incubated for 30 minutes at 4°C. Samples were washed twice and an antibody cocktail containing FITC-conjugated anti-kappa chain antibody, as well as anti-CD3 (BV711, OKT3), anti-CD4 (BV605, OKT4) and anti-CD8a (PE, RPA-T8) antibodies, all at a concentration of 2, 5 μg/ml, diluted in buffer (PBS, 2 mm EDTA, 0.5% BSA, pH 7.2), was added to each sample and incubated for 30 minutes at 4°C. Samples were washed twice and analyzed on an LSRFortessa flow cytometer (BD Biosciences). Flow cytometry plots were analyzed using a combination of FACS DIVA and WEHI Weasel software.

As shown in FIG. 4, all light chain optimized variants tested in this study exhibited stronger binding to activated human CD8+ T cells compared to the parent antibody pab2085.

Next, the binding of anti-TIM-3 antibody pab2188 to primary cynomolgus monkey cells was examined.

Cryopreserved PBMC isolated from cynomolgus monkeys (Worldwide Primates, Inc.) were thawed, washed and then subjected to flow cytometric analysis. Prior to antibody incubation, these cells were treated with 10% cynomolgus monkey serum (Abcam) for 15 minutes at room temperature to reduce non-specific binding. Anti-TIM-3 antibodies or IgG isotype control antibodies (10-point dose titration, 20,000 ng/ml to 0.6 ng/ml) were added to individual samples and incubated for 30 min at 4°C. Samples were washed twice and an antibody cocktail containing FITC-conjugated anti-kappa chain antibody and anti-CD11b antibody (BV785, M1/70) at a concentration of 2.5 μg/ml, diluted in buffer (PBS, 2 mM EDTA, 0.5 % BSA, pH 7.2) was added to each sample and incubated for 30 min at 4°C. Samples were washed twice and analyzed on an LSRFortessa flow cytometer (BD Bio-75 043100 sciences). Flow cytometry plots were analyzed using a combination of FACS DIVA and WEHI Weasel software.

As shown in FIG. 5, the anti-TIM-3 antibody pab2188 bound cynomolgus monkey primary myeloid cells in a dose-dependent manner.

7.1.6. Ligand-blocking activity of antibodies against TIM-3.

Anti-TIM-3 antibodies were tested for their ability to block the binding of recombinant human or cynomolgus TIM-3 to phosphatidylserine-expressing irradiated WR19L mouse lymphoma cells. Anti-TIM-3 antibodies or IgG isotype control antibodies (9-point dose titration, 20,000 to 70 ng/mL in humans; or 6-point dose titration, 20,000 to 625 ng/mL in cynomolgus monkey) were incubated with recombinant TIM-3 Fc human (R&D Systems, #2365-TM) or recombinant cynomolgus TIM-3 Fc (R&D Systems, #7914-TM) (10,000 ng/mL) in 1x Annexin-V binding buffer (10 mM Hepes, pH adjusted to 7.4, 140 mM NaCl and 2.5 mM CaCl ₂ ) for 30 min at room temperature. WR19L cells irradiated at 20 Gy and resuspended in 1x Annexin-V binding buffer were added to an anti-TIM-3:TIM-3-Fc antibody cocktail to a final density of 1x106 cells/mL and incubated at room temperature for 45 min. Samples were washed once and an antibody cocktail containing PE-conjugated anti-Fc antibody (1:100 dilution) and viability dye (Biolegend, near-infrared channel; dilution 1:1000) diluted in 1x Annexin binding buffer -V was added to each sample and incubated for 20 minutes at room temperature. Samples were then washed once in 1x Annexin-V binding buffer, resuspended in 150 μl of buffer (PBS, 2 mM EDTA, 0.5% BSA, pH 7.2) and analyzed on an LSRFortessa flow cytometer (BD Biosciences). Flow cytometry plots were analyzed using FACS DIVA.

Anti-TIM-3 antibodies pab2085 and pab2188 blocked the binding of recombinant human TIM-3 (FIG. 6A) and recombinant cynomolgus TIM-3 (FIG. 6B) to phosphatidylserine.

7.1.7. Effect of anti-TIM-3 antibodies on human PBMC stimulated with staphylococcus enterotoxin A (SEA).

The functional activity of the optimized light chain variants against primary human PBMCs was evaluated after stimulation with staphylococcus enterotoxin A (SEA). Briefly, cryopreserved human PBMCs (Research Blood Components) were plated at 1x105 cells/well in RPMI1640 supplemented with normocin (Normocin™, Invivogen, Cat. No. ant-nr) and 10% heat-inactivated PBS (Gibco, Invitrogen Corporation) at 96- well plate coated with NUNCLON-delta surface (NUNC™). Cells were cultured in the presence of 5 µg/ml pembrolizumab anti-PD-1 antibody (lot 7002688300, Myoderm), anti-TIM-3 antibody (10 µg/ml) and SEA superantigen (100 ng/ml, Toxin Technologies) for 6 days at 37 °C and 5% CO ₂ . Cell-free supernatant was collected and stored at -80°C until analysis. IFNy levels were determined using AlphaLISA (Perkin Elmer).

In combination with the anti-PD-1 antibody pembrolizumab, light chain optimized variants enhanced IFN-γ production in this assay with primary human PBMCs (FIG. 7).

The functional activity of pab2188w was analyzed in the SEA stimulation assay using a modified protocol. Cryopreserved human PBMCs (Research Blood Components) were plated at 1x105 cells/well in RPMI1640 supplemented with normocin (Normocin™, Invivogen KaT. no. ant-nr) and 10% heat-inactivated PBS (Gibco, Invitrogen Corporation) in a 96-well plate coated with NUNCLON -delta surface (NUNC™). Cells were cultured in the presence of 5 µg/ml pembrolizumab anti-PD-1 antibody (lot 7002688300, Myoderm), anti-TIM-3 antibody (10 µg/ml) and SEA superantigen (100 ng/ml, Toxin Technologies) for 9 days at 37 °C and 5% CO2. The cells were then washed once and re-stimulated with fresh SEA and antibody for two days. Cell-free supernatant was collected and stored at -80°C until analysis. IFNy levels were determined using AlphaLISA (Perkin Elmer).

As shown in FIG. 8A-8F, anti-TIM-3 antibody pab2188w (IgG1 N297A), alone or in combination with anti-PD-1 antibody pembrolizumab, increased IFN-y production in human PBMC from multiple donors in this SEA challenge assay.

7.2. Example 2 Optimization of anti-TIM-3 antibodies using CDR mutagenesis.

To increase binding affinity, the anti-TIM-3 antibody pab2188w was modified using site-directed mutagenesis of heavy and light chain variable region CDR residues. Briefly, six Fab phage display libraries were generated from the parent antibody pab2188w, each containing a CDRH or CDRL region modified using NNK degenerate codon randomization. The Fab phage libraries were selected based on affinity for recombinant human and cynomolgus TIM-3 antigens. Nine clones, designated AM-1, AM-2, AM-3, AM-4, AM-5, AM-6, AM-7, AM-8, and AM-9, were selected based on binding and dissociation rate measurements. . Information about the sequences of the variable regions of these nine clones is summarized in Table 1. 4. All of these variants include the pab2188w light chain but contain mutations in the heavy chain CDR1. AM-1 - AM-9 were expressed as full-length antibodies containing the Fc region of IgG1 N297A and analyzed in the experiments described below.

7.2.1. Binding of anti-TIM-3 antibodies to TIM-3 expressing cells.

Binding of AM-1-AM-9 antibodies to Jurkat cells ectopically expressing human TIM-3 was compared to that of the parent pab2188w antibody in a flow cytometric analysis. As shown in FIG. 9A, all variants bound to TIM-3 expressing Jurkat cells, and AM-2 and AM-6 showed stronger binding than the parent antibody pab2188w. Binding of AM2 and AM-6 was further analyzed by flow cytometry in the human acute myeloid leukemia cell line Kasumi-3 (ATCC® CRL-2725™) endogenously expressing TIM-3 (FIG. 9B) as well as human CD8+ T cells, staphylococcal enterotoxin A (SEA) stimulated (FIG. 9C), and cynomolgus monkey CD8+ T cells stimulated with SEA (FIG. 9D). In case of binding to human CD8+ T cells, human PBMC cells isolated in ficoll gradient from buffy coat from healthy donors (Research Blood Components, LLC) were activated with SEA (100 ng/mL) for 8 days in RPMI supplemented with 10 % heat-inactivated FBS at 37°C and 5% CO ₂ . After activation, cells were incubated with a human Fc receptor blocker for 15 min at room temperature to reduce non-specific binding (FcR blocker, Biolegend). Isotype control antibodies against TIM-3 or IgG (12-point dose titration, from 10,000 ng/ml to 0.06 ng/ml) were added to individual samples and incubated for 30 min at 4°C. Similarly, in case of binding to cynomolgus macaque CD8+ T cells, isolated cynomolgus macaque PBMCs from frozen base cultures (Worldwide Primates Inc.) were thawed and activated with SEA (100 ng/mL) for five days in RPMI supplemented with 10% inactivated heating FBS at 37°C and 5% CO ₂ . Activated cynomolgus PBMCs were incubated with a combination of a human Fc receptor blocker (FcR blocker, Biolegend) and cynomolgus monkey serum (Abcam) for 15 minutes at room temperature to reduce non-specific binding. Phycoerythrin-conjugated AM-2 antibody or isotype control antibody (Biolegend, PE conjugation, dotted dose titration, 10,000 to 41 ng/mL) and a cocktail of anti-CD4 antibody (BV605, OKT4) and anti-CD8a antibody (PE, SK1), each at a concentration of 2.5 μg/ml, diluted in buffer (PBS, 2 mm EDTA, 0.5% BSA, pH 7.2), added to each sample and incubated for 30 min at 4°C. Samples were washed twice and an antibody cocktail containing FITC-conjugated anti-kappa antibody as well as anti-CD3 (BV711, OKT3), anti-CD4 (BV605, OKT4) and anti-CD8a (PE, RPA-T8) antibodies, all at a concentration of 2.5 µg/ml, diluted in buffer (PBS, 2 mM EDTA, 0.5% BSA, pH 7.2), was added to each sample and incubated for 30 min at 4°C. Samples were washed twice and analyzed on an LSRFortessa flow cytometer (BD Biosciences). Flow cytometry plots were analyzed using a combination of FACS DIVA and WEHI Weasel software. Both AM-2 and AM-6 showed binding to Kasumi-3 cells (FIG. 9B) and activated human CD8+ T cells (FIG. 9C). AM-2 also showed binding to activated cynomolgus monkey CD8+ T cells (FIG. 9D).

Further, in a similar assay, binding to primary myeloid CD14+ human and cynomolgus monkey cells was analyzed by flow cytometry using phycoerythrin (PE) conjugated pab2188w, AM-2, or an isotype control antibody. Briefly, cryopreserved PBMC isolated from human or cynomolgus monkey (Worldwide Primates, Inc.) were thawed, washed, and then subjected to flow cytometric analysis. Prior to antibody incubation, cells were treated with 10% cynomolgus monkey serum (Abcam, cat# ab155109) for 15 minutes at room temperature to reduce non-specific binding. PE-conjugated anti-TIM-3 antibody or IgG isotype control antibody (12-point dose titration, 10,000 ng/mL to 0.05 ng/mL for human PBMC and 100,000 ng/mL to 0.5 ng/mL for cynomolgus PBMC ) was added to individual samples in an antibody cocktail containing anti-CD14 antibody (APC, M5E2) and reversible viability marker Zombie Green™ and then incubated for 30 min at 4°C. Additional samples were kept for correction control for individual dyes (CD45-FITC, CD45-PE and CD45-APC; clone MB4-6D6, Miltenyi). Samples were washed twice in buffer and analyzed on an LSRFortessa flow cytometer (BD Biosciences). Flow cytometry plots were analyzed using a combination of FACS DIVA and WEHI Weasel software. AM-2 showed stronger binding to CD14+ human (FIG. 9E) and cynomolgus monkey (FIG. 9F) myeloid cells than the parent antibody pab2188w.

7.2.2. Analysis of the selectivity of antibodies against TIM-3.

The selectivity of AM-2 and AM-6 for TIM-3 was assessed using suspension microarray technology. Luminex® microspheres were coupled with recombinant human TIM-3 His (Sino Biological, no. 10390-H08H), recombinant cynomolgus macaque TIM-3 Fc (Sino Biological, no. 90312-C02H), recombinant mouse TIM-3 Fc (R&D Systems, no. 1529 -TM), recombinant human TIM-1 His (R&D Systems, no. 1750-TM), recombinant human TIM-4 His (R&D, no. 2929-TM), recombinant

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Human OX40 His (Sino Biological, No. 10481-H08H), recombinant human GITR Fc (R&D Systems, No. 689-GR), recombinant human DR3 Fc (R&D Systems, No. 943-D3), and recombinant human CD137 Fc (in-house produced material ) by amine coupling with COOH on the surface of the granules. Purified pab2188w (IgG1 n297A), AM-2 (IgG1 N297A), AM-6 (IgG ₁ N297A), and IgG1 N297A isotype control antibody were diluted in assay buffer (Roche 11112589001) titrated from 10,000 to 0.1 ng/mL. Each dilution (25 μl) was incubated in the dark (20° C., 650 rpm) with 1500 Luminex® microspheres in 5 μl assay buffer for 1 hour in 96-well half-well filter plates (Millipore, MABVN1250). Detection was performed using 60 μl of goat anti-human F(ab) ₂ IgG labeled with R-phycoerythrin (2.5 μg/ml; JIR 109-116-097) and incubated for another hour (20° C., 650 rpm). The plates were analyzed using the Luminex® 200 system (Millipore). A total of 100 pellets per well were counted in a sample volume of 48 µl. PE MFI values were used to determine specific or non-specific binding to recombinant proteins.

Anti-TIM-3 antibodies pab2188w (FIG. 10B), AM-2 (FIG. 10C), and AM-6 (FIG. 10D) showed specific binding to human and cynomolgus TIM-3; at the concentrations tested, no significant binding was detected to mouse TIM-3, human TIM-1, human TIM-4, human OX40, human GITR, human DR3, or human CD137.

7.2.3. Ligand-blocking activity of antibodies against TIM-3.

Additionally, the ability of anti-TIM-3 antibodies AM-2 and AM-6 to block the binding of phosphatidylserine to human or cynomolgus TIM-3 was analyzed. Briefly, anti-TIM-3 antibodies or IgG isotype control antibodies (10 point dose titration, 40,000 ng/mL - 1,000 ng/mL) were incubated with recombinant human TIM-3-Fc (R&D Systems, #2365-TM) or recombinant TIM -3Fc cynomolgus monkey (R&D Systems, #7914-TM) (10,000 ng/mL) in 1x Annexin-V binding buffer (10 mM Hepes adjusted to pH 7.4, 140 mM NaCl and 2.5 mM CaCl ₂ ) for 30 min at room temperature. WR19L cells irradiated at 20 Gy and resuspended in 1x Annexin-V binding buffer were added to an anti-TIM-3:TIM-3-Fc antibody cocktail to a final density of 1 x 10 ⁶ cells/ml and incubated at room temperature for 45 min. The samples were washed once; an antibody cocktail containing PE-conjugated anti-Fc antibody (1:100 dilution) and viability dye (Biolegend, near-infrared channel; dilution 1:1000) diluted in 1x Annexin binding buffer was added to each sample -V, and incubated for 20 min at room temperature. Samples were then washed once in 1x Annexin-V binding buffer and analyzed on an LSRFortessa flow cytometer (BD Biosciences). Flow cytometry plots were analyzed using FACS DIVA.

As shown in FIG. 11A and 11B, pab218 8w, AM-2, and AM-6 anti-TIM-3 antibodies effectively blocked human or cynomolgus TIM-3 binding to phosphatidylserine-expressing cells.

7.2.4. Effect of anti-TIM-3 antibodies on human PBMC stimulated with staphylococcus enterotoxin A (SEA).

The functional activity of pab2188w variants was analyzed using primary human PBMC stimulated with staphylococcus enterotoxin A (SEA). Briefly, cryopreserved human PBMCs (Research Blood Components) were plated at 1 x 10 ⁵ cells/well in RPMI1640 supplemented with normocin (Normocin™, Invivogen cat. no. ant-nr) and 10% heat-inactivated PBS (Gibco, Invitrogen Corporation) at 96- well plates coated with NUNCLON-delta surface (NUNC™). Cells were cultured in the presence of 5 µg/ml pembrolizumab anti-PD-1 antibody (lot 7002688300, Myoderm), anti-TIM-3 antibody (10 µg/ml) and SEA superantigen (100 ng/ml, Toxin Technologies) for 9 days at 37 °C and 5% CO ₂ . The cells were then washed once and re-stimulated with fresh SEA and antibody for two days. Cell-free supernatant was collected and stored at -80°C until analysis. IFNy levels were determined using AlphaLISA (Perkin Elmer).

As shown in FIG. 12A and 12B, many pab2188w variants, alone or in combination with the anti-PD-1 antibody pembrolizumab, enhanced IFN-y production in human PBMC from two different donors.

7.2.5. Effect of anti-TIM-3 antibodies on cytokine production by tumor-infiltrating lymphocytes.

Additionally, the ability of anti-TIM-3 antibodies to stimulate cytokine production by activated primary tumor-infiltrating lymphocytes (IOLs), alone or in combination with an anti-PD-1 antibody, was evaluated. Single cell suspensions from fresh tumors of non-small cell lung cancer (NSCLC) (stage II), adenocarcinoma of the gallbladder (stage IV) or breast cancer (stage II) (UMass Medical School, Worcester, MA) were isolated by mechanical microdissection. In some cases, depending on the level of fibrosis, enzymatic digestion was necessary (liberase and DNase-I, Roche). Cells were left for 1 day in 96-well NUNCLON-delta coated (NUNC™) plates in RPMI1640 supplemented with

- 78 043100 normocin (Normocin™, Invivogen Cat. No. ant-nr), recombinant human IL-2 (20 U/ml, R&D Systems) and 10% heat-inactivated PBS (Gibco, Invitrogen Corporation) at a density of 5x10 ⁴ cells/well . The next day, samples were centrifuged and fresh culture medium containing antibodies of interest (20 µg/mL anti-TIM-3 antibody and 5 µg/mL anti-PD-1 antibody pembrolizumab) and anti-CD3/CD28 microbeads (at a ratio of granules to cells 1:1), to a final volume of 100 μl and left for incubation for 3 days at 37°C and 5% CO ₂ . Cell-free supernatant was collected and stored at -80°C until analysis. IFNy and TNFa levels were determined using AlphaLISA (Perkin Elmer).

As shown in FIG. 13A-13F, anti-TIM-3 antibodies enhanced IFN-γ and TNFα production by activated primary IOLs from NSCLC tumors, gallbladder adenocarcinoma, or breast cancer.

7.2.6. Internalization of anti-TIM-3 antibodies after binding.

In this example, the internalization of anti-TIM-3 antibodies in cells was analyzed. In the first set of experiments, the internalization of the anti-TIM-3 antibody was evaluated using aHFc-NC-DM1 (anti-human IgG Fc conjugated to DM1 maytansinoid by a non-cleavable linker, Moradec LLC). Said aHFc-NCDM1 secondary antibody drug conjugate binds to the test antibody (eg, anti-TIM-3 antibody) resulting in the release of a cytotoxic DM1 load into the cytoplasm of the cell upon internalization. In the second set of experiments, internalization was assessed using antibodies against TIM-3 pab218 8w (IgG1 N2 97A) and Hum11 (IgG ₄ S228P) directly conjugated to monomethylauristatin E (MMAE). A similar drug:antibody ratio (DAR; isotype control=3.5, pab2188w=4.0, Hum11=3.0) was observed for all antibodies, maintaining an equivalent level of antibody drug conjugate (ADC) delivery. after internalization. In the third group of experiments, internalization was assessed by the subcellular localization of the TIM-3 protein labeled with a fluorescent dye that does not penetrate cells.

Briefly, Kasumi-3 (ATCC® CRL-2725™), an acute myeloid leukemia cell line endogenously expressing TIM-3, and a Jurkat cell line engineered to overexpress TIM-3 were seeded in white bottom tissue culture plates at a density of 2x10 ⁴ per hole. In the first set of experiments using a secondary antibody-drug conjugate aHFc-NC-DM1, 8-point titrated doses (3.333 to 1 ng/mL) of either anti-TIM-3 antibody or IgG isotype control antibody, together with aHFc, were added to cells. -NC-DM1 (ratio to primary antibody 1:1) to a final volume of 100 µl/well. Cells were incubated with primary antibodies and secondary antibody-drug conjugate at 37°C and 5% CO2 for 72 h.

Anti-TIM-3 antibodies pab2188w (IgG1 N297A), AM-2 (IgG1 N297A), and AM-6 (IgG ₁ N297A) internalized TIM-3 expressed on Jurkat cells (Fig. 14A) and Kasumi-3 cells (Fig. 14B), in experiments with aHFc-NC-DM1 is more effective than the reference antibodies against TIM-3 Hum11 (IgG ₄ S228P) and pab1944w (IgG1 N297A), judging by the greater decrease in cell survival over a wide range of antibody concentrations.

In the second set of experiments, antibodies pab2188w (IgG1 N297A) and Hum11 (reference antibody, IgG4 S228P) were directly conjugated to MMAE at similar concentrations to account for potential differences in the propensity of the secondary antibody drug conjugate (aHFc-NCDM1) to bind different Fc regions. antibodies. A 9-point titrated dose (6.666 to 1 ng/mL) of either MMAE-conjugated anti-TIM-3 antibody or MMAE-conjugated isotype control IgG antibody was added to the cells to a final volume of 100 μl/well. Cells were incubated with conjugated antibodies at 37°C and 5% CO ₂ for 72 h. After incubation, 90 µl of reconstituted Cell Titer Glo (Promega) was added to each well, and cells were incubated at room temperature for 5 min. The resulting luminescence was recorded using an Envision instrument (Perkin Elmer).

As shown in FIG. 14C, pab2188w (IgG1 N297A) induced a greater reduction in cell survival than Hum11 (reference antibody, IgG4 S228P), indicating that the effect observed for the secondary antibody drug conjugate (e.g., shown in FIG. 14A) was due to the internalization potential of each of the anti-TIM-3 antibodies.

In the third group of experiments, the internalization of antibodies against TIM-3 was analyzed using confocal fluorescence microscopy on living cells. Jurkat cells expressing the HaloTag-TIM-3 fusion protein were first incubated with 1 μM Violet Proliferation Dye 450 (BD) for 30 min at 37°C and 5% CO ₂ . After incubation, cells were washed in PBS and resuspended in cell culture medium. To detect the extracellular domain of TIM-3, Jurkat HaloTag-TIM-3 cells were stained with HaloTag Alexa Fluor 488 (Promega, 1 μM), which does not penetrate membranes, for 15 min at 37°C and 5% CO ₂ . Cells were then resuspended in fresh culture medium and plated in a 384-well microplate (15,000 cells/well) with either anti-TIM-3 AM-2 antibody (IgG1 N297A) or isotype control (each antibody in conc.

- 79 043100 10 µg/ml). Real-time images were taken on an ImageXpress Micro Confocal High-Content microscope (Molecular Devices) under controlled environmental conditions (37°C and 5% CO2); images were captured every 30 minutes for 3.5 hours. Image analysis was performed using MetaXpress analysis software (Molecular Devices). Jurkat cells were identified by the DAPI channel (Violet Proliferation Dye 450) and the magnitude of the internalized TIM-3 signal per cell was quantified by the FITC channel (HaloTag Alexa Fluor 488).

As shown in FIG. 15, cells incubated with anti-TIM-3 antibody AM-2 showed an increase in TIM-3 internalization over time compared to cells incubated with isotype control antibody. Specifically, after 3.5 hours, treatment with AM-2 antibody resulted in a doubling of the percentage of TIM-3 positive cells, showing greater TIM-3 internalization compared to TIM-3 positive cells treated with an isotype control antibody (namely, 15.1% internalization versus 7.2% internalization, respectively). In addition, the internalization signal observed in AM-2 treated cells after 3.5 hours was significantly higher compared to the signal in cells treated with isotype control antibody (p=0.00027, one-way T-test). There was no statistically significant difference at time zero (p=0.91, one-tailed t-test).

7.3. Example 3 Epitope Mapping of Anti-TIM-3 Antibodies.

In this example, the epitope of anti-TIM-3 antibodies pab2188 (IgG1 variant), pab2187 (IgG1 variant), and AM-2 (IgG1 N297A) was characterized.

7.3.1. Epitope mapping of anti-TIM-3 antibodies using alanine scanning.

The binding characteristics of the anti-TIM-3 antibodies pab2188 (IgG1 variant) and pab2187 (IgG1 variant) were assessed using alanine scanning. Briefly, Agilent Technologies' QuikChange HT Protein Engineering System (Cat. No. G5901A) was used to generate human TIM-3 mutants with alanine substitutions in the extracellular domain. These human TIM-3 mutants were expressed on the surface of mouse 1624-5 B cell progenitors by retroviral transduction. Transduction efficiency, or the percentage of cells expressing human TIM-3, was kept below 5% to ensure that the majority of cells did not express two or more different TIM-3 mutants.

Thereafter, cells expressing correctly folded human TIM-3 mutants, as assessed by flow cytometry binding to an anti-TIM-3 polyclonal antibody (R&D Systems, cat. no. AF2365), were selected to obtain a subpopulation expressing human TIM-3 mutants, not binding anti-TIM-3 monoclonal antibody pab2188 (IgG1 variant) or pab2187 (IgG1 variant). Cells that showed specific binding to the antibody were separated from the population of non-binding cells using a preparative speed FACS sort (FACSAriaII, BD Biosciences). The pooled pools of cells capable or incapable of reacting with the antibody were replicated in tissue culture; cycles of antibody-guided cell sorting and tissue culture expansion were repeated until a population of cells clearly detectably incapable of reacting with anti-TIM-3 antibody (pab2188 (IgG1 variant) or pab2187 (IgG1 variant)) was obtained. This population of cells unable to react with anti-TIM-3 antibody (pab2188 (IgG1 variant) or pab2187 (IgG1 variant)) was subjected to the last step of single cell sorting or general sorting. After a few days of expansion, cells that had undergone single cell sorting or overall sorting were again tested for binding to anti-TIM-3 polyclonal antibody and no binding to pab2188 (IgG1 variant) or pab2187 (IgG1 variant) monoclonal antibody using flow cytometry.

To compare the phenotype with the genotype, next generation sequencing (NGS) was performed on cells that had undergone a general sorting and expressed human TIM-3 mutants. Sequence analysis demonstrated that cells capable of reacting with the anti-TIM-3 polyclonal antibody but not with the anti-TIM-3 monoclonal antibody pab2188 (IgG1 variant) or pab2187 (IgG1 variant) expressed the human TIM-3 mutant in which the mutation occurred. at position 40 with Phe replaced by Ala, in accordance with the sequence numbering of SEQ ID NO: 79.

7.3.2. Epitope mapping of anti-TIM-3 antibodies using hydrogen-deuterium exchange (HDX) with mass spectroscopy.

In the first study, the interaction of pab2188 (an IgG1 variant) with human TIM-3 was investigated using hydrogen-deuterium exchange (HDX) with mass spectroscopy.

For the deglycosylation treatment, 250 μg of recombinant human TIM-3/Fc chimera (R&D Systems cat. no. 2365-TM) were incubated with 4 μl of PNase F at 37° C. for 3 hours. The human TIM-3 /Fc chimera contains the amino acid sequence, SEQ ID NO: 102, fused to human IgG1.

For pepsin digestion, 6.9 µg of native or deglycosylated chimeric human TIM-3/Fc in 115 µl of control buffer (50 mM phosphate, 100 mM sodium chloride, pH 7.4) was denatured by adding 115 µl of 4 M guanidine hydrochloride, 0.85 M TCEP buffer (final pH 2.5)

- 80 043100 and incubation of the specified mixture for 3 min at 10°C. This mixture was then subjected to column pepsin digestion using a self-filled pepsin column; the resulting peptides were analyzed using an UPLC-MS system consisting of a Waters Acquity UPLC UPLC system coupled to a Q Exactive™ Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo). The peptides were separated on a 50 mm x 1 mm C8 column in a 20.5 minute gradient of 2-32% solvent B (0.1% formic acid in acetonitrile). Peptide identification was performed by MS/MS search with human TIM-3 sequence matching using Mascot software. The mass tolerance for precursor and product ions was 20 ppm. and 0.05 Yes, respectively.

µl native or deglycosylated chimeric human TIM-3/Fc (6.9 µg), 10 µl native chimeric human TIM-3/Fc and antibody mixture (6.9 µg:12.9 µg), or 10 µl deglycosylated chimeric TIM-3/Fc 3 /Fc human and antibody mixture (6.9 μg: 12.9 μg) were incubated with 105 μl deuterium oxide labeling buffer (50 mM phosphate, 100 mM sodium chloride, pD 7.4) for 0.60.300 , 1800, 7200, 14400 and 28800 s. Deuterium exchange was performed either at 10° C. for native chimeric human TIM-3/Fc and its complex with antibody, or at 4° C. for deglycosylated chimeric human TIM-3/Fc and its complex with antibody. Deuterium exchange was stopped by adding 115 μl of 4 M guanidine hydrochloride, 0.85 M TCEP buffer (final pH 2.5). After stopping the exchange, the sample was subjected to pepsin digestion on a column and LC-MS analysis was carried out as described above. Mass spectra were recorded in the MS only mode. To calculate the incorporation of deuterium, the mass spectra of a certain peptide were combined with the peaks in the extraction ion chromatograms and a weighted average m/z was calculated. The increase in weight relative to the weight of the native peptide (0 minutes) to the weighted average weight corresponds to the level of inclusion of deuterium.

Sequence coverage for native and deglycosylated human TIM-3 was 71.6% and 98.4%, respectively. Most human TIM-3 peptides showed identical or similar levels of deuterium in the presence or absence of anti-human TIM-3 antibody, however, a number of peptide segments were found to have significantly lower incorporation of deuterium upon antibody binding. Both native and deglycosylated human TIM-3 showed a significant reduction in deuterium incorporation when bound to the anti-human TIM-3 antibody pab2188 (IgG1 variant) in the region consisting of the amino acid sequence shown in SEQ ID NO: 94 (VCWGKGACPVFECGNWL) as well as in the region consisting of the amino acid sequence shown in SEQ ID NO: 95 (RIQIPGIMND). The most pronounced decrease in deuterium incorporation was observed in the region consisting of the amino acid sequence shown in SEQ ID NO: 93 (PVFECGN).

Next, an interaction study of AM-2 (IgG1 N297A) with human TIM-3 was performed using hydrogen-deuterium exchange mass spectrometry, similar to the study described above. Briefly, deglycosylated chimeric human TIM-3/Fc was incubated with deuterium oxide either alone or in combination with anti-human TIM-3 antibody AM-2 (IgG1 N297A). Deuterium exchange was carried out at 10°C for 0, 60, 300, 1800, 7200, and 14400 s. The exchange reaction was stopped by low pH; after stopping, the samples were digested with pepsin/protease XIII or protease XVIII on a column, and LC-MS analysis was carried out as described above. Raw MS data were processed using HDX WorkBench, H/D exchange MS data analysis software (source: J. Am. Soc. Mass Spectrom. 2012, 23 (9), 1512-1521, incorporated herein in its entirety by reference ). Deuterium levels were calculated based on the average mass difference between the deuterated peptide and its native form (t ₀ ).

For deglycosylated human TIM-3, 100% sequence coverage was achieved. Anti-TIM-3 antibody AM-2 (IgG1 N297A) showed a binding pattern similar to that observed for pab2188 (IgG1 variant). In two regions, the first, consisting of the amino acid sequence shown in SEQ ID NO: 94 (VCWGKGACPVFECGNWL) and the second, consisting of the amino acid sequence shown in SEQ ID NO: 96 (RIQIPGIMNDEKFNLKL), a pronounced protection against deuterium incorporation was observed at binding of deglycosylated human TIM-3 to anti-TIM-3 antibody AM-2 (IgG1 N2 97A). The most pronounced decrease was observed in the region consisting of the amino acid sequence presented in the sequence of SEQ ID NO: 93 (PVFECGN).

7.3.3. Epitope mapping of anti-TIM-3 antibody using Pepscan assay.

The binding of anti-TIM-3 antibody pab2188 (IgG1 variant) to synthetic TIM-3 related peptide fragments, presented as an array of chip-bound peptides, was measured. The analysis was performed at Pepscan Presto BV, Lelystad, The Netherlands. Briefly, a peptide library was synthesized to reconstruct human TIM-3 epitopes. An amino-functionalized polypropylene support was prepared by grafting a proprietary hydrophilic polymer formulation and then reacting with tert-butyloxycarbonyl-hexamethylenediamine (BocHMDA) using dicyclohexylcarbodiimide (DCC) with N-hydroxybenzotriazole (HOBt) and then resolving

- 81 043100 cleavage of Boc groups with trifluoroacetic acid (TFA). For the synthesis of peptides on an amino-functionalized solid support, standard Fmoc-peptide synthesis was used with specially modified JANUS liquid dosing stations (Perkin Elmer). Synthesis of structural mimetics was carried out using Pepscan's CLIPS (Chemically Linked Peptides on Scaffolds) technology. CLIPS technology allows structuring peptides into single loops, double loops, triple loops, fold-like folds, helical folds, and combinations of the above. The binding of the antibody to each of the synthesized peptides was tested in a PEPSCAN-based ELISA assay. The peptide chips were incubated with the primary antibody solution overnight at 4°C. After washing, the peptide chips were incubated with a goat anti-human Ig HRP conjugate (Southern Biotech cat. no. 2010-05) for one hour at 25°C. After washing, peroxidase substrate 2, 2'-azino-di-3-ethylbenzothiazoline sulfonate (ABTS) and 2 μl/ml 3% H ₂ O ₂ were added. After 1 hour, color development was measured and quantified using a charge-coupled device (CCD) camera and an image processing system.

The Pepscan study showed that the anti-TIM-3 antibody pab2188 (IgG1 variant) recognized stretches of human TIM-3, including the region consisting of the amino acid sequence shown in SEQ ID NO: 99 (GKGACPVFE) and the region consisting of the sequence amino acids shown in SEQ ID NO: 100 (DFTAAFPR).

The scope of the present invention is not limited to the specific embodiments described in the document. Thus, various modifications of the present invention other than those described will be apparent to those skilled in the art upon reading the above description and the accompanying drawings. Such modifications are within the scope of the appended claims.

All sources (e.g., publications, or patents, or patent applications) cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual source (e.g., publication , or patent or patent application) is specifically incorporated by reference in its entirety and for any purpose.

Other implementation options are included in the following claims.

Claims (18)

1. An isolated antibody that specifically binds to human TIM-3, where the antibody contains a heavy chain variable region containing complementarity-determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region containing complementarity-determining regions CDRL1, CDRL2 and CDRL3, where: (a) CDRH1 contains the amino acid sequence X ₁ X ₂ X ₃ X ₄ X ₅ S (SEQ ID NO: 48), where Xi is R, S, A, G, K, M or T, X ₂ is Q, S, A, G, R or T, X ₃ is N, Y, G or Q, X ₄ is A or Q, and X ₅ represents W, M, A, S or T; (b) CDRH2 contains the amino acid sequence of SEQ ID NO: 2; (c) CDRH3 contains the amino acid sequence of SEQ ID NO: 3; (d) CDRL1 contains the amino acid sequence X1ASQSVX2SSYLA (SEQ ID NO: 52), where X1 is R or G, and X2 is absent or represents S; (e) CDRL2 contains the amino acid sequence X1ASX2RAT (SEQ ID NO: 53), where X1 is D or G, and X2 is N, S or T; and (f) CDRL3 contains the amino acid sequence QQYGSSPX1T (SEQ ID NO: 54), where X1 is L or I. 2. The selected antibody according to claim 1, where (a) CDRH1 contains the amino acid sequence X1X2NAWS (SEQ ID NO: 49), where X1 is R or A; and X ₂ is Q or R; (b) CDRH1 contains the amino acid sequence X1X2GQX3S (SEQ ID NO: 50), where X1 is K, M or G; X2 is A or S; and X3 is S or T; (c) CDRH1 contains the amino acid sequence X1X2QQAS (SEQ ID NO: 51), where X1 is S, R, T or G; and X2 is A, S, T or G; (d) CDRH1 contains an amino acid sequence selected from the group consisting of the sequences of SEQ ID NO: 1 and 4-12; (e) CDRL1 contains an amino acid sequence selected from the group consisting of the sequences of SEQ ID NO: 13-16; - 82 043100 (f) CDRL2 contains an amino acid sequence selected from the group consisting of the sequences of SEQ ID NO: 17-21; (g) CDRL3 contains an amino acid sequence selected from the group consisting of the sequences of SEQ ID NO: 22 and 23; (h) CDRH1, CDRH2 and CDRH3 contain the amino acid sequences CDRH1, CDRH2 and CDRH3, respectively, the sequence of SEQ ID NO: 1, 2 and 3; 4, 2 and 3; 5, 2 and 3; 6, 2 and 3; 7, 2 and 3; 8, 2 and 3; 9, 2 and 3; 10, 2 and 3; 11, 2 and 3; or 12, 2 and 3, respectively; (i) CDRL1, CDRL2 and CDRL3 contain the amino acid sequences CDRL1, CDRL2 and CDRL3, respectively, the sequence of SEQ ID NO: 13, 17 and 22; 14, 17 and 22; 15, 18 and 22; 14, 19 and 22; 14, 20 and 22; 14, 21 and 22; 16, 20 and 22; or 14, 17 and 23; and/or (j) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 contain sequences 1, 2, 3, 14, 21 and 22; 4, 2, 3, 14, 21 and 22; 5, 2, 3, 14, 21 and 22; 6, 2, 3, 14, 21 and 22; 7, 2, 3, 14, 21 and 22; 8, 2, 3, 14, 21 and 22; 9, 2, 3, 14, 21 and 22; 10, 2, 3, 14, 21 and 22; 11, 2, 3, 14, 21 and 22; or 12, 2, 3, 14, 21 and 22, respectively. 3. An isolated antibody according to claim 1 or 2, wherein the antibody contains: (a) a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 55; or 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: 24-35; and/or (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 56; or an amino acid sequence at least 75%, 80%, 85%, 90%, 95% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 36-47. 4. An isolated antibody that specifically binds to human TIM-3, wherein the antibody contains: (a) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of the sequences of SEQ ID NOs: 24-35; and a light chain variable region comprising an amino acid sequence selected from the group consisting of the sequences of SEQ ID NOs: 36-47; (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 58, 61 or 65 and a light chain containing the amino acid sequence of SEQ ID NO: 69; (c) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region, respectively, comprise the amino acid sequences of SEQ ID NOs: 24 and 36; 24 and 38; 26 and 42; 24 and 42; 24 and 46; 24 and 43; 26 and 43; 26 and 46; 26 and 41; 24 and 41; 25 and 39; 24 and 47; 25 and 40; 26 and 47; 25 and 37; 25 and 45; 25 and 44; 25 and 46; 25 and 42; 25 and 41; 25 and 43; 25 and 47; 27 and 46; 28 and 46; 29 and 46; 30 and 46; 31 and 46; 32 and 46; 33 and 46; 34 and 46; or 35 and 46; or (d) a heavy chain and a light chain, wherein the heavy chain and light chain, respectively, comprise the amino acid sequences of SEQ ID NOs: 58 and 69, 61 and 69, 65 and 69. 5. An isolated antibody according to any one of the preceding claims, wherein the antibody comprises: (a) a heavy chain constant region selected from the group consisting of human IgG1, IgG ₂ , IgG ₃ , IgG4, IgA1 and IgA ₂ , optionally wherein the heavy chain constant region is: (i) a wild-type human IgG heavy chain constant region variant, wherein said human IgG heavy chain constant region variant binds to the human Fc-γ receptor with an affinity lower than that of the wild-type human IgG heavy chain constant region to the receptor The human Fcy, optionally said human Fc-γ receptor is selected from the group consisting of FcyRI, FcyRII and FcyRIII; (ii) IgG1 containing the N297A mutation according to the EU numbering system, optionally containing the amino acid sequence of SEQ ID NO: 72; (iii) IgG1 containing the N297Q mutation according to the EU numbering system; (iv) non-fucosylated IgG1; and/or (v) an IgG4 containing the S228P mutation according to the EU numbering system, optionally containing the amino acid sequence of SEQ ID NO: 74, and/or (b) a light chain constant region selected from the group consisting of a human light chain and a human λ light chain, optionally wherein the light chain constant region comprises the amino acid sequence of SEQ ID NO: 76. 6. An isolated antibody according to any one of the preceding claims, wherein the antibody: (a) (i) binds specifically to the TIM-3 protein variant of the amino acid sequence of SEQ ID NO: 101 with lower affinity compared to binding to the wild-type TIM-3 protein of the amino acid sequence of SEQ ID NO: 79, or (ii) specifically does not bind to the TIM-3 protein variant with the amino acid sequence of SEQ ID NO: 101; (b) binds to residue 40 of SEQ ID NO: 79; (c) binds to an epitope located within the human TIM-3 region, consisting of - 83 043100 amino acid sequence of SEQ ID NO: 93; (d) binds to an epitope located within the human TIM-3 region, consisting of the amino acid sequence of SEQ ID NO: 94; (e) binds to an epitope located within the human TIM-3 region, consisting of the amino acid sequence of SEQ ID NO: 95; (f) binds to an epitope located within the human TIM-3 region, consisting of the amino acid sequence of SEQ ID NO: 96; (g) binds to an epitope located within the human TIM-3 region, consisting of the amino acid sequence of SEQ ID NO: 97; (h) binds to an epitope located within the human TIM-3 region, consisting of the amino acid sequence of SEQ ID NO: 98; (i) binds to an epitope located within the human TIM-3 region, consisting of the amino acid sequence of SEQ ID NO: 99; and/or (j) binds to an epitope located within the human TIM-3 region, consisting of the amino acid sequence of SEQ ID NO: 100. 7. An isolated antibody according to any one of the preceding claims, wherein the antibody: (a) is a human antibody; (b) is antagonistic to human TIM-3; (c) deactivates, reduces or inhibits the activity of human TIM-3; (d) inhibits the binding of human TIM-3 to phosphatidylserine; (e) induces the production of IFNy by peripheral blood mononuclear cells (PBMC) stimulated with staphylococcal enterotoxin A (SEA); and/or (f) induces the production of IFNy or TNFa by tumor infiltrating lymphocytes (TILs) stimulated with anti-CD3 and anti-CD28 antibodies. 8. An isolated antibody according to any one of the preceding claims, wherein the antibody is conjugated to a cytotoxic agent, cytostatic agent, toxin, or radionuclide. 9. An isolated antibody according to any one of the preceding claims, wherein the antibody is conjugated to a detectable label. 10. A pharmaceutical composition comprising an antibody according to any of the preceding claims and a pharmaceutically acceptable carrier or excipient. 11. An isolated polynucleotide encoding a heavy chain variable region or an antibody heavy chain according to any one of claims 1-9. 12. An isolated polynucleotide encoding a light chain variable region or an antibody light chain according to any one of claims 1-9. 13. A vector containing a polynucleotide according to claim 11 or 12. 14. Recombinant host cell containing: (a) a polynucleotide according to claim 11 or 12; (b) a vector according to claim 13; (c) a polynucleotide encoding a heavy chain variable region and a light chain variable region or a heavy chain or a light chain of an antibody according to any one of claims 1 to 9; (d) a first polynucleotide encoding a heavy chain variable region or a heavy chain of an antibody according to any one of claims 1 to 9 and a second polynucleotide encoding a light chain variable region or a light chain of an antibody according to any one of paragraphs 1 to 9; (e) a vector containing a polynucleotide encoding a heavy chain variable region and a light chain variable region or a heavy chain or light chain of an antibody according to any one of claims 1 to 9; or (f) a first vector containing a first polynucleotide encoding a heavy chain variable region or a heavy chain of an antibody according to any one of paragraphs. 1-9 and a second vector containing a second polynucleotide encoding a light chain variable region and a light chain of an antibody according to any one of claims 1-9. 15. A method for producing an antibody that binds to human TIM-3, wherein the method includes culturing a recombinant host cell containing: (a) a polynucleotide encoding a heavy chain variable region and a light chain variable region or a heavy chain or light chain of an antibody according to any one of claims 1 to 9; (b) a first polynucleotide encoding a heavy chain variable region or a heavy chain of an antibody according to any one of claims 1 to 9 and a second polynucleotide encoding a light chain variable region or a light chain of an antibody according to any one of paragraphs 1 to 9; (c) a vector containing a polynucleotide encoding a heavy chain variable region and a light chain variable region, or a heavy chain or light chain of an antibody according to any one of claims 1 to 9; or (d) a first vector containing a first polynucleotide encoding a heavy chain variable region or a heavy chain of an antibody according to any one of claims 1 to 9 and a second vector containing a second polynucleotide encoding a light chain variable region and a light chain of an antibody according to any one of paragraphs. 1-9 such that the polynucleotide is expressed and an antibody is produced. - 84 043100 16. A method of increasing T cell activation in response to an antigen in an individual, wherein the method comprises administering to said individual an effective amount of an isolated antibody according to any one of claims 1 to 9 or a pharmaceutical composition according to claim 10. 17. A method of treating cancer in an individual, wherein the method comprises administering to said individual an effective amount of an isolated antibody according to any one of claims 1 to 9 or a pharmaceutical composition according to claim 10. 18. A method of treating an infectious disease in an individual, wherein the method comprises administering to said individual an effective amount of an isolated antibody according to any one of claims 1 to 9 or a pharmaceutical composition according to claim 10.