HK40113626A - Anti-tim-3 antibodies and methods of use thereof - Google Patents

Description

Anti-TIM-3 antibody and its usage

This application is a divisional application of patent application No. 201780046441.7, filed on May 26, 2017, entitled "Anti-TIM-3 Antibody and Method of Using Thereof".

1. Cross-referencing of related applications

This application claims the benefits of U.S. Provisional Application No. 62/342,610, filed May 27, 2016, and U.S. Provisional Application No. 62/420,276, filed November 10, 2016, each of which is incorporated herein by reference in its entirety.

2. Technical Field

This disclosure relates to antibodies that specifically bind to TIM-3 (e.g., human TIM-3) and methods of using them.

3. Background Technology

T-cell immunoglobulin and mucin domain-3 (TIM-3) is a type I membrane protein of the immunoglobulin (Ig) superfamily. It has an extracellular Ig variable-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 type 1 helper T (Th1) lymphocytes and CD8 ⁺ T (Tc1) lymphocytes, 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 plays a role in suppressing T cell, myeloid cell, and NK cell-mediated responses and promoting immune tolerance. For example, TIM-3IgV peptide, which fuses with an immunoglobulin domain and binds to and neutralizes TIM-3 ligands, induces excessive proliferation of Th1 cells and release of Th1 cytokines in immunized mice (Sabatos et al. (2003) Nat Immunol 4:1102-10). In fact, in vivo administration of anti-TIM-3 antibodies enhances the pathological severity of experimental autoimmune encephalomyelitis (i.e., an animal model of multiple sclerosis) (Monney et al. (2002) Nature 415:536-41). Moreover, TIM-3 expression is upregulated in CD8 ⁺ T cells of cancer patients. For example, approximately 30% of NY-ESO-1-specific CD8+ T cells in patients with advanced melanoma show upregulated TIM-3 expression (Fourcade et al. (2010) J Exp Med 207:2175–86).

Given the significant role of human TIM-3 in regulating immune responses, therapeutic agents designed to antagonize TIM-3 signaling show promise for treating diseases involving TIM-3-mediated immunosuppression.

4. Summary of the Invention

This disclosure provides antibodies that specifically bind to and antagonize TIM-3 (e.g., human TIM-3), such as TIM-3-mediated immunosuppression. Pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for preparing these antibodies, and methods for treating subjects using these antibodies are also provided. The antibodies disclosed herein are particularly useful for increasing T cell activation in response to antigens (e.g., tumor antigens or infectious disease antigens) and/or reducing Treg-mediated immunosuppression, and are therefore particularly useful for treating cancer in subjects or for treating or preventing infectious diseases in subjects.

Therefore, in one aspect, this disclosure provides an antibody or isolated antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions CDRH1, CDRH2, and CDRH3, and the light chain variable region comprises complementarity-determining regions CDRL1, CDRL2, and CDRL3, wherein:

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

_X1 is R, S, A, G, K, M, or T.

X ₂ can be Q, S, A, G, R, or T.

X ₃ can be N, Y, G, or Q.

X ₄ is either A or Q, and

X ₅ can be 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 X ₁ ASQSVX ₂ SSYLA (SEQ ID NO:52), in which

_X1 is R or G, and

_X2 either does not exist or is S;

(e) CDRL2 contains the amino acid sequence X ₁ ASX ₂ RAT (SEQ ID NO:53).

in

_X1 is either D or G, and

_X2 is N, S, or T; and

(f) CDRL3 contains the amino acid sequence QQYGSSPX ₁ T (SEQ ID NO:54), where X ₁ is L or I.

On the other hand, this disclosure provides an antibody or isolated antibody that specifically binds to human TIM-3, said antibody comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising complementarity-determining regions CDRH1, CDRH2, and CDRH3, said light chain variable region comprising complementarity-determining regions CDRL1, CDRL2, and CDRL3, wherein:

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

_X1 is R, S, A, G, K, M, or T.

X ₂ can be Q, S, A, G, R, or T.

X ₃ can be N, Y, G, or Q.

X ₄ is either A or Q, and

X ₅ can be 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 X ₁ ASQSVX ₂ SSYLA (SEQ ID NO:52), in which

_X1 is R or G, and

_X2 either does not exist or is S;

(e) CDRL2 contains the amino acid sequence X ₁ ASX ₂ RAT (SEQ ID NO:53).

in

_X1 is either D or G, and

_X2 is N, S, or T; and

(f) CDRL3 contains the amino acid sequence QQYGSSPX ₁ T (SEQ ID NO:54), where X ₁ is L or I.

On the other hand, this disclosure provides an antibody or isolated antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having complementarity-determining regions CDRH1, CDRH2 and CDRH3, the light chain variable region having complementarity-determining regions CDRL1, CDRL2 and CDRL3, wherein the antibody is internalized upon binding to cells expressing human TIM-3, and wherein CDRH3 contains the amino acid sequence AKGGDYGGNYFD (SEQ ID NO:3).

On the other hand, this disclosure provides an antibody or isolated antibody that specifically binds to human TIM-3, the antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having complementarity-determining regions CDRH1, CDRH2 and CDRH3, the light chain variable region having complementarity-determining regions CDRL1, CDRL2 and CDRL3, wherein the antibody is internalized upon binding to cells expressing human TIM-3, and wherein CDRH3 contains the amino acid sequence AKGGDYGGNYFD (SEQ ID NO:3).

In some implementation schemes:

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

_X1 is R, S, A, G, K, M, or T.

X ₂ can be Q, S, A, G, R, or T.

X ₃ can be N, Y, G, or Q.

X ₄ is either A or Q, and

X ₅ can be 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 X ₁ ASQSVX ₂ SSYLA (SEQ ID NO:52), in which

_X1 is R or G, and

_X2 either does not exist or is S;

(d) CDRL2 contains the amino acid sequence X ₁ ASX ₂ RAT (SEQ ID NO:53).

in

_X1 is either D or G, and

_X2 is N, S, or T; and

(e)CDRL3 contains the amino acid sequence QQYGSSPX ₁ T (SEQ ID NO:54), where X ₁ is L or I.

In some embodiments, CDRH1 comprises the amino acid sequence _{X1 X2} NAWS (SEQ ID NO:49), wherein _X1 is R or A; and _X2 is Q or R. In some embodiments, CDRH1 comprises the amino acid sequence _{X1 X2 GQX3} S (SEQ ID NO:50), wherein _X1 is K, M, or G; _X2 is A or S; and _X3 is S or T. In some embodiments, CDRH1 comprises the amino acid sequence _{X1 X2} QQAS (SEQ ID NO:51), wherein _X1 is S, R, T, or G; and _X2 is A, S, T, or G. In some embodiments, CDRH1 comprises an amino acid sequence selected from SEQ ID NO:1 and 4-12.

In some embodiments, CDRL1 comprises an amino acid sequence selected from SEQ ID NO:13-16. In some embodiments, CDRL2 comprises an amino acid sequence selected from SEQ ID NO:17-21. In some embodiments, CDRL3 comprises an amino acid sequence selected from SEQ ID NO:22 and 23.

In some embodiments, CDRH1, CDRH2, and CDRH3 comprise the amino acid sequences of CDRH1, CDRH2, and CDRH3 listed in 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.

In some embodiments, CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences of CDRL1, CDRL2, and CDRL3 listed in 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.

In some embodiments, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences listed in SEQ ID NO: 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.

On the other hand, this disclosure provides an antibody or isolated antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions CDRH1, CDRH2, and CDRH3, and the light chain variable region comprises complementarity-determining regions CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 respectively comprise the amino acid sequences listed in SEQ ID NO: 1, 2, 3, 14, 21, and 22.

On the other hand, this disclosure provides 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 heavy chain variable region comprises complementarity-determining regions CDRH1, CDRH2, and CDRH3, and the light chain variable region comprises complementarity-determining regions CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 respectively comprise the amino acid sequences listed in SEQ ID NO: 1, 2, 3, 14, 21, and 22.

On the other hand, this disclosure provides an antibody or isolated antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions CDRH1, CDRH2, and CDRH3, and the light chain variable region comprises complementarity-determining regions CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 respectively comprise the amino acid sequences listed in SEQ ID NO: 5, 2, 3, 14, 21, and 22.

On the other hand, this disclosure provides 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 heavy chain variable region comprises complementarity-determining regions CDRH1, CDRH2, and CDRH3, and the light chain variable region comprises complementarity-determining regions CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 respectively comprise the amino acid sequences listed in SEQ ID NO: 5, 2, 3, 14, 21, and 22.

On the other hand, this disclosure provides an antibody or isolated antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions CDRH1, CDRH2, and CDRH3, and the light chain variable region comprises complementarity-determining regions CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 respectively comprise the amino acid sequences listed in SEQ ID NO: 9, 2, 3, 14, 21, and 22.

On the other hand, this disclosure provides 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 heavy chain variable region comprises complementarity-determining regions CDRH1, CDRH2, and CDRH3, and the light chain variable region comprises complementarity-determining regions CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 respectively comprise the amino acid sequences listed in SEQ ID NO: 9, 2, 3, 14, 21, and 22.

On the other hand, this disclosure provides an antibody or isolated antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions CDRH1, CDRH2, and CDRH3, and the light chain variable region comprises complementarity-determining regions CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 respectively comprise the amino acid sequences listed in SEQ ID NO: 1, 2, 3, 15, 18, and 22.

On the other hand, this disclosure provides 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 heavy chain variable region comprises complementarity-determining regions CDRH1, CDRH2, and CDRH3, and the light chain variable region comprises complementarity-determining regions CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 respectively comprise the amino acid sequences listed in SEQ ID NO: 1, 2, 3, 15, 18, and 22.

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

On the other hand, this disclosure provides an antibody or isolated antibody that specifically binds to human TIM-3, wherein the antibody is internalized after binding to cells expressing human TIM-3.

In some embodiments, in an assay comprising the steps of: (a) seeding human TIM-3-expressing cells at 2 x ^10⁴ cells per well in a tissue culture plate; (b) adding 1111 ng/ml of αHFc- _NC -DM1 and 1111 ng/ml of the antibody or pab1944w (IgG 1 N297A) to a final volume of 100 μl/well; (c) incubating at 37°C and 5% _CO₂ for 72 h; (d) measuring the viability of the human TIM- ₃ -expressing cells; and (e) calculating the percentage of cell survival relative to untreated human TIM-3-expressing cells. In some embodiments, the percentage of cell survival in the presence of the antibody is at least 50% lower than the percentage of cell survival in the presence of pab1944w (IgG ₁ N297A). In some embodiments, the cells expressing human TIM-3 are Kasumi-3 cells. In some embodiments, the cells expressing human TIM-3 are Kasumi-3 cells (CRL-2725 ^™ ). In some embodiments, the cells expressing human TIM-3 are Jurkat cells engineered to express human TIM-3.

In some embodiments, in an assay comprising the steps of: (a) seeding the human TIM-3-expressing cells at 2 x ^10⁴ cells per well in a tissue culture plate; (b) adding 1111 ng/ml of αHFc _- NC-DM1 and 1111 ng/ml of the antibody or Hum11 (IgG ₄ S228P) to a final volume of 100 μl/well; (c) incubating at 37°C and 5% _CO₂ for 72 h; (d) measuring the survival of the human TIM-3-expressing cells; and (e) calculating the percentage of cell survival relative to untreated human TIM-3-expressing cells. In some embodiments, the percentage of cell survival in the presence of the antibody is at least 50% lower than the percentage of cell survival in the presence of Hum11 (IgG ₄ S228P). In some embodiments, the cells expressing human TIM-3 are Kasumi-3 cells. In some embodiments, the cells expressing human TIM-3 are Kasumi-3 cells (CRL-2725 ^™ ). In some embodiments, the cells expressing human TIM-3 are Jurkat cells engineered to express human TIM-3.

In some embodiments, the antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO:55. In some embodiments, the antibody comprises a heavy chain variable region containing at least 75%, 80%, 85%, 90%, 95%, or 100% identical amino acid sequences selected from SEQ ID NO:24-35. In some embodiments, the heavy chain variable region contains amino acid sequences selected from SEQ ID NO:24-35. In some embodiments, the heavy chain variable region contains the amino acid sequence of SEQ ID NO:25. In some embodiments, the heavy chain variable region contains the amino acid sequence of SEQ ID NO:28. In some embodiments, the heavy chain variable region contains the amino acid sequence of SEQ ID NO:32. In some embodiments, the N-terminal glutamic acid (E) residue of the heavy chain variable region of the antibody as described herein is replaced by a pyroglutamic acid (pE) residue.

In some embodiments, the antibody comprises a light chain variable region containing the amino acid sequence of SEQ ID NO:56. In some embodiments, the antibody comprises a light chain variable region containing at least 75%, 80%, 85%, 90%, 95%, or 100% identical amino acid sequences to those selected from SEQ ID NO:36-47. In some embodiments, the light chain variable region contains amino acid sequences selected from SEQ ID NO:36-47. In some embodiments, the light chain variable region contains the amino acid sequence of SEQ ID NO:46. In some embodiments, the N-terminal glutamic acid (E) residue of the light chain variable region of the antibody as described herein is replaced by a pyroglutamic acid (pE) residue.

On the other hand, this disclosure provides an antibody or isolated antibody comprising a heavy chain variable region containing an amino acid sequence selected from SEQ ID NO:24-35. In some embodiments, the heavy chain variable region contains an amino acid sequence of SEQ ID NO:25. In some embodiments, the heavy chain variable region contains an amino acid sequence of SEQ ID NO:28. In some embodiments, the heavy chain variable region contains an amino acid sequence of SEQ ID NO:32. In some embodiments, the antibody comprises a heavy chain containing an amino acid sequence of SEQ ID NO:58. In some embodiments, the antibody comprises a heavy chain containing an amino acid sequence of SEQ ID NO:61. In some embodiments, the antibody comprises a heavy chain containing an amino acid sequence of SEQ ID NO:65. In some embodiments, the antibody comprises a heavy chain containing an amino acid sequence of SEQ ID NO:70, 71, 72, 73, 74, or 75. In some embodiments, the N-terminal glutamic acid (E) residue of the antibody heavy chain as described herein is replaced by a pyroglutamic acid (pE) residue.

On the other hand, this disclosure provides an antibody or isolated antibody that specifically binds to human TIM-3, said antibody comprising a heavy chain variable region containing an amino acid sequence selected from SEQ ID NO: 24-35. In some embodiments, the heavy chain variable region contains an amino acid sequence of SEQ ID NO: 25. In some embodiments, the heavy chain variable region contains an amino acid sequence of SEQ ID NO: 28. In some embodiments, the heavy chain variable region contains an amino acid sequence of SEQ ID NO: 32. In some embodiments, the antibody comprises a heavy chain containing an amino acid sequence of SEQ ID NO: 58. In some embodiments, the antibody comprises a heavy chain containing an amino acid sequence of SEQ ID NO: 61. In some embodiments, the antibody comprises a heavy chain containing an amino acid sequence of SEQ ID NO: 65. In some embodiments, the antibody comprises a heavy chain containing an amino acid sequence of SEQ ID NO: 70, 71, 72, 73, 74, or 75.

On the other hand, this disclosure provides an antibody or isolated antibody comprising a light chain variable region containing an amino acid sequence selected from SEQ ID NO:36-47. In some embodiments, the light chain variable region comprises an amino acid sequence of SEQ ID NO:46. In some embodiments, the light chain variable region comprises an amino acid sequence of SEQ ID NO:69. In some embodiments, the antibody comprises a light chain containing an amino acid sequence of SEQ ID NO:76 or 77. In some embodiments, the N-terminal glutamic acid (E) residue of the antibody light chain as described herein is replaced by a pyroglutamic acid (pE) residue.

On the other hand, this disclosure provides an antibody or isolated antibody that specifically binds to human TIM-3, said antibody comprising a light chain variable region containing an amino acid sequence selected from SEQ ID NO:36-47. In some embodiments, the light chain variable region contains an amino acid sequence of SEQ ID NO:46. In some embodiments, the light chain variable region contains an amino acid sequence of SEQ ID NO:69. In some embodiments, the antibody comprises a light chain containing an amino acid sequence of SEQ ID NO:76 or 77.

On the other hand, this disclosure provides an antibody or isolated antibody comprising 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 listed in 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, the heavy chain variable region and the light chain variable region comprise the amino acid sequences listed in SEQ ID NO:25 and 46, respectively. In some embodiments, the heavy chain variable region and the light chain variable region comprise the amino acid sequences listed in SEQ ID NO:28 and 46, respectively. In some embodiments, the heavy chain variable region and the light chain variable region comprise the amino acid sequences listed in SEQ ID NO:32 and 46, respectively. In some embodiments, the N-terminal glutamate (E) residue of the antibody heavy chain variable region, as described herein, is replaced by a pyroglutamate (pE) residue and/or the N-terminal glutamate (E) residue of the antibody light chain variable region is replaced by a pyroglutamate (pE) residue.

On the other hand, this disclosure provides an antibody or isolated antibody comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region are respectively composed of the amino acid sequences listed in 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, the amino acid sequences of the heavy chain variable region and the light chain variable region consist of the amino acid sequences listed in SEQ ID NO: 25 and 46, respectively. In some embodiments, the amino acid sequences of the heavy chain variable region and the light chain variable region consist of the amino acid sequences listed in SEQ ID NO: 28 and 46, respectively. In some embodiments, the amino acid sequences of the heavy chain variable region and the light chain variable region consist of the amino acid sequences listed in SEQ ID NO: 32 and 46, respectively.

On the other hand, this disclosure provides an antibody or isolated antibody that specifically binds to human TIM-3, the antibody comprising 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 listed in 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, the heavy chain variable region and the light chain variable region respectively comprise the amino acid sequences listed in SEQ ID NO: 25 and 46. In some embodiments, the heavy chain variable region and the light chain variable region respectively comprise the amino acid sequences listed in SEQ ID NO: 28 and 46. In some embodiments, the heavy chain variable region and the light chain variable region respectively comprise the amino acid sequences listed in SEQ ID NO: 32 and 46.

On the other hand, this disclosure provides an antibody or isolated antibody that specifically binds to human TIM-3, said antibody 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 are respectively defined by 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; The amino acid sequences comprised of SEQ ID NO: 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 the heavy chain variable region and the light chain variable region comprised of the amino acid sequences listed in SEQ ID NO: 25 and 46, respectively. In some embodiments, the amino acid sequences of the heavy chain variable region and the light chain variable region comprised of the amino acid sequences listed in SEQ ID NO: 28 and 46, respectively. In some embodiments, the amino acid sequences of the heavy chain variable region and the light chain variable region consist of the amino acid sequences listed in SEQ ID NO:32 and 46, respectively.

On the other hand, this disclosure provides an antibody or isolated antibody 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.

On the other hand, this disclosure provides an antibody or isolated antibody that specifically binds to human TIM-3, said antibody 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.

On the other hand, this disclosure provides an antibody or isolated antibody 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.

On the other hand, this disclosure provides an antibody or isolated antibody that specifically binds to human TIM-3, said antibody 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.

On the other hand, this disclosure provides an antibody or isolated antibody 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.

On the other hand, this disclosure provides an antibody or isolated antibody that specifically binds to human TIM-3, said antibody 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 antibody heavy chain, as described herein, is replaced by a pyroglutamate (pE) residue and/or the N-terminal glutamate (E) residue of the antibody light chain is replaced by a pyroglutamate (pE) residue.

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

On the other hand, this disclosure provides an antibody or isolated antibody that specifically binds to human TIM-3, said antibody comprising a heavy chain variable region having an amino acid sequence derived from a human IGHV3-23 germline sequence and a light chain variable region having an amino acid sequence derived from a human germline sequence selected from IGKV1-27, IGKV3-11, IGKV3-20 and IGKV3D-20.

In some embodiments, the antibody comprises a heavy chain constant region selected from human _IgG1 , _IgG2 , _IgG3 , _IgG4 , _IgA1 , and _IgA2 . In some embodiments, the heavy chain constant region is _IgG1 . In some embodiments, the amino acid sequence of _IgG1 contains the N297A mutation numbered according to the EU numbering system. In some embodiments, the antibody comprises a heavy chain constant region containing the amino acid sequence of SEQ ID NO:72. In some embodiments, the amino acid sequence of _IgG1 contains the N297Q mutation numbered according to the EU numbering system. In some embodiments, the _IgG1 is non-fucosylated _IgG1 . In some embodiments, the heavy chain constant region is _IgG4 . In some embodiments, the amino acid sequence of _IgG4 contains the S228P mutation numbered according to the EU numbering system. In some embodiments, the antibody comprises a heavy chain constant region containing the amino acid sequence of SEQ ID NO:74.

In some embodiments, the antibody comprises a light chain constant region selected from human IgGκ and IgGλ. In some embodiments, the light chain constant region is IgGκ. In some embodiments, the antibody comprises a light chain constant region containing the amino acid sequence SEQ ID NO:76. In some embodiments, the light chain constant region is IgGλ.

On the other hand, this disclosure provides an antibody or isolated antibody that cross-competes with the antibody disclosed herein to bind human TIM-3. In some embodiments, this disclosure provides an antibody or isolated antibody that cross-competes with an antibody comprising the heavy chain and light chain variable region amino acid sequences as listed in SEQ ID NO: 55 and 56, respectively. In some embodiments, this disclosure provides an antibody or isolated antibody that cross-competes with an antibody comprising the heavy chain and light chain variable region amino acid sequences as listed in SEQ ID NO: 25 and 46, respectively. In some embodiments, this disclosure provides an antibody or isolated antibody that cross-competes with an antibody comprising the heavy chain and light chain variable region amino acid sequences as listed in SEQ ID NO: 28 and 46, respectively. In some embodiments, this disclosure provides an antibody or isolated antibody that cross-competes with an antibody comprising the heavy chain and light chain variable region amino acid sequences as listed in SEQ ID NO: 32 and 46, respectively.

On the other hand, this disclosure provides an antibody or isolated antibody that binds to the same epitope of human TIM-3 as the antibodies disclosed herein. In some embodiments, this disclosure provides an antibody or isolated antibody that binds to the same epitope of human TIM-3 as antibodies comprising the heavy chain and light chain variable region amino acid sequences as listed in SEQ ID NO: 55 and 56, respectively. In some embodiments, this disclosure provides an antibody or isolated antibody that binds to the same epitope of human TIM-3 as antibodies comprising the heavy chain and light chain variable region amino acid sequences as listed in SEQ ID NO: 25 and 46, respectively. In some embodiments, this disclosure provides an antibody or isolated antibody that binds to the same epitope of human TIM-3 as antibodies comprising the heavy chain and light chain variable region amino acid sequences as listed in SEQ ID NO: 28 and 46, respectively. In some embodiments, this disclosure provides an antibody or isolated antibody that binds to the same epitope of human TIM-3 as antibodies comprising the heavy chain and light chain variable region amino acid sequences as listed in SEQ ID NO: 32 and 46, respectively.

On the other hand, this disclosure provides an antibody or isolated antibody that specifically binds to human TIM-3, wherein the antibody specifically binds to a variant TIM-3 protein having the amino acid sequence of SEQ ID NO:101 with a lower affinity than wild-type TIM-3 protein having the amino acid sequence of SEQ ID NO:79.

On the other hand, this disclosure provides an antibody or isolated antibody that binds to the same epitope of human TIM-3 as the antibody of the present invention. In one embodiment, the antibody specifically binds to a variant TIM-3 protein having the amino acid sequence of SEQ ID NO: 101 with a lower affinity than that of wild-type TIM-3 protein having the amino acid sequence of SEQ ID NO: 79.

On the other hand, this disclosure provides an antibody or isolated antibody that specifically binds to human TIM-3, wherein the antibody does not specifically bind to a variant TIM-3 protein having the amino acid sequence of SEQ ID NO:101.

On the other hand, this disclosure provides an antibody or isolated antibody that specifically binds to the same epitope of human TIM-3 as any antibody of the present invention. In one embodiment, the antibody does not specifically bind to a variant TIM-3 protein having the amino acid sequence of SEQ ID NO: 101.

On the other hand, this disclosure provides an antibody or isolated antibody that specifically binds to human TIM-3, wherein the binding of the antibody to a variant TIM-3 protein having the amino acid sequence of SEQ ID NO: 101 is significantly weakened relative to the binding between the antibody and a wild-type TIM-3 protein having the amino acid sequence of SEQ ID NO: 79.

On the other hand, this disclosure provides an antibody or isolated antibody that specifically binds to the same epitope of human TIM-3 as any antibody of the present invention. In one embodiment, the binding of the antibody to a variant TIM-3 protein having the amino acid sequence of SEQ ID NO: 101 is significantly weakened relative to the binding between the antibody and wild-type TIM-3 protein having the amino acid sequence of SEQ ID NO: 79.

On the other hand, this disclosure provides an antibody or isolated antibody that specifically binds to human TIM-3, wherein, compared with binding to wild-type TIM-3 protein having the amino acid sequence of SEQ ID NO:79, the antibody exhibits reduced or absent binding to variant TIM-3 protein having the amino acid sequence of SEQ ID NO:101.

On the other hand, this disclosure provides an antibody or isolated antibody that specifically binds to the same epitope of human TIM-3 as any antibody of the present invention. In one embodiment, the antibody exhibits reduced or absent binding to variant TIM-3 protein having the amino acid sequence of SEQ ID NO: 101, compared to binding to wild-type TIM-3 protein having the amino acid sequence of SEQ ID NO: 79.

On the other hand, this disclosure provides an antibody or isolated antibody that binds (e.g., specifically binds) to an epitope of human TIM-3. In some embodiments, the antibody binds to residue 40 of SEQ ID NO:79.

On the other hand, this disclosure provides an antibody or isolated antibody that specifically binds to the same epitope of human TIM-3 as any antibody of the present invention. In some embodiments, the antibody binds to residue 40 of SEQ ID NO:79.

On the other hand, this disclosure provides an antibody or isolated antibody that binds (e.g., specifically binds) to an epitope of human TIM-3. In some embodiments, the antibody binds to an epitope located within the human TIM-3 region consisting of the amino acid sequence of SEQ ID NO: 93. In some embodiments, the antibody binds to an epitope located within the human TIM-3 region consisting of the amino acid sequence of SEQ ID NO: 94. In some embodiments, the antibody binds to an epitope located within the human TIM-3 region consisting of the amino acid sequence of SEQ ID NO: 95. In some embodiments, the antibody binds to an epitope located within the human TIM-3 region consisting of the amino acid sequence of SEQ ID NO: 96. In some embodiments, the antibody binds to an epitope located within the human TIM-3 region consisting of the amino acid sequence of SEQ ID NO: 97. In some embodiments, the antibody binds to an epitope located within the human TIM-3 region consisting of the amino acid sequence of SEQ ID NO: 98. In some embodiments, the antibody binds to an epitope located within the human TIM-3 region consisting of the amino acid sequence of SEQ ID NO: 99. In some embodiments, the antibody binds to an epitope located within the human TIM-3 region consisting of the amino acid sequence of SEQ ID NO: 100.

On the other hand, this disclosure provides an antibody or isolated antibody that specifically binds to the same epitope of human TIM-3 as any antibody of the present invention. In some embodiments, the antibody binds to an epitope located within the human TIM-3 region consisting of the amino acid sequence of SEQ ID NO: 93. In some embodiments, the antibody binds to an epitope located within the human TIM-3 region consisting of the amino acid sequence of SEQ ID NO: 94. In some embodiments, the antibody binds to an epitope located within the human TIM-3 region consisting of the amino acid sequence of SEQ ID NO: 95. In some embodiments, the antibody binds to an epitope located within the human TIM-3 region consisting of the amino acid sequence of SEQ ID NO: 96. In some embodiments, the antibody binds to an epitope located within the human TIM-3 region consisting of the amino acid sequence of SEQ ID NO: 97. In some embodiments, the antibody binds to an epitope located within the human TIM-3 region consisting of the amino acid sequence of SEQ ID NO: 98. In some embodiments, the antibody binds to an epitope located within the human TIM-3 region consisting of the amino acid sequence of SEQ ID NO: 99. In some embodiments, the antibody binds to an epitope located within the human TIM-3 region consisting of the amino acid sequence of SEQ ID NO: 100.

On the other hand, this disclosure provides an antibody that, as determined by hydrogen/deuterium determination, reduces hydrogen/deuterium exchange in the region consisting of the amino acid sequence listed in SEQ ID NO:93 when binding to a human TIM-3 protein or a fragment thereof containing the amino acid sequence of SEQ ID NO:102, relative to the hydrogen/deuterium exchange in the region consisting of the amino acid sequence listed in SEQ ID NO:94 when the antibody is absent. On the other hand, this disclosure provides an antibody that, as determined by hydrogen/deuterium determination, reduces hydrogen/deuterium exchange in the region consisting of the amino acid sequence listed in SEQ ID NO:94 when binding to a human TIM-3 protein or a fragment thereof containing the amino acid sequence of SEQ ID NO:102, relative to the hydrogen/deuterium exchange in the region consisting of the amino acid sequence listed in SEQ ID NO:94 when the antibody is absent. On the other hand, this disclosure provides an antibody that, as determined by hydrogen/deuterium determination, reduces hydrogen/deuterium exchange in the region consisting of the amino acid sequence listed in SEQ ID NO:95 when binding to a human TIM-3 protein or a fragment thereof containing the amino acid sequence of SEQ ID NO:102, relative to the hydrogen/deuterium exchange in the region consisting of the amino acid sequence listed in SEQ ID NO:96 when the antibody is absent. On the other hand, this disclosure provides an antibody that, as determined by hydrogen/deuterium determination, reduces hydrogen/deuterium exchange in the region consisting of the amino acid sequence listed in SEQ ID NO:96 when binding to a human TIM-3 protein or a fragment thereof containing the amino acid sequence of SEQ ID NO:102, relative to the hydrogen/deuterium exchange in the region consisting of the amino acid sequence listed in SEQ ID NO:96 when the antibody is absent. On the other hand, this disclosure provides an antibody that, as determined by a hydrogen/deuterium assay, reduces hydrogen/deuterium exchange in the region consisting of the amino acid sequence listed in SEQ ID NO:97 when bound to a human TIM-3 protein or a fragment thereof containing the amino acid sequence of SEQ ID NO:102, relative to hydrogen/deuterium exchange in the region consisting of the amino acid sequence listed in SEQ ID NO:98 when bound, as determined by a hydrogen/deuterium assay, relative to hydrogen/deuterium exchange in the region consisting of the amino acid sequence listed in SEQ ID NO:98 when bound to a human TIM-3 protein or a fragment thereof containing the amino acid sequence of SEQ ID NO:102. In some embodiments, such as as described herein in the examples, the reduction in hydrogen/deuterium exchange is measured using a hydrogen-deuterium exchange method (HDX).

On the other hand, this disclosure provides an antibody or isolated antibody that specifically binds to the same epitope of human TIM-3 as any antibody of the present invention. In some embodiments, as determined by hydrogen/deuterium assay, the antibody reduces hydrogen/deuterium exchange in the region consisting of the amino acid sequence listed in SEQ ID NO:93 when binding to human TIM-3 protein or a fragment thereof containing the amino acid sequence of SEQ ID NO:102, relative to hydrogen/deuterium exchange in the region consisting of the amino acid sequence listed in SEQ ID NO:94 when binding to human TIM-3 protein or a fragment thereof containing the amino acid sequence of SEQ ID NO:102, as determined by hydrogen/deuterium assay. In some embodiments, the antibody reduces hydrogen/deuterium exchange in the region consisting of the amino acid sequence listed in SEQ ID NO:94 when binding to human TIM-3 protein or a fragment thereof containing the amino acid sequence of SEQ ID NO:102, relative to hydrogen/deuterium exchange in the region consisting of the amino acid sequence listed in SEQ ID NO:94 when binding to human TIM-3 protein or a fragment thereof containing the amino acid sequence of SEQ ID NO:102, as determined by hydrogen/deuterium assay. In some embodiments, as determined by hydrogen/deuterium determination, the antibody reduces hydrogen/deuterium exchange in the region comprising the amino acid sequence listed in SEQ ID NO:95 when binding to human TIM-3 protein or a fragment thereof containing the amino acid sequence of SEQ ID NO:102, relative to hydrogen/deuterium exchange in the region comprising the amino acid sequence listed in SEQ ID NO:95 when the antibody is absent. In some embodiments, as determined by hydrogen/deuterium determination, the antibody reduces hydrogen/deuterium exchange in the region comprising the amino acid sequence listed in SEQ ID NO:96 when binding to human TIM-3 protein or a fragment thereof containing the amino acid sequence of SEQ ID NO:102, relative to hydrogen/deuterium exchange in the region comprising the amino acid sequence listed in SEQ ID NO:96 when the antibody is absent. In some embodiments, as determined by hydrogen/deuterium assay, the antibody reduces hydrogen/deuterium exchange in the region comprising the amino acid sequence listed in SEQ ID NO:97 upon binding to a human TIM-3 protein or a fragment thereof containing the amino acid sequence of SEQ ID NO:102, relative to hydrogen/deuterium exchange in the region comprising the amino acid sequence listed in SEQ ID NO:97 when the antibody is absent. In some embodiments, as determined by hydrogen/deuterium assay, the antibody reduces hydrogen/deuterium exchange in the region comprising the amino acid sequence listed in SEQ ID NO:98 upon binding to a human TIM-3 protein or a fragment thereof containing the amino acid sequence of SEQ ID NO:102, relative to hydrogen/deuterium exchange in the region comprising the amino acid sequence listed in SEQ ID NO:98 when the antibody is absent. In some embodiments, the reduction in hydrogen/deuterium exchange is measured using a hydrogen-deuterium exchange method (HDX), for example as described herein in the examples.

On the other hand, this disclosure provides an antibody or isolated antibody that binds (e.g., specifically binds) to the same epitope of human TIM-3 as any antibody of the present invention, wherein the epitope is determined by, for example, the hydrogen-deuterium exchange method (HDX) described in the examples, by, for example, the peptide scanning analysis described in the examples, or by, for example, the alanine scanning method described in the examples.

In some embodiments, the antibody comprises a human IgG heavy chain constant region that is a variant of the wild-type human IgG heavy chain constant region, wherein the variant human IgG heavy chain constant region has a lower affinity for binding to the human Fcγ receptor than the wild-type human IgG heavy chain constant region. In some embodiments, the human Fcγ receptor is selected from FcγRI, FcγRII, and FcγRIII. In some embodiments, the variant human IgG heavy chain constant region is an IgG ₁ constant region containing the N297A mutation.

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

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

On the other hand, this disclosure provides antibodies or isolated antibodies conjugated to cytotoxic agents as disclosed herein.

On the other hand, this disclosure provides antibodies or isolated antibodies conjugated to cell inhibitors as disclosed herein.

On the other hand, this disclosure provides antibodies conjugated to toxins, as disclosed herein, or isolated antibodies.

On the other hand, this disclosure provides antibodies or isolated antibodies conjugated to radionuclides as disclosed herein.

On the other hand, this disclosure provides antibodies or isolated antibodies conjugated to detectable markers as disclosed herein.

On the other hand, this disclosure provides a pharmaceutical composition comprising an antibody as disclosed herein and a pharmaceutically acceptable carrier or excipient.

On the other hand, this disclosure provides a polynucleotide or isolated polynucleotide encoding the heavy and/or light chains of an antibody as disclosed herein. On the other hand, this disclosure provides a vector comprising said polynucleotide. On the other hand, this disclosure provides a recombinant host cell comprising said polynucleotide. On the other hand, this disclosure provides a recombinant host cell comprising said vector. On the other hand, this disclosure provides a method for generating an antibody as disclosed herein, the method comprising culturing the host cell to express the polynucleotide and generate the antibody. In one embodiment, the method is an in vitro method.

In one embodiment, the present invention relates to the antibody of the present invention used as a drug, or the pharmaceutical composition of the present invention, or the polynucleotide of the present invention, or the vector of the present invention, or the recombinant host cell of the present invention.

In one embodiment, the present invention relates to the antibody of the present invention used as a diagnostic agent, or the pharmaceutical composition of the present invention, or the polynucleotide of the present invention, or the vector of the present invention, or the recombinant host cell of the present invention.

On the other hand, this disclosure provides a method for increasing T-cell activation in response to a subject antigen, the method comprising administering to the subject an effective amount of an antibody or pharmaceutical composition as disclosed herein. On the other hand, this disclosure provides a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of an antibody or pharmaceutical composition as disclosed herein. In some embodiments of the foregoing methods, the antibody or pharmaceutical composition is administered subcutaneously. In some embodiments of the foregoing methods, the antibody or pharmaceutical composition is administered intravenously. In some embodiments of the foregoing methods, the antibody or pharmaceutical composition is administered intratumorally. In some embodiments of the foregoing methods, the antibody or pharmaceutical composition is delivered to a tumor-draining lymph node. In some embodiments of the foregoing methods, the antibody or pharmaceutical composition is administered intra-arterially.

On one hand, the present invention relates to antibodies, polynucleotides, vectors, recombinant host cells and/or pharmaceutical compositions of the present invention in methods for increasing T cell activation in response to antigens.

On one hand, the present invention relates to antibodies, polynucleotides, vectors, recombinant host cells and/or pharmaceutical compositions of the present invention used in methods for increasing T cell activation in response to subject antigens.

On one hand, the present invention relates to antibodies, polynucleotides, vectors, recombinant host cells and/or pharmaceutical compositions of the present invention used in methods for increasing T cell activation in response to a subject antigen, said methods comprising administering an effective amount of the antibodies, polynucleotides, vectors, recombinant host cells and/or pharmaceutical compositions of the present invention to said subject.

On one hand, the present invention relates to antibodies, polynucleotides, vectors, recombinant host cells and/or pharmaceutical compositions of the present invention in methods for treating cancer.

On one hand, the present invention relates to antibodies, polynucleotides, vectors, recombinant host cells and/or pharmaceutical compositions of the present invention in methods for treating cancer in subjects.

On one hand, the present invention relates to antibodies, polynucleotides, vectors, recombinant host cells and/or pharmaceutical compositions of the present invention in a method for treating cancer in a subject, the method comprising administering an effective amount of the antibodies, polynucleotides, vectors, recombinant host cells and/or pharmaceutical compositions of the present invention to the subject.

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

In some embodiments, the aforementioned method further includes administering an additional therapeutic agent to the subject. Therefore, in one embodiment of the antibody, polynucleotide, vector, recombinant host cell, and/or pharmaceutical composition used in the method of the present invention, the method further includes administering an additional therapeutic agent to the subject.

On the one hand, the present invention relates to (a) antibodies, polynucleotides, carriers, recombinant host cells and/or pharmaceutical compositions of the present invention and (b) adjunctive therapeutic agents used as pharmaceuticals.

On one hand, the present invention relates to (a) the antibodies, polynucleotides, vectors, recombinant host cells and/or pharmaceutical compositions of the present invention and (b) adjunctive therapeutic agents in methods for treating cancer.

On one hand, the present invention relates to pharmaceutical compositions, kits or dispensing kits comprising (a) antibodies, polynucleotides, carriers, recombinant host cells and/or pharmaceutical compositions of the present invention and (b) additional therapeutic agents.

In some embodiments, the additional therapeutic agent is a chemotherapy agent. In some embodiments, the additional therapeutic agent is a radiotherapy agent.

In some embodiments, the additional therapeutic agent is a checkpoint target. In some embodiments, the checkpoint target is selected from antagonist anti-PD-1 antibodies, antagonist anti-PD-L1 antibodies, antagonist anti-PD-L2 antibodies, antagonist anti-CTLA-4 antibodies, antagonist anti-TIM-3 antibodies, antagonist anti-LAG-3 antibodies, antagonist anti-CEACAM1 antibodies, agonist anti-CD137 antibodies, antagonist anti-TIGIT antibodies, antagonist anti-VISTA antibodies, agonist anti-GITR antibodies, and agonist anti-OX40 antibodies. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is pembrolizumab. In some embodiments, the anti-PD-1 antibody is nivolumab.

In some embodiments, the additional therapeutic agent is an indoleamine-2,3-dioxygenase (IDO) inhibitor. In some embodiments, the inhibitor is selected from epacadostat, F001287, indoximod, and NLG919. In some embodiments, the inhibitor is epacadostat. In some embodiments, the inhibitor is F001287. In some embodiments, the inhibitor is indoximod. In some embodiments, the inhibitor is NLG919.

In some embodiments, the adjunctive therapeutic agent is a vaccine. In some embodiments, the vaccine comprises a heat shock protein peptide complex (HSPPC) containing a heat shock protein complexed with an antigenic peptide. In some embodiments, the heat shock protein is hsc70 and complexed with a tumor-associated antigenic peptide. In some embodiments, the heat shock protein is gp96 protein and complexed with a tumor-associated antigenic peptide, wherein the HSPPC is derived from a tumor obtained from a subject. In some embodiments, the adjunctive therapeutic agent comprises a TCR. In some embodiments, the adjunctive therapeutic agent is a soluble TCR. In some embodiments, the adjunctive therapeutic agent is a cell expressing a TCR. In some embodiments, the adjunctive therapeutic agent is a cell expressing a chimeric antigen receptor. In some embodiments, the adjunctive therapeutic agent is an antibody that specifically binds to a peptide-MHC complex. In some embodiments, the adjunctive therapeutic agent is an adjuvant. On one hand, the present invention relates to (a) antibodies, polynucleotides, vectors, recombinant host cells, and/or pharmaceutical compositions of the present invention, and (b) vaccines, used as pharmaceuticals, such as in methods for treating cancer, optionally wherein said vaccine comprises a heat shock protein peptide complex (HSPPC) containing a heat shock protein complexed with an antigenic peptide. On the other hand, the present invention relates to a pharmaceutical composition, kit, or dispensing kit comprising (a) antibodies, polynucleotides, vectors, recombinant host cells, and/or pharmaceutical compositions of the present invention, and (b) a vaccine, optionally wherein said vaccine comprises a heat shock protein peptide complex (HSPPC) containing a heat shock protein complexed with an antigenic peptide.

5. Brief description of the attached diagram

Figure 1 is a set of histograms showing the binding of anti-TIM-3 antibodies pab2085 (IgG ₁ ) and pab2088 (IgG ₁ ) or isotype control antibodies to wild-type mouse 1624-5 cells or engineered 1624-5 cells expressing human TIM-3, as measured by flow cytometry.

Figures 2A and 2B are a pair of graphs showing the binding of anti-TIM-3 antibodies pab2085 (IgG ₁ ) (Figure 2A) and pab2088 (IgG ₁ ) (Figure 2B) to recombinant human TIM-1His (rhTIM-1His), recombinant human TIM-4His (rhTIM-4His), recombinant human TIM-3His (rhTIM-3His), recombinant human TIM-3Fc (rhTIM-3Fc), and recombinant cynomolgus monkey TIM-3Fc (rcmTIM-3Fc), as measured by Luminex assays. Median fluorescence intensity (MFI) values were plotted based on antibody concentrations.

Figures 3A, 3B, 3C, and 3D are a set of histograms showing the binding of anti-TIM-3 antibodies or allotype control antibodies to mouse 1624-5 cells engineered to express human TIM-3 (Figures 3A and 3B) or cynomolgus monkey TIM-3 (Figures 3C and 3D), as measured by flow cytometry. The anti-TIM-3 antibodies tested in this study included 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 the IgG ₁ Fc region.

Figure 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 allotype control antibodies to human primary CD8+ T cells activated with anti-CD3 and anti-CD28 antibodies, as measured by flow cytometry. The light chain optimized variants contain the Fc region of the _IgG1 variant. MFI value curves were plotted based on a series of antibody concentrations tested.

Figure 5 is a graph showing the binding of anti-TIM-3 antibody pab2188 (IgG ₁ variant) or isotype control antibody to primary CD11b+ bone marrow cells of cynomolgus monkeys, as measured by flow cytometry. MFI value curves were plotted based on antibody concentrations.

Figures 6A and 6B are graphs showing the percentage of binding between irradiated WR19L mouse lymphoma cells expressing _{phosphatidylserine} and recombinant human TIM-3Fc (Figure 6A) or recombinant cynomolgus monkey TIM-3Fc (Figure 6B) at dose titration in the presence of anti-TIM-3 antibody or IgG 1 isotype control antibody. The anti-TIM-3 antibodies tested in this study were pab2085 (IgG ₁ ) and pab2188 (IgG ₁ variant).

Figure 7 is a bar chart showing the IFNγ production induced by a combination of anti-TIM-3 antibody or IgG ₁ isotype control antibody and anti-PD-1 antibody pembrolizumab in human peripheral blood mononuclear cells (PBMCs) after stimulation with staphylococcal enterotoxin A (SEA). The anti-TIM-3 antibodies tested in this study included light chain optimized variants pab2175 (IgG ₁ ), pab2176 (IgG ₁ ), pab2180 (IgG ₁ ), pab2182 (IgG ₁ ), pab2183 (IgG ₁ variant), pab2184 (IgG ₁ variant), pab2186 (IgG ₁ variant), pab2187 (IgG ₁ variant), pab2188 (IgG ₁ variant), pab2189 (IgG ₁ variant), pab2190 (IgG ₁ variant), pab2191 (IgG ₁ variant), and pab2192 (IgG ₁ variant).

Figures 8A, 8B, 8C, 8D, 8E, and 8F are a series of bar graphs showing the IFNγ production induced by anti-TIM-3 antibody pab2188w (IgG ₁ N297A) or IgG ₁ N297A isotype control antibody (alone or in combination with anti-PD-1 antibody pembrolizumab) in human PBMCs from six different donors after SEA stimulation. The protocol depicted in Figures 8A-8F is a modification of the protocol depicted in Figure 7.

Figures 9A, 9B, 9C, 9D, 9E, and 9F are graphs or histograms showing the binding of anti-TIM-3 antibodies to TIM-3-expressing cells. In Figures 9A, 9B, 9E, and 9F, MFI value curves are plotted based on a series of antibody concentrations tested. Figures 9C and 9D are a set of histograms showing the binding of anti-TIM-3 antibodies to TIM-3-expressing cells. The anti-TIM-3 antibodies tested included pab2188w (IgG ₁ N297A), AM-1 (IgG ₁ N297A), AM-2 (IgG ₁ N297A), AM-3 (IgG ₁ N297A), AM-4 (IgG ₁ N297A), AM-5 (IgG ₁ N297A), AM-6 (IgG ₁ N297A), AM-7 (IgG ₁ N297A), AM-8 (IgG ₁ N297A), and AM-9 (IgG ₁ N297A). The 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), cynomolgus monkey CD8+ T cells stimulated with SEA (Fig. 9D), and primary CD14+ bone marrow cells from humans (Fig. 9E) and cynomolgus monkeys (Fig. 9F).

Figures 10A, 10B, 10C, and 10D are graphs showing the binding of anti-TIM-3 antibody or IgG ₁ N297A isotype control antibody to recombinant human TIM-3His (rhTIM-3His), recombinant cynomolgus monkey TIM-3Fc (rcmTIM-3Fc), recombinant mouse TIM-3Fc (rmTIM-3Fc), recombinant human TIM-1His (rhTIM-1His), recombinant human TIM-4His (rhTIM-4His), recombinant human OX40His (rhOX40His), recombinant human GITR Fc (rhGITR Fc), recombinant human DR3 Fc (rhDR3 Fc), and recombinant human CD137 Fc (rhCD137 Fc), as measured by Luminex assay. MFI curves were plotted based on antibody concentrations. The anti-TIM-3 antibodies tested in this study included pab2188w (IgG ₁ N297A) (Figure 10B), AM-2 (IgG ₁ N297A) (Figure 10C), and AM-6 (IgG ₁ N297A) (Figure 10D).

Figures 11A and 11B are graphs showing the percentage of binding of recombinant human TIM-3Fc (Figure 11A) or recombinant cynomolgus monkey TIM-3Fc (Figure 11B) to WR19L cells expressing phosphatidylserine at dose titration in the presence of anti-TIM- ₃ antibody or IgG 1 N297A isotype control antibody. The anti-TIM-3 antibodies tested in this study included pab2188w (IgG ₁ N297A), AM-2 (IgG ₁ N297A), and AM-6 (IgG ₁ N297A).

Figures 12A and 12B are bar charts showing the IFNγ production induced by anti-TIM-3 antibody or IgG ₁ N297A isotype control antibody (alone or in combination with anti-PD-1 antibody pembrolizumab) in human PBMCs from two different donors after SEA stimulation. The anti-TIM-3 antibodies tested included pab2188w (IgG ₁ N297A), AM-1 (IgG ₁ N297A), AM-2 (IgG ₁ N297A), AM-3 (IgG ₁ N297A), AM-4 (IgG ₁ N297A), AM-5 (IgG ₁ N297A), AM-6 (IgG ₁ N297A), AM-7 (IgG ₁ N297A), and AM-8 (IgG ₁ N297A).

Figures 13A, 13B, 13C, 13D, 13E, and 13F are graphs showing the production of IFNγ or TNFα in primary tumor-infiltrating lymphocytes (TILs) induced by anti-TIM-3 antibody or IgG ₁ N297A isotype control antibody (alone or in combination with the anti-PD-1 antibody pembrolizumab). TILs were isolated from non-small cell lung cancer (NSCLC) (Figures 13A and 13B), gallbladder adenocarcinoma (Figures 13C and 13D), or breast cancer (Figures 13E and 13F) tumors and activated with anti-CD3/CD28 microbeads. The anti-TIM-3 antibodies tested in this study included pab2188w (IgG ₁ N297A), AM-2 (IgG ₁ N297A), and AM-6 (IgG ₁ N297A).

Figures 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 IgG ₁ N297A isotype control antibody. Figures 14A and 14B show treatment with the specified antibody in combination with the secondary antibody drug conjugate αHFc-NC-DM1. The cells tested were Jurkat cells engineered to overexpress TIM-3 (Figure 14A) or Kasumi-3 cells, an acute myeloid leukemia cell line endogenously expressing TIM-3 (Figure 14B). Figure 14C shows treatment with the specified antibody as a conjugate with monomethyl auristatin E (MMAE). The anti-TIM-3 antibodies tested in this study included pab2188w (IgG ₁ N297A), AM-2 (IgG ₁ N297A), AM-6 (IgG ₁ N297A), and reference antibodies Hum11 (IgG ₄ S228P) and pab1944w (IgG ₁ N297A).

Figure 15 is a series of graphs showing the internalization of TIM-3 in Jurkat cells expressing the HaloTag-TIM-3 fusion protein, as measured by live-cell confocal fluorescence microscopy at different time points (i.e., 0–3.5 h) when incubated with 10 μg/mL anti-TIM-3 antibody AM-2 or an isotype control antibody. Black dots represent the average fluorescence level observed at a given time point for each condition.

6. Detailed Description of the Invention

This disclosure provides antibodies that specifically bind to and antagonize TIM-3 (e.g., human TIM-3), such as TIM-3-mediated immunosuppression. Pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for preparing these antibodies, and methods for treating subjects using these antibodies are also provided. The antibodies disclosed herein are particularly useful for increasing T cell activation in response to antigens (e.g., tumor antigens or infectious disease antigens), and are therefore particularly useful for treating cancer in subjects or for treating or preventing infectious diseases in subjects. Furthermore, all instances of “isolated antibodies” described herein are considered as antibodies that can be isolated, but do not require isolation. Furthermore, all instances of “isolated polynucleotides” described herein are considered as polynucleotides that can be isolated, but do not require isolation. Furthermore, all instances of “antibodies” described herein are considered as antibodies that can be isolated, but do not require isolation. Furthermore, all instances of “polynucleotides” described herein are considered as polynucleotides that can be isolated, but do not require isolation.

6.1 Definition

As used herein, the terms “about” and “approximately”, when used to modify numerical values or ranges, indicate that a deviation of 5% to 10% (e.g., 5% to 10% higher) and 5% to 10% (e.g., 5% to 10% lower) from the value or range still falls within the intended meaning of the enumerated value or range.

As used herein, the term "TIM-3" refers to T-cell immunoglobulin and mucin domain-3 (also known as the protein containing T-cell immunoglobulin and mucin domain-3 or hepatitis A virus cell receptor 2 (HAVCR2)) encoded by the HAVCR2 gene in humans. An exemplary human TIM-3 amino acid sequence is provided in Swiss-Prot accession number Q8TDQ0-1. The immature amino acid sequence of human TIM-3 is provided as SEQ ID NO:78. The mature amino acid sequence of human TIM-3 is provided as SEQ ID NO:79. As used herein, the term "human TIM-3" refers to TIM-3 containing the amino acid sequence of SEQ ID NO:79.

As used herein, the terms “antibody” and “(multiple) antibodies” include full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules containing antibody CDRs, VH regions, or VL regions. Examples of antibodies include monoclonal antibodies, recombinant antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetramers comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabody antibodies, heteroconjugate antibodies, antibody-drug conjugates, single-domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fv (scFv), camelified antibodies, affybody, Fab fragment, F(ab') ₂ fragment, disulfide-linked Fv (sdFv), anti-idiotype (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), and antigen-binding fragments of any of the above. In some embodiments, the antibodies described herein refer to a population of polyclonal antibodies. Antibodies can be any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., _IgG1 , _IgG2 , _IgG3 , _IgG4 , _IgA1 , or _IgA2 ), or any subclass (e.g., _IgG2a or _IgG2b ) of immunoglobulin molecules. In some embodiments, the antibodies described herein are IgG antibodies or their classifications (e.g., human _IgG1 or _IgG4 ) or their subclasses. In a specific embodiment, the antibody is a humanized monoclonal antibody. In another specific embodiment, the antibody is a human monoclonal antibody.

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

As used herein, the term “CDR” or “complementarity-determining region” refers to a non-adjacent antigen-binding site found within the variable region of both the heavy and light chain polypeptides. These specific regions have been described in Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977), Kabat et al., Sequences of protein of immunological interest. (1991), Chothia et al., J. Mol. Biol. 196: 901-917 (1987), and MacCallum et al., J. Mol. Biol. 262: 732-745 (1996), all of which are incorporated herein by reference in their entirety, where definitions include overlaps or subgroups of amino acids when compared to one another. In some embodiments, the term "CDR" is the CDR defined by MacCallum et al., J. Mol. Biol. 262:732-745 (1996) and Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann, and Dübel ed., pp. 422-439, Chapter 31, Springer-Verlag, Berlin (2001). In some embodiments, the term "CDR" is the CDR defined by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991). In some embodiments, different conventions are used to define the heavy chain CDR and light chain CDR of the antibody. For example, in some implementations, heavy chain CDRs are defined according to MacCallum (ibid.) and light chain CDRs are defined according to Kabat (ibid.). CDRH1, CDRH2, and CDRH3 refer to heavy chain CDRs, and CDRL1, CDRL2, and CDRL3 refer to light chain CDRs.

As used herein, the term “framework (FR) amino acid residue” refers to an amino acid in the immunoglobulin chain backbone region. The terms “framework region” or “FR region” as used herein include amino acid residues that are part of the variable region but not part of the CDR (e.g., as defined using the Kabat or MacCallum of the CDR).

As used herein, the terms “variable region” and “variable domain” are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, usually a portion of the light or heavy chain, typically about 110 to 120 or 110 to 125 amino acids from the amino terminus of the mature heavy chain and about 90 to 115 amino acids from the mature light chain, which are widely sequence-differentiated between antibodies and are responsible for the binding and specificity of a particular antibody to its specific antigen. Sequence variability is concentrated in those regions called complementarity-determining regions (CDRs), while more highly conserved regions within a variable domain are called frame regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are the primary cause of antibody-antigen interactions and specificity. In some embodiments, the variable region is a human variable region. In some embodiments, the variable region comprises a rodent or mouse CDR and a human frame region (FR). In certain embodiments, the variable region is a primate (e.g., non-human primate) variable region. In some implementations, the variable region includes the rodent or mouse CDR and the primate (e.g., non-human primate) frame region (FR).

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

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

As used herein, the terms “constant region” and “constant domain” are used interchangeably and are common in the art. A constant region is an antibody portion, such as a light chain and/or heavy chain portion, that does not directly participate in antibody-antigen binding but can exhibit various effector functions, such as the carboxyl-terminal portion that interacts with Fc receptors (e.g., Fcγ receptors). The constant regions of immunoglobulin molecules typically have a more conserved amino acid sequence than the variable domains of immunoglobulins.

As used herein, the term “heavy chain” in relation to antibodies can refer to any different type based on a constant domain amino acid sequence, such as alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), which produce IgA, IgD, IgE, IgG, and IgM antibodies, including IgG subclasses such as _IgG1 , _IgG2 , _IgG3 , and _IgG4 .

As used herein, the term "light chain" in relation to antibody use can refer to any different type based on a constant domain amino acid sequence, such as kappa(κ) or lambda(λ). Light chain amino acid sequences are well known in the art. In a specific embodiment, the 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 Edelman, G.M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Dept. Health and Human Services, 5th edition, 1991, each of which is incorporated herein by reference in its entirety.

"Binding affinity" generally refers to the total strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y is generally expressed as a dissociation constant ( _KD ). Affinity can be measured and/or expressed in a variety of ways known in the art, including, but not limited to, the equilibrium dissociation constant ( _KD ) and the equilibrium association constant ( _KA ). _KD is calculated by the quotient of _koff / _kon , while _KA is calculated by the quotient of _kon / _koff . _kon refers to, for example, the association rate constant between an antibody and an antigen, while _koff refers to, for example, the dissociation rate constant between an antibody and an antigen. _kon and _koff can be determined by techniques known to those skilled in the art, such as KinExA. As used herein, "lower affinity" refers to a larger _KD .

As used herein, the terms “specific binding,” “specific recognition,” “immunospecific binding,” and “immunospecific recognition” are similar terms in the context of antibodies and refer to the binding of a molecule to an antigen (e.g., an epitope or immune complex) in a manner as understood by those skilled in the art. For example, a molecule that specifically binds to an antigen may bind to other peptides or polypeptides, typically with lower affinity, as determined by, for example, an immunoassay, a KinEx A3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art. In one specific embodiment, a molecule that specifically binds to an antigen binds to that antigen with a K _{_A} at least 2 log (e.g., a factor of 10), 2.5 log, 3 log, 4 log, or more greater than the K_{_A} of the molecule when it nonspecifically binds to another antigen.

In another specific embodiment, the molecule that binds specifically to the antigen does not cross-react with other proteins under similar binding conditions. In another specific embodiment, the molecule that binds specifically to TIM-3 does not cross-react with other non-TIM-3 proteins. In one specific embodiment, this document provides an antibody that binds to TIM-3 (e.g., human TIM-3) with a higher affinity than to another unrelated antigen. In some embodiments, this document provides an antibody that binds to TIM-3 (e.g., human TIM-3) with an affinity 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or higher than to another unrelated antigen, as measured by, for example, radioimmunoassay, surface proton resonance, or kinetic exclusion assay. In one specific embodiment, measured by, for example, radioimmunoassay, the binding of the anti-TIM-3 antibody provided herein to unrelated non-TIM-3 proteins is 10%, 15%, or 20% lower than the binding of the antibody to the TIM-3 protein.

As used herein, the term "unfucosylated" or "non-fucosylated" in the context of Fc refers to a substantial lack of fucose directly or indirectly covalently linked to the corresponding residue in human IgG1 _Fc region residue 297 or non- _IgG1 or non-human _IgG1 immunoglobulin numbered according to the EU numbering system. Therefore, in compositions comprising multiple unfucosylated antibodies, at least 70% of the antibody is not directly or indirectly fucosylated at residue 297 of the antibody Fc region (e.g., by inserting sugar), and in some embodiments, at least 80%, 85%, 90%, 95%, or 99% is not directly or indirectly fucosylated at residue 297 of the Fc region.

As used herein, “epitope” is a term in the art and refers to a localized region of an antigen that an antibody can specifically bind to. An epitope can be, for example, a series of amino acids of a polypeptide (linear or continuous epitope), or an epitope can be, for example, an aggregation of two or more discontinuous regions of one or more polypeptides (conformal, nonlinear, discontinuous, or non-continuous epitope). In some embodiments, the antibody-bound epitope can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography, ELISA assay, hydrogen/deuterium exchange combined mass spectrometry (e.g., liquid chromatography-electrospray ionization mass spectrometry), array-based oligopeptide scanning assays (e.g., binding peptides to discontinuous or conformational epitopes using CLIPS (peptide-to-scaffold chemical linkage)), and/or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be performed using any known method in the art (e.g., Giegé R et al., (1994) Acta Crystallogr D Biol Crystallogr 50 (Pt4):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 is incorporated herein by reference in its entirety). Antibody: Antigen crystals can be studied using well-known X-ray diffraction techniques and can be refined using computer software such as X-PLOR (Yale University, 1992, sponsored by Molecular Simulations, Inc.; see, for example, Meth Enzymol (1985) Vols. 114 and 115, edited by Wyckoff HW et al.; U.S. 2004/0014194) and BUSTER (Bricogne G (1993) A Acta Crystallogr D Biol Crystallogr 49(Pt 1):37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, edited by Carter CW; Roversi P et al. (2000) Acta Crystallogr D Biol Crystallogr 56(Pt10):1316-1323), each incorporated herein by reference in its entirety. Mutagenesis mapping studies can be performed using any method known to those skilled in the art. For a description of mutagenesis techniques, including alanine scanning mutagenesis, see, for example, Champe M et al. (1995) J Biol Chem 270:1388-1394 and Cunningham BC & Wells JA (1989) Science 244:1081-1085, each incorporated herein by reference in its entirety. CLIPS (Chemical Linkage of Peptides to Scaffolds) is a technique that presents one or more peptides in a structurally constrained conformation to function as functional mimics of complex protein domains. See, for example, U.S. Publications US2008/0139407 A1 and US2007/099240 A1 and U.S. Patent No. 7,972,993, each of which is incorporated herein by reference in its entirety. In one embodiment, an alanine scanning mutagenesis study is used to determine the epitope of the antibody. In one embodiment, hydrogen/deuterium exchange coupled mass spectrometry is used to determine the epitope of the antibody. In one embodiment, CLIPS epitope mapping technology from PepscanTherapeutics is used to determine the epitope of the antibody.

As used herein, the term "epitope located within the human TIM-3" region, consisting of a specific amino acid sequence or set of amino acid residues, refers to an epitope comprising one or more amino acid residues of a designated region, wherein the designated region includes the first and last designated amino acid residues of the human TIM-3 region. In some embodiments, the epitope comprises every amino acid residue located within the designated region. In some embodiments, one or more additional amino acid residues of human TIM-3 outside the designated region bind to an antibody having an epitope located within the designated region.

As used herein, the terms “T cell receptor” and “TCR” are used interchangeably and refer to a full-length heterodimer αβ or γδ TCR, an antigen-binding fragment of a full-length TCR, and a molecule containing a TCR CDR or variable region. Examples of TCRs include, but are not limited to, full-length TCRs, antigen-binding fragments of full-length TCRs, soluble TCRs lacking transmembrane and cytoplasmic regions, single-chain TCRs containing variable regions linked by flexible linkers, TCR chains linked by engineered disulfide bonds, monospecific TCRs, multispecific TCRs (including bispecific TCRs), TCR fusions, human TCRs, humanized TCRs, chimeric TCRs, recombinant TCRs, and synthetic TCRs. The term encompasses wild-type TCRs and genetically engineered TCRs (e.g., chimeric TCRs containing chimeric TCR chains, which include a first portion of a TCR from a first species and a second portion of a TCR from a second species).

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

As used herein, the term “peptide-MHC complex” refers to an MHC molecule (MHC class I or MHC class II) that has peptide binding in a pocket of MHC peptides recognized in the art.

As used herein, the term “treatment” refers to the therapeutic or preventative measures described herein. A method of “treatment” involves administering antibodies to a subject who has a disease or condition or is susceptible to such a disease or condition, in order to prevent, cure, delay, reduce the severity of the disease or condition, improve one or more symptoms of the disease or condition, or reduce the recurrence of the disease or condition, or to prolong the subject’s survival beyond the expected survival in the absence of such treatment.

As used in this article, the term "effective amount" in the context of administering a therapy to a subject refers to the therapeutic amount required to achieve the desired preventive or therapeutic effect.

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

The determination of “percentage identity” between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using mathematical algorithms. A specific, non-limiting example of a mathematical algorithm used to compare two sequences is the algorithm of Karlin S & Altschul SF (1990) PNAS 87:2264-2268, modified as in Karlin S & Altschul SF (1993) PNAS 90:5873-5877, each incorporated herein by reference in its entirety. Such algorithms are also incorporated in the NBLAST and XBLAST procedures of Altschul SF et al. (1990) J Mol Biol 215:403, which are also incorporated herein by reference in their entirety. BLAST nucleotide searches can be performed using the NBLAST nucleotide procedure parameter set, e.g., score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using the XBLAST program parameter set, such as a score of 50 and a word length of 3, to obtain amino acid sequences homologous to the protein molecules described herein. For comparison purposes, gap-filled alignments can be obtained using GappedBLAST as described in Altschul SF et al. (1997) Nuc Acids Res 25:3389-3402, which is incorporated herein by reference in its entirety. Alternatively, iterative searches can be performed using PSI BLAST to detect distant relationships between molecules (ibid.). When using BLAST, Gapped BLAST, and PSI BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, e.g., National Center for Biotechnology Information (NCBI) on the World Wide Web, ncbi.nlm.nih.gov). Another specific, non-limiting example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17, which is incorporated herein by reference in its entirety. Such algorithms are incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, the PAM120 weighted residual table, a gap length penalty of 12, and a gap penalty of 4 can be used.

Percentage identity between two sequences can be determined using techniques similar to those described above (with or without gaps). When calculating percentage identity, typically only exact matches are considered.

As used in this article, the term "internalization" or "internalization" refers to the process by which an antibody binds to an antigen expressed on the cell surface and is then taken up into the intracellular compartment.

6.2 Anti-TIM-3 antibody

On the one hand, this disclosure provides antibodies that specifically bind to TIM-3 (e.g., human TIM-3) and antagonize TIM-3 function. The amino acid sequences of exemplary antibodies are listed in Tables 1-4 herein.

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

*Heavy chain CDRs are defined according to the MacCallum numbering system, while light chain CDRs are defined according to the Kabat numbering system.

Table 2. Exemplary heavy chain CDR amino acid sequences of anti-TIM-3 antibodies.

*Based on the MacCallum numbering system definition.

Table 3. Exemplary light chain CDR amino acid sequences of anti-TIM-3 antibodies.

*Based on the Kabat numbering system definition.

Table 4. Exemplary anti-TIM-3 antibodies.

Table 5. Recent Germ Genetics

Table 6. Exemplary sequences for TIM-3.

In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), said antibody comprising a VH domain containing one, two, or all three CDRs of the VH domains listed in Table 1 herein. In some embodiments, the antibody comprises CDRH1 of one of the VH domains listed in Table 1. In some embodiments, the antibody comprises CDRH2 of one of the VH domains listed in Table 1. In some embodiments, the antibody comprises CDRH3 of one of the VH domains listed in Table 1.

In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), said antibody comprising a VL domain containing one, two, or all three CDRs of the VL domains listed in Table 1 herein. In some embodiments, the antibody comprises CDRL1 of one of the VL domains listed in Table 1. In some embodiments, the antibody comprises CDRL2 of one of the VL domains listed in Table 1. In some embodiments, the antibody comprises CDRL3 of one of the VL domains listed in Table 1.

In some embodiments, the CDR of the antibody is determined according to MacCallum RM et al., (1996) JMol Biol 262:732-745, which is incorporated herein by reference in its entirety. See also, for example, Martin A. “Protein Sequence and Structure Analysis of Antibody Variable Domains,” in Antibody Engineering, edited by Kontermann and Dübel, Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001), which is incorporated herein by reference in its entirety. In some embodiments, the heavy chain CDR of the antibody is determined according to MacCallum, while the light chain CDR of the antibody is determined according to different methods.

In some embodiments, the antibody CDR is determined 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, the antibody light chain CDR is determined according to Kabat and the antibody heavy chain CDR is determined according to MacCallum (ibid.).

In some implementations, the antibody CDR can be determined according to the Chothia numbering scheme, which involves the localization of the immunoglobulin structural loop (see, for example, Chothia C and Lesk AM, (1987), J Mol Biol 196:901-917; Al-Lazikani B et al., (1997) J Mol Biol 273:927-948; Chothia C et al., (1992) J Mol Biol 227:799-817; Tramontano A et al., (1990) J Mol Biol 215(1):175-82; and U.S. Patent No. 7,709,226, all of which are incorporated herein by reference in their entirety). Typically, when using Kabat numbering, the Chothia CDRH1 ring is located at positions 26 to 32, 33, or 34 of the heavy chain amino acids; the Chothia CDRH2 ring is located at positions 52 to 56 of the heavy chain amino acids; and the Chothia CDRH3 ring is located at positions 95 to 102 of the heavy chain amino acids. Conversely, the Chothia CDRL1 ring is located at positions 24 to 34 of the light chain amino acids; the Chothia CDRL2 ring is located at positions 50 to 56 of the light chain amino acids; and the Chothia CDRL3 ring is located at positions 89 to 97 of the light chain amino acids. When the end of the Chothia CDRH1 ring is numbered using the Kabat numbering scheme, it varies between H32 and H34 depending on the length of the ring (this is because the Kabat numbering scheme has insertions at H35A and H35B; if neither 35A nor 35B exists, the ring ends at 32; if only 35A exists, the ring ends at 33; if both 35A and 35B exist, the ring ends at 34).

In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), said antibody comprising the Chothia VH CDR of VH disclosed in Table 1 herein. In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), said antibody comprising the Chothia VL CDR of VL disclosed in Table 1 herein. In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), said antibody comprising the Chothia VH CDR and Chothia VL CDR of the antibodies disclosed in Table 1 herein. In some embodiments, the antibody that specifically binds to TIM-3 (e.g., human TIM-3) comprises one or more CDRs, wherein the Chothia and Kabat CDRs have the same amino acid sequence. In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) and comprises a combination of Kabat CDR and Chothia CDR.

In some implementations, the CDR of the antibody can be determined according to the IMGT numbering system 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 to 35, CDRH2 at positions 51 to 57, CDRH3 at positions 93 to 102, CDRL1 at positions 27 to 32, CDRL2 at positions 50 to 52, and CDRL3 at positions 89 to 97.

In some embodiments, this disclosure provides antibodies that specifically bind to TIM-3 (e.g., human TIM-3) and the antibodies comprise, for example, the antibody CDRs disclosed in Table 1, as determined by the IMGT numbering system, as described above by Lefranc M-P (1999) and Lefranc M-P et al. (1999).

In some embodiments, the CDR of the antibody can be determined according to the AbM numbering scheme, which involves the AbM hypervariable region representing a trade-off between the Kabat CDR and the Chothia structural loop, and is used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.), which is incorporated herein by reference in its entirety. In one particular embodiment, this disclosure provides an antibody that specifically binds to TIM-3 (e.g., human TIM-3) and the antibody comprises the antibody CDR disclosed in Table 1 as determined by the AbM numbering scheme.

In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), wherein the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequences of the CDRH1, CDRH2, and CDRH3 regions of the VH domain listed in SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35, and the light chain variable region comprising the amino acid sequences of the CDRL1, CDRL2, and CDRL3 regions of the VL domain listed in SEQ ID NO: 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47, wherein each CDR is defined according to the MacCallum definition, the Kabat definition, the Chothia definition, a combination of the Kabat and Chothia definitions, the IMGT numbering system, or the AbM definition of the CDR.

In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), said antibody comprising:

(a) CDRH1, containing the amino acid sequence _{X1 X2 X3 X4 X5} S (SEQ ID NO:48), wherein

_X1 is R, S, A, G, K, M, or T.

X ₂ can be Q, S, A, G, R, or T.

X ₃ can be N, Y, G, or Q.

X ₄ is either A or Q, and

X ₅ is W, M, A, S, or T; and/or

(b) CDRH2, containing the amino acid sequence WVSAISGSGGSTY (SEQ ID NO:2); and/or

(c) CDRH3, containing the amino acid sequence AKGGDYGGNYFD (SEQ ID NO:3); and/or

(d) CDRL1, containing the amino acid sequence X ₁ ASQSVX ₂ SSYLA (SEQ ID NO:52), wherein

_X1 is R or G, and

_X2 does not exist or is S; and/or

(e)CDRL2, containing the amino acid sequence X ₁ ASX ₂ RAT (SEQ ID NO:53),

in

_X1 is either D or G, and

_X2 is N, S, or T; and/or

(f)CDRL3, containing the amino acid sequence QQYGSSPX ₁ T (SEQ ID NO:54), wherein X ₁ is L or I.

In some embodiments, CDRH1 comprises the amino acid sequence _{X1 X2} NAWS (SEQ ID NO:49), wherein _X1 is R or A; and _X2 is Q or R. In some embodiments, CDRH1 comprises the amino acid sequence _{X1 X2 GQX3} S (SEQ ID NO:50), wherein _X1 is K, M, or G; _X2 is A or S; and _X3 is S or T. In some embodiments, CDRH1 comprises the amino acid sequence _{X1 X2} QQAS (SEQ ID NO:51), wherein _X1 is S, R, T, or G; and _X2 is A, S, T, or G. In some embodiments, CDRH1 comprises amino acid sequences selected from SEQ ID NO:1 and 4-12. In some embodiments, CDRL1 comprises amino acid sequences selected from SEQ ID NO:13-16. In some embodiments, CDRL2 comprises amino acid sequences selected from SEQ ID NO:17-21. In some embodiments, CDRL3 comprises an amino acid sequence selected from SEQ ID NO:22 and 23.

In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), wherein the VH domain of said antibody comprises the amino acid sequences of CDRH1, CDRH2, and CDRH3 listed in 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. In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), wherein the VH domain of said antibody comprises the amino acid sequences of CDRH1, CDRH2, and CDRH3 listed in SEQ ID NO: 1, 2, and 3, respectively. In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), wherein the VH domain of said antibody comprises the amino acid sequences CDRH1, CDRH2, and CDRH3 listed in SEQ ID NO: 5, 2, and 3, respectively. In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), wherein the VH domain of said antibody comprises the amino acid sequences CDRH1, CDRH2, and CDRH3 listed in SEQ ID NO: 9, 2, and 3, respectively.

In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), wherein the VL domain of said antibody comprises the amino acid sequences CDRL1, CDRL2, and CDRL3 listed in 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. In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), wherein the VL domain of said antibody comprises the amino acid sequences CDRL1, CDRL2, and CDRL3 listed in SEQ ID NO: 14, 21, and 22, respectively.

In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), wherein the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising CDRH1, CDRH2, and CDRH3 regions, and the light chain variable region comprising CDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions comprise SEQ ID NO. NO: 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 the amino acid sequences listed in 12, 2, 3, 14, 21 and 22 respectively. In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), wherein the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising CDRH1, CDRH2, and CDRH3 regions, and the light chain variable region comprising CDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions comprise the amino acid sequences listed in SEQ ID NO: 1, 2, 3, 14, 21, and 22, respectively. In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), wherein the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising CDRH1, CDRH2, and CDRH3 regions, and the light chain variable region comprising CDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions comprise the amino acid sequences listed in SEQ ID NO: 5, 2, 3, 14, 21, and 22, respectively. In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), wherein the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising CDRH1, CDRH2, and CDRH3 regions, and the light chain variable region comprising CDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions comprise the amino acid sequences listed in SEQ ID NO: 9, 2, 3, 14, 21, and 22, respectively.

In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:55. In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), wherein the heavy chain variable region comprises 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%) of the amino acid sequence listed in SEQ ID NO:24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In some embodiments, the antibody comprises a heavy chain variable region having an amino acid sequence listed in SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In some embodiments, the antibody comprises a heavy chain variable region having an amino acid sequence listed in SEQ ID NO: 24. In some embodiments, the antibody comprises a heavy chain variable region having an amino acid sequence listed in SEQ ID NO: 25. In some embodiments, the antibody comprises a heavy chain variable region having an amino acid sequence listed in SEQ ID NO: 26. In some embodiments, the antibody comprises a heavy chain variable region having an amino acid sequence listed in SEQ ID NO: 27. In some embodiments, the antibody comprises a heavy chain variable region having an amino acid sequence listed in SEQ ID NO: 28. In some embodiments, the antibody comprises a heavy chain variable region having an amino acid sequence listed in SEQ ID NO: 29. In some embodiments, the antibody comprises a heavy chain variable region having an amino acid sequence listed in SEQ ID NO: 30. In some embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence listed in SEQ ID NO:31. In some embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence listed in SEQ ID NO:32. In some embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence listed in SEQ ID NO:33. In some embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence listed in SEQ ID NO:34. In some embodiments, the antibody comprises a heavy chain variable region having the amino acid sequence listed in SEQ ID NO:35. In some embodiments, the N-terminal glutamic acid (E) residue of the antibody heavy chain variable region as described herein is replaced by a pyroglutamic acid (pE) residue.

In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:56. In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), wherein the light chain variable region comprises 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%) of the amino acid sequence listed in SEQ ID NO:36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47. In some embodiments, the antibody comprises a light chain variable region having an amino acid sequence listed in SEQ ID NO: 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47. In some embodiments, the antibody comprises a light chain variable region having an amino acid sequence listed in SEQ ID NO: 36. In some embodiments, the antibody comprises a light chain variable region having an amino acid sequence listed in SEQ ID NO: 37. In some embodiments, the antibody comprises a light chain variable region having an amino acid sequence listed in SEQ ID NO: 38. In some embodiments, the antibody comprises a light chain variable region having an amino acid sequence listed in SEQ ID NO: 39. In some embodiments, the antibody comprises a light chain variable region having an amino acid sequence listed in SEQ ID NO: 40. In some embodiments, the antibody comprises a light chain variable region having an amino acid sequence listed in SEQ ID NO: 41. In some embodiments, the antibody comprises a light chain variable region having an amino acid sequence listed in SEQ ID NO: 42. In some embodiments, the antibody comprises a light chain variable region having the amino acid sequence listed in SEQ ID NO:43. In some embodiments, the antibody comprises a light chain variable region having the amino acid sequence listed in SEQ ID NO:44. In some embodiments, the antibody comprises a light chain variable region having the amino acid sequence listed in SEQ ID NO:45. In some embodiments, the antibody comprises a light chain variable region having the amino acid sequence listed in SEQ ID NO:46. In some embodiments, the antibody comprises a light chain variable region having the amino acid sequence listed in SEQ ID NO:47. In some embodiments, the N-terminal glutamic acid (E) residue of the antibody light chain variable region as described herein is replaced by a pyroglutamic acid (pE) residue.

In some embodiments, this disclosure 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 the amino acid sequence of SEQ ID NO:56. In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), comprising a heavy chain variable region comprising at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95) of the amino acid sequences listed in SEQ ID NO:24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. The amino acid sequence is identical to that of SEQ ID NO: 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47 by 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%). In some embodiments, the antibody comprises a heavy chain variable region having an amino acid sequence listed in SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35, and a light chain variable region having an amino acid sequence listed in SEQ ID NO: 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having amino acid sequences listed in SEQ ID NO: 24 and 36; 24 and 38; 26 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 antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences listed in SEQ ID NO:24 and 36, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences listed in SEQ ID NO:24 and 38, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences listed in SEQ ID NO:26 and 42, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences listed in SEQ ID NO:24 and 46, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences listed in SEQ ID NO:24 and 43, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences listed in SEQ ID NO:26 and 43, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences listed in SEQ ID NO:26 and 46, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences listed in SEQ ID NO:26 and 41, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences listed in SEQ ID NO:24 and 41, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences listed in SEQ ID NO:25 and 39, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences listed in SEQ ID NO:24 and 47, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences listed in SEQ ID NO: 25 and 40, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences listed in SEQ ID NO: 26 and 47, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences listed in SEQ ID NO: 25 and 37, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences listed in SEQ ID NO: 25 and 45, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences listed in SEQ ID NO: 25 and 44, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences listed in SEQ ID NO: 25 and 46, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences listed in SEQ ID NO: 25 and 42, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences listed in SEQ ID NO: 25 and 41, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences listed in SEQ ID NO: 25 and 43, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences listed in SEQ ID NO: 25 and 47, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences listed in SEQ ID NO: 27 and 46, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences listed in SEQ ID NO: 28 and 46, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences listed in SEQ ID NO: 29 and 46, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences listed in SEQ ID NO: 30 and 46, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences listed in SEQ ID NO: 31 and 46, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences listed in SEQ ID NO: 32 and 46, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences listed in SEQ ID NO: 33 and 46, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences listed in SEQ ID NO: 34 and 46, respectively. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region having the amino acid sequences listed in SEQ ID NO:35 and 46, respectively. In some embodiments, the N-terminal glutamate (E) residue of the antibody heavy chain variable region as described herein is replaced by a pyroglutamate (pE) residue and/or the N-terminal glutamate (E) residue of the antibody light chain variable region as described herein is replaced by a pyroglutamate (pE) residue.

In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), comprising a heavy chain variable region having an amino acid sequence derived from a human IGHV3-23 germline sequence (e.g., IGHV3-23*04, such as the amino acid sequence having SEQ ID NO: 84). One or more regions selected from frames 1, 2, 3, CDRH1, and CDRH2 (e.g., two, three, four, or five of these regions) may be derived from a human IGHV3-23 germline sequence (e.g., IGHV3-23*04, such as the amino acid sequence having SEQ ID NO: 84). In one embodiment, frames 1, 2, 3, CDRH1, and CDRH2 are all derived from a human IGHV3-23 germline sequence (e.g., IGHV3-23*04, such as the amino acid sequence having SEQ ID NO: 84).

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

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

In some embodiments, this disclosure provides an isolated antibody that cross-competitively binds to TIM-3 (e.g., human TIM-3) with an antibody comprising the heavy and light chain variable region amino acid sequences listed in SEQ ID NO: 24 and 36; 24 and 38; 26 and 42; 24 and 46; 24 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, this disclosure provides an isolated antibody that binds to the same or overlapping epitopes of TIM-3 (e.g., epitopes of human TIM-3) as the antibody described herein, such as comprising 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 Antibodies containing the amino acid sequences of the heavy and light chain variable regions listed in 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 epitopes of the antibodies can be determined by, for example, NMR spectroscopy, surface plasmon resonance X-ray diffraction crystallography, ELISA assay, hydrogen/deuterium exchange combined mass spectrometry (e.g., liquid chromatography-electrospray mass spectrometry), array-based oligopeptide scanning assays, and/or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be performed using any known method in the art (e.g., Giegé R et al., (1994) Acta Crystallogr D Biol Crystallogr 50 (Pt 4):339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen NE (1997) Structure 5:1269-1274; McPherson A (1976) J Biol Chem 251:6300-6303, all of which are incorporated herein by reference in their entirety). Antibody: Antigen crystals can be studied using known X-ray diffraction techniques and can be refined using computer software such as X-PLOR (Yale University, 1992, sponsored by Molecular Simulations, Inc.; see, for example, MethEnzymol (1985), vols. 114 and 115, edited by Wyckoff HW et al.; U.S. Patent Application No. 2004/0014194) and BUSTER (Bricogne G (1993) A). Acta Crystallogr D Biol Crystallogr 49(Pt 1):37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, edited by Carter CW; Roversi P et al. (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10):1316-1323, all of which are incorporated herein by reference in their entirety. Mutagenesis mapping studies can be performed using any method known to those skilled in the art. For a description of mutagenesis techniques (including alanine scanning mutagenesis), see, for example, Champe M et al. (1995), ibid. and Cunningham BC & Wells JA (1989), ibid. In one particular embodiment, alanine scanning mutagenesis is used to determine the epitopes of the antibody. Additionally, antibodies that recognize and bind to the same or overlapping epitopes of TIM-3 (e.g., human TIM-3) can be identified using conventional techniques, such as immunoassays, for example by demonstrating the ability of one antibody to block the binding of another antibody to the target antigen, i.e., competitive binding assays. Competitive binding assays can also be used to determine whether two antibodies have similar binding specificity to the epitope. Competitive binding can be measured in assays that specifically bind to a reference immunoglobulin inhibitory antibody being tested against a common antigen, such as TIM-3 (e.g., human TIM-3). Many types of competitive binding assays are known, such as: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competitive assay (see Stahli C et al., (1983) Methods Enzymol 9:242-253); solid-phase direct biotin-avidin EIA (see Kirkland TN et al., (1986) J Immunol 137:3614-9); solid-phase direct labeling assay, solid-phase direct labeling sandwich assay (see Harlow E & Lane D, (1988) Antibodies: A Labor atory Manual, Cold Spring Harbor Press); solid-phase direct-labeled RIA using I-125 label (see Morel GA et al., (1988) Mol Immunol 25(1):7-15); solid-phase direct biotin-avidin EIA (see Cheung RC et al., (1990) Virology 176:546-52); and direct-labeled RIA (see Moldenhauer G et al., (1990) Scand J Immunol 32:77-82), all of which are incorporated herein by reference in their entirety. Typically, such assays involve the use of purified antigens (e.g., TIM-3, such as human TIM-3) bound to a solid surface or cell bearing any of these (i.e., unlabeled test immunoglobulins and labeled reference immunoglobulins). Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cell in the presence of the test immunoglobulin. An excess of test immunoglobulin is usually present. Typically, the presence of excessive competitive antibodies will inhibit the specific binding of the reference antibody to the common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or more. Competitive binding assays can be configured in numerous different forms using labeled antigens or labeled antibodies. In a common form of this assay, the antigen is immobilized on a 96-well plate. Radiolabeling or enzyme labeling is then used to measure the ability of the unlabeled antibody to block the binding of the labeled antibody to the antigen. For further details, see, for example, Wagener C et al., (1983) J Immunol 130:2308-2315; Wagener C et al., (1984) J Immunol Methods 68:269-274; Kuroki M 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, edited by Harlow E and Lane D, ibid., pp. 386-389, all of which are incorporated herein by reference in their entirety.

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

In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), said antibody comprising a light chain containing the amino acid sequence listed in SEQ ID NO:69. In some embodiments, the N-terminal glutamic acid (E) residue of the light chain of the antibody as described herein is replaced with a pyroglutamic acid (pE) residue.

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

Any Ig constant region may be used in the antibodies disclosed herein. In some embodiments, the Ig region is a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class (e.g., _IgG1 , _IgG2 , _IgG3 , _IgG4 , _IgA1 , and _IgA2 ) or any subclass (e.g., _IgG2a and _IgG2b ) of immunoglobulin molecule.

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

In some embodiments, one, two or more mutations (e.g., amino acid substitutions) numbered according to the EU numbering system are introduced into the Fc region (e.g., the _CH2 domain ( _residues 231-340 of human IgG1) and/or the CH3 domain (residues 341-447 of human IgG1) and/or the hinge region of the antibody described herein to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cytotoxicity.

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

In one specific embodiment, one, two, or more amino acid mutations (e.g., substitution, insertion, or deletion) are introduced into the constant domain of IgG or its FcRn-binding fragment (preferably Fc or hinge-Fc domain fragment) to alter (decrease or increase) the half-life of the antibody in vivo. Examples of mutations that alter (decrease or increase) the half-life of the antibody in vivo are found, for example, International Publications WO 02/060919, WO 98/23289, and WO 97/34631; and U.S. Patents 5,869,046, 6,121,022, 6,277,375, and 6,165,745, all of which are incorporated herein by reference in their entirety. In some embodiments, one, two, or more amino acid mutations (e.g., substitution, insertion, or deletion) are introduced into the constant domain of IgG or its FcRn-binding fragment (preferably Fc or hinge-Fc domain fragment) to decrease the half-life of the antibody in vivo. In other embodiments, one, two, or more amino acid mutations (e.g., substitutions, insertions, or deletions) are introduced into the constant domain of IgG or its FcRn-binding fragment (preferably an Fc or hinge-Fc domain fragment) to increase the half-life of the antibody in vivo. In one specific embodiment, the antibody may have one or more amino acid mutations (e.g., substitutions) numbered according to the EU numbering system at the second constant (CH2) domain (residues 231-340 of human IgG ₁ ) and/or the third constant (CH3) domain (residues 341-447 of human IgG ₁ ). In one specific embodiment, the constant region of IgG ₁ of the antibody described herein comprises a methionine (M) to tyrosine (Y) substitution at position 252 according to the EU numbering system, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256. See U.S. Patent No. 7,658,921, which is incorporated herein by reference in its entirety. This type of mutant IgG, known as the “YTE mutant,” has been shown to exhibit a fourfold increase in half-life compared to the wild-type form of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281:23514-24, which is incorporated herein by reference in its entirety). In some embodiments, the antibody comprises one, two, three, or more amino acid 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) numbered according to the EU numbering system are introduced into the Fc region of the antibody described _herein (e.g., the CH2 domain (residues 231-340 of human IgG1) and/or the _CH3 domain (residues 341-447 of human IgG1) and/or the hinge region to increase or decrease the antibody's affinity for an Fc receptor (e.g., an activated Fc receptor) on the surface of an effector cell. Mutations in the antibody Fc region that decrease or increase the antibody's affinity for an Fc receptor and techniques for introducing such mutations into the Fc receptor or fragments thereof are known to those skilled in the art. Examples of mutations that can be generated in the antibody Fc region to alter the antibody's affinity for an Fc receptor are, for example, Smith P et al., (2012) PNAS 109:6181-6186, U.S. Patent No. 6,737,056, and International Publications Nos. WO 02/060919, WO 98/23289, and WO As described in document 97/34631, which is incorporated herein by reference in its entirety.

In another embodiment, one, two, or more amino acid substitutions are introduced into the Fc region of the IgG constant domain to alter the effector function of the 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 can be substituted with different amino acid residues, thereby altering the antibody's affinity for the effector ligand while maintaining the antigen-binding ability of the parent antibody. The effector ligand whose affinity is altered can be, for example, an Fc receptor or a complement C1 component. This method is described in more detail in U.S. Patents 5,624,821 and 5,648,260, each of which is incorporated herein by reference in its entirety. In some embodiments, deletion or inactivation of the constant domain (through point mutation or other means) can reduce the binding of circulating antibodies to the Fc receptor, thereby increasing tumor localization. For descriptions of mutations that result in the loss or inactivation of constant domains and thereby increase tumor localization, see, for example, U.S. Patent Nos. 5,585,097 and 8,591,886, each of which is incorporated herein by reference in its entirety. In some embodiments, one or more amino acid substitutions may be introduced into the Fc region of the antibody described herein to remove potential glycosylation sites on the Fc region, thereby reducing Fc receptor binding (see, for example, Shields RL et al. (2001) J Biol Chem 276:6591-604, which is incorporated herein by reference in its entirety). In various embodiments, one or more of the following mutations, numbered according to the EU numbering system, may be generated in the constant region of the antibody described herein: N297A substitution; N297Q substitution; L235A and L237A substitution; L234A and L235A substitution; E233P substitution; L234V substitution; L235A substitution; C236 deletion; P238A substitution; D265A substitution; A327Q substitution; or P329A substitution. In some embodiments, mutations selected from D265A, P329A, and combinations thereof, numbered according to the EU numbering system, may be generated in the constant region of the antibody described herein.

In one specific embodiment, the antibody described herein comprises a constant _IgG1 domain having an amino acid substitution of N297Q or N297A, according to EU numbering system. In one embodiment, the antibody described herein comprises a constant IgG1 domain having a mutation selected from D265A, P329A, and combinations thereof, according to EU numbering system. In another embodiment, the antibody described herein comprises a _{constant IgG1} domain having a mutation selected from L234A, L235A, and combinations thereof, according to EU numbering system. In some embodiments, the amino acid residues at the positions corresponding to EU numbering system positions L234, L235, and _D265 in the human IgG1 heavy chain in the constant region of the antibody described herein 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 one particular implementation, according to the EU numbering system, the amino acids corresponding to positions L234, L235, and D265 in the human _IgG1 heavy chain are F, E, and A, respectively; or A, A, and A.

In some embodiments, one or more amino acid residues selected from amino acid residues 329, 331, and 322 in the constant region of the antibody described herein, according to EU numbering system designations, may be substituted with different amino acid residues, thereby altering the C1q binding of the antibody and/or reducing or eliminating complement-dependent cytotoxicity (CDC). This method is further described in detail in U.S. Patent No. 6,194,551 (Idusogie et al.), which is incorporated herein by reference in its entirety. In some embodiments, one or more amino acid residues at positions 231 to 238 in the N-terminal region of the CH2 domain of the antibody described herein are altered, according to EU numbering system designations, thereby altering the antibody's ability to fix complement. This method is further described in International Publication No. WO 94/29351, which is incorporated herein by reference in its entirety. In some embodiments, the Fc region of the antibody described herein is modified by mutating (e.g., introducing amino acid substitutions) one or more amino acid positions numbered according to the EU numbering system at the following locations to increase the antibody's ability to mediate antibody-dependent cytotoxicity (ADCC) and/or increase the antibody's affinity for the Fcγ receptor: 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. This method is further described in International Publication WO 00/42072, which is incorporated herein by reference in its entirety.

In some embodiments, the antibody described herein comprises the constant region of an IgG ₄ antibody and the serine residue at heavy chain amino acid residue 228, numbered according to the EU numbering system, is replaced by a proline. In some embodiments, this disclosure provides isolated antibodies that specifically bind to TIM-3 (e.g., human TIM-3), said antibodies comprising a heavy chain constant region containing the amino acid sequence of SEQ ID NO:74. In some embodiments, this disclosure provides isolated antibodies that specifically bind to TIM-3 (e.g., human TIM-3), said antibodies comprising a heavy chain constant region containing the amino acid sequence of SEQ ID NO:75.

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

In some embodiments, this disclosure provides isolated antibodies that specifically bind to TIM-3 (e.g., human TIM-3) and act as antagonists.

In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) and, when evaluated by methods described herein and/or known to those skilled in the art, 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% relative to the TIM-3 (e.g., human TIM-3) activity in the absence of any antibody or in the presence of an irrelevant antibody (e.g., an antibody that does not specifically bind to TIM-3 (e.g., human TIM-3)). In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) and, when evaluated by methods described herein and/or known to those skilled in the art, reduces TIM-3 (e.g., human TIM-3) activity by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold, relative to the TIM-3 (e.g., human TIM-3) activity in the absence of any antibody or in the presence of an irrelevant antibody (e.g., an antibody that does not specifically bind to TIM-3 (e.g., human TIM-3)). Non-limiting examples of TIM-3 (e.g., human TIM-3) activity include TIM-3 (e.g., human TIM-3) signaling, TIM-3 (e.g., human TIM-3) binding to a TIM-3 (e.g., human TIM-3) ligand (e.g., phosphatidylserine), and inhibition of cytokine production (e.g., IFN-γ and/or TNF-α). In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) and inactivates, reduces, or inhibits TIM-3 (e.g., human TIM-3) activity. In specific embodiments, as described in the examples below, the reduction of TIM-3 (e.g., human TIM-3) activity is evaluated.

In specific embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) and, when evaluated by methods described herein (see examples below) or known to those skilled in the art, reduces the binding of TIM-3 (e.g., human TIM-3) to its ligand (e.g., phosphatidylserine) 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% compared to the binding of TIM-3 (e.g., human TIM-3) to its ligand (e.g., phosphatidylserine) with the presence of no antibody or irrelevant antibody (e.g., an antibody that does not specifically bind to TIM-3 (e.g., human TIM-3)). In a specific embodiment, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) and, when evaluated by methods described herein (see examples below) or known to those skilled in the art, reduces the binding of TIM-3 (e.g., human TIM-3) to its ligand (e.g., phosphatidylserine) by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold compared to the binding of TIM-3 (e.g., human TIM-3) to its ligand (e.g., phosphatidylserine) with the presence of no antibody or irrelevant antibody (e.g., an antibody that does not specifically bind to TIM-3 (e.g., human TIM-3)).

In specific embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) and, when evaluated by methods described herein (see examples below) or known to those skilled in the art, 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% compared to the absence of any antibody or the presence of irrelevant antibodies (e.g., antibodies that do not specifically bind to TIM-3 (e.g., human TIM-3)). In specific embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) and, when evaluated by methods described herein (see examples below) or known to those skilled in the art, increases cytokine (e.g., IFN-γ and/or TNF-α) production by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold, relative to cytokine production in the absence of any antibody or in the presence of irrelevant antibodies (e.g., antibodies that do not specifically bind to TIM-3 (e.g., human TIM-3)).

In a specific embodiment, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) and, alone or in combination with an anti-PD-1 antibody (e.g., pembrolizumab or nivolumab), by methods described herein (see examples below) or known to those skilled in the art, increases IFN-γ production in human peripheral blood mononuclear cells (PBMCs) in response to staphylococcal enterotoxin A (SEA) stimulation by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold, relative to IFN-γ production in the absence of any antibody or in the presence of irrelevant antibodies (e.g., antibodies that do not specifically bind to TIM-3 (e.g., human TIM-3)).

In some embodiments, in the presence of an antibody that specifically binds to TIM-3 (e.g., human TIM-3) as described herein, human peripheral blood mononuclear cells (PBMCs) stimulated with staphylococcal enterotoxin A (SEA) were evaluated by methods described herein (see examples below) or known to those skilled in the art, and found that, compared with PBMCs stimulated only with SEA in the absence of any antibody or with irrelevant antibodies (e.g., antibodies that do not specifically bind to TIM-3 (e.g., human TIM-3)), IFN-γ production was increased by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold.

In a specific embodiment, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) and, alone or in combination with an anti-PD-1 antibody (e.g., pembrolizumab or nivolumab), by methods described herein (see examples below) or known to those skilled in the art, increases IFN-γ and/or TNFα production in tumor-infiltrating lymphocytes (TILs) in response to stimulation by anti-CD3 and anti-CD28 antibodies by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold, relative to the absence of an antibody specifically binding to TIM-3 (e.g., human TIM-3). In one embodiment, the TIL originates from a non-small cell lung cancer (NSCLC) tumor. In another embodiment, the TIL originates from a gallbladder adenocarcinoma tumor. In yet another embodiment, the TIL originates from a breast cancer tumor.

In some embodiments, in the presence of antibodies that specifically bind to TIM-3 (e.g., human TIM-3) as described herein, tumor-infiltrating lymphocytes (TILs) stimulated with anti-CD3 and anti-CD28 antibodies, as assessed by methods described herein (see examples below) or known to those skilled in the art, showed an increase in IFN-γ and/or TNFα production of at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold in TILs stimulated only with anti-CD3 and anti-CD28 antibodies without antibodies specifically binding to TIM-3 (e.g., human TIM-3), by methods described herein (see examples below) or known to those skilled in the art. In one embodiment, the TILs are derived from non-small cell lung cancer (NSCLC) tumors. In another embodiment, the TIL originates from a gallbladder adenocarcinoma tumor. In yet another embodiment, the TIL originates from a breast cancer tumor.

In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3) and is internalized upon binding to cells expressing TIM-3 (e.g., 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% of the antibody described herein is internalized upon binding to cells expressing TIM-3 (e.g., human TIM-3) by methods described herein (see examples below) or known to those skilled in the art. In some embodiments, in an assay including the following steps, the percentage of surviving TIM-3-expressing (e.g., human TIM-3) cells is lower in the presence of the antibody described herein compared to the presence of a reference anti-TIM-3 (e.g., human TIM-3) antibody:

(a) Plate cells expressing TIM-3 (e.g., human TIM-3) in tissue culture plates at a density of 2 x ^10⁴ cells per well;

(b) Add the same concentration of αHFc-NC-DM1 and the antibody described herein or a reference anti-TIM-3 (e.g., human TIM-3) antibody (e.g., 1.5 ng/ml, 4.6 ng/ml, 13.7 ng/ml, 41.2 ng/ml, 123.5 ng/ml, 370 ng/ml, 1111 ng/ml or 3333 ng/ml) to a final volume of 100 μl/well;

(c) _Incubate at 37°C and 5% CO2 for 72 hours;

(d) Measure the survival of cells expressing TIM-3 (e.g., human TIM-3); and

(e) Calculate the percentage of cell survival relative to untreated cells expressing TIM-3 (e.g., human TIM-3).

In some embodiments, the percentage of cell survival in the presence of the 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 (e.g., human TIM-3). In some embodiments, the percentage of cell survival in the presence of the antibody described herein is at least about 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times lower than the percentage of cell survival in the presence of a reference anti-TIM-3 (e.g., human TIM-3) antibody. In some embodiments, the reference anti-TIM-3 (e.g., human TIM-3) antibody is pab1944w (IgG ₁ N297A). In some embodiments, the reference anti-TIM-3 (e.g., human TIM-3) antibody is Hum11 (IgG ₄ S228P). In some embodiments, the cells expressing TIM-3 (e.g., human TIM-3) are Kasumi-3 cells. In some embodiments, the cells expressing TIM-3 (e.g., human TIM-3) are Kasumi-3 cells (CRL-2725 ^™ ). In some embodiments, the cells expressing TIM-3 (e.g., human TIM-3) are Jurkat cells engineered to express TIM-3 (e.g., human TIM-3).

In some embodiments, in an assay including the following steps, up to 50% of the TIM-3-expressing (e.g., human TIM-3) cells survive in the presence of the antibody described herein, relative to untreated TIM-3-expressing cells (e.g., human TIM-3):

(a) Plate cells expressing TIM-3 (e.g., human TIM-3) in tissue culture plates at a density of 2 x ^10⁴ cells per well;

(b) Add the same concentration of αHFc-NC-DM1 and the antibody described herein (e.g., 1.5 ng/ml, 4.6 ng/ml, 13.7 ng/ml, 41.2 ng/ml, 123.5 ng/ml, 370 ng/ml, 1111 ng/ml or 3333 ng/ml) to a final volume of 100 μl/well;

(c) _Incubate at 37°C and 5% CO2 for 72 hours;

(d) Measure the survival of cells expressing TIM-3 (e.g., human TIM-3); and

(e) Calculate the percentage of cell survival relative to untreated cells expressing TIM-3 (e.g., human TIM-3).

In some embodiments, up to 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of TIM-3-expressing cells (e.g., human TIM-3) survive in the presence of the antibody described herein, relative to untreated TIM-3-expressing cells (e.g., human TIM-3). In some embodiments, αHFc-NC-DM1 at a concentration of 1111 ng/ml and the antibody described herein are added, and in the presence of the antibody described herein, up to 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of TIM-3-expressing cells (e.g., human TIM-3) survive in the presence of the antibody described herein, relative to untreated TIM-3-expressing cells (e.g., human TIM-3). In some embodiments, αHFc-NC-DM1 at a concentration of 1111 ng/ml and the antibody described herein are added, and up to 50% of the TIM-3-expressing (e.g., human TIM-3) cells survive in the presence of the antibody described herein, relative to untreated TIM-3-expressing cells (e.g., human TIM-3).

In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), wherein 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 antibody, as evaluated by methods described herein (see examples below) or known to those skilled in the art, is internalized after binding to cells expressing TIM-3 (e.g., human TIM-3), and wherein said antibody comprises CDRH1 containing the amino acid sequence X ₁ X ₂ X ₃ X ₄ X ₅ S (SEQ ID NO:48), wherein:

_X1 is R, S, A, G, K, M, or T.

X ₂ can be Q, S, A, G, R, or T.

X ₃ can be N, Y, G, or Q.

X ₄ is either A or Q, and

X ₅ can be W, M, A, S or T.

In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), wherein 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 antibody, as evaluated by methods described herein (see examples below) or known to those skilled in the art, is internalized after binding to cells expressing TIM-3 (e.g., human TIM-3), and wherein said antibody comprises CDRH1 containing the amino acid sequence X ₁ X ₂ NAWS (SEQ ID NO:49), wherein

_X1 is R or A; and

X ₂ is either Q or R.

In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), wherein 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 antibody, as evaluated by methods described herein (see examples below) or known to those skilled in the art, is internalized after binding to cells expressing TIM-3 (e.g., human TIM-3), and wherein said antibody comprises CDRH1 containing the amino acid sequence X ₁ X ₂ GQX ₃ S (SEQ ID NO: 50), wherein

_X1 is K, M, or G;

_X2 is either A or S; and

X ₃ is S or T.

In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), wherein 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 antibody, as evaluated by methods described herein (see examples below) or known to those skilled in the art, is internalized after binding to cells expressing TIM-3 (e.g., human TIM-3), and wherein said antibody comprises CDRH1 containing the amino acid sequence X ₁ X ₂ QQAS (SEQ ID NO: 51), wherein

_X1 is S, R, T, or G; and

X ₂ can be A, S, T or G.

In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), wherein 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 antibody is internalized after binding to cells expressing TIM-3 (e.g., human TIM-3), and wherein said antibody comprises CDRH2 containing the amino acid sequence WVSAISGSGGSTY (SEQ ID NO: 2).

In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), wherein 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 antibody is internalized after binding to cells expressing TIM-3 (e.g., human TIM-3), and wherein said antibody comprises CDRH3 containing the amino acid sequence AKGGDYGGNYFD (SEQ ID NO:3).

In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), wherein 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 antibody, as evaluated by methods described herein (see examples below) or known to those skilled in the art, is internalized after binding to cells expressing TIM-3 (e.g., human TIM-3), and wherein said antibody comprises CDRL1 containing the amino acid sequence _{X1 ASQSVX2} SSYLA (SEQ ID NO: 52), wherein

_X1 is R or G; and

_X2 does not exist or is S.

In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), wherein, as evaluated by methods described herein (see examples below) or known to those skilled in the art, 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 antibody is internalized after binding to cells expressing TIM-3 (e.g., human TIM-3), and wherein said antibody comprises CDRL2 containing the amino acid sequence X ₁ ASX ₂ RAT (SEQ ID NO: 53), wherein

_X1 is either D or G, and

_X2 is N, S or T.

In some embodiments, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), wherein, as evaluated by methods described herein (see examples below) or known to those skilled in the art, 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 antibody is internalized after binding to cells expressing TIM-3 (e.g., human TIM-3), and wherein said antibody comprises CDRL3 containing the amino acid sequence QQYGSSPX ₁ T (SEQ ID NO: 54), wherein

_X1 is either L or I.

In some embodiments, this disclosure provides a segregated antibody that specifically binds to TIM-3 (e.g., human TIM-3), wherein, as evaluated by methods described herein (see examples below) or known to those skilled in the art, 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 antibody is internalized after binding to cells expressing TIM-3 (e.g., human TIM-3), and wherein said antibody is internalized with a variable region comprising heavy and light chain amino groups as listed below, respectively. Antibodies with amino acid sequences cross-competitively bind to TIM-3 (e.g., human TIM-3): 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, this disclosure provides an isolated antibody that specifically binds to TIM-3 (e.g., human TIM-3), wherein 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 antibody, as evaluated by methods described herein (see examples below) or known to those skilled in the art, is internalized after binding to cells expressing TIM-3 (e.g., human TIM-3), and wherein the antibody, like the antibody described herein, comprises, for example, variable heavy and light chains as listed below. Antibodies targeting the amino acid sequence of TIM-3 (e.g., human TIM-3) bind to the same or overlapping epitopes of TIM-3: 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, this disclosure provides a separated antibody that specifically binds to TIM-3 (e.g., human TIM-3), wherein 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 antibody is internalized after binding to cells expressing TIM-3 (e.g., human TIM-3), as evaluated by methods described herein (see examples below) or known to those skilled in the art, and wherein said antibody comprises a human IgG heavy chain constant region variant of the wild-type human IgG heavy chain constant region, wherein said variant human IgG heavy chain constant region binds to the human Fcγ receptor with a lower affinity than the wild-type human IgG heavy chain constant region binding to the human Fcγ receptor. In some embodiments, the human Fcγ receptor is selected from FcγRI, FcγRII, and FcγRIII. In some implementations, the variant human IgG heavy chain constant region is an IgG ₁ constant region containing the N297A mutation numbered according to the EU numbering system.

6.3 Pharmaceutical Compositions

This article provides compositions of the described anti-TIM-3 (e.g., human TIM-3) antibodies containing the desired purity in physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the doses and concentrations used and include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residuals). Polypeptides (based on hydroxyl groups); 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 dextrin; chelating agents such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and/or nonionic surfactants, such as TWEEN ^™ , PLURONICS ^™ , or polyethylene glycol (PEG).

In one embodiment, the pharmaceutical composition comprises an anti-TIM-3 (e.g., human TIM-3) antibody as described herein and optionally one or more additional prophylactic or therapeutic agents in a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises an effective amount of an anti-TIM-3 (e.g., human TIM-3) antibody as described herein and optionally one or more additional prophylactic or therapeutic agents in a pharmaceutically acceptable carrier. In some embodiments, the antibody is the only active ingredient included in the pharmaceutical composition. The pharmaceutical compositions described herein can be used to inhibit TIM-3 (e.g., human TIM-3) activity and treat conditions such as cancer or infectious diseases. In one embodiment, the invention relates to a pharmaceutical composition comprising the anti-TIM-3 antibody of the invention used as a medicament. In another embodiment, the invention relates to a pharmaceutical composition of the invention used in a method for treating cancer or infectious diseases. In yet another embodiment, the invention relates to the use of the pharmaceutical compositions of the invention in the preparation of a medicament for treating cancer or infectious diseases.

Pharmaceutically acceptable carriers used in parenteral preparations include aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifiers, multivalent chelating agents or chelating agents, and other pharmaceutically acceptable substances. Examples of aqueous media include sodium chloride injection, Ringer's injection, isotonic glucose injection, sterile water injection, and glucose and lactated Ringer's injection. Non-aqueous parenteral media include non-volatile plant-derived oils, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents at antibacterial or antifungal concentrations can be added to parenteral preparations packaged in multi-dose containers, including phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methylparaben, thimerosal, benzalkonium chloride, and benzyl chloride. Isotonic agents include sodium chloride and dextran. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspensions and dispersants include sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Emulsifiers include polysorbate 80 (80). Multivalent chelating agents or chelating agents for metal ions include EDTA. Drug carriers also include ethanol, polyethylene glycol, and propylene glycol for water-miscible media; and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

Pharmaceutical compositions can be formulated for use in any route of administration to a subject. Specific examples of routes of administration include intranasal, oral, pulmonary, transdermal, intradermal, and parenteral administration. Parenteral administration, characterized by subcutaneous, intramuscular, or intravenous injection, is also considered herein. Injectable formulations can be prepared in conventional forms, such as liquid solutions or suspensions, solid forms suitable for dissolution or suspension in a liquid prior to injection, or emulsions. Injectables, solutions, and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextran, glycerol, or ethanol. Additionally, if desired, the pharmaceutical composition to be administered may contain small amounts of non-toxic excipients, such as wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers, and other such agents, such as sodium acetate, sorbitol monolaurate, triethanolamine oleate, and cyclodextrin.

Preparations for parenteral administration of antibodies include sterile solutions ready for injection; sterile, dried soluble products, such as lyophilized powders, including subcutaneous tablets, prepared for combination with a solvent before use; sterile, ready-to-use suspensions; sterile, dried insoluble products prepared for combination with a medium before use; and sterile emulsions. The solutions may be aqueous or non-aqueous solutions.

If administered intravenously, suitable carriers include physiological saline or phosphate-buffered saline (PBS), as well as solutions containing thickeners and solubilizers such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.

Prepare the antibody-containing topical mixture as described for local and systemic application. The resulting mixture may be a solution, suspension, emulsion, etc., and may be formulated into creams, gels, ointments, lotions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, rinses, sprays, suppositories, bandages, skin patches, or any other formulation suitable for local application.

The anti-TIM-3 (e.g., human TIM-3) antibodies described herein can be formulated as aerosols for topical application, such as by inhalation (see, for example, U.S. Patent Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for delivering steroids useful for treating inflammatory diseases, particularly asthma, and are incorporated herein by reference in their entirety). These formulations for administration to the respiratory tract can be in the form of an aerosol or solution for use in nebulizers, or as a fine powder for inhalation, alone or in combination with an inert carrier such as lactose. In such cases, the particles of the formulation will have a diameter of less than 50 micrometers in one embodiment and less than 10 micrometers in another embodiment.

The anti-TIM-3 (e.g., human TIM-3) antibodies described herein can be formulated into gel, cream, and lotion forms for local or topical application, such as for application to the skin and mucous membranes, including the eyes, and can be formulated for ocular application or intracranial or intraspinal application. Consideration is given to local administration, transdermal delivery, and also for ocular or mucous membrane administration, or for inhalation therapy. Nasal solutions containing the antibody alone or in combination with other pharmaceutically acceptable excipients can also be administered.

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

In some embodiments, the pharmaceutical composition comprising the antibody described herein is a lyophilized powder, which can be reconstituted into solutions, emulsions, and other mixtures for administration. It can also be reconstituted and formulated into a solid or gel. The lyophilized powder is prepared by dissolving the antibody described herein or a pharmaceutically acceptable derivative thereof in a suitable solvent. In some embodiments, the lyophilized powder is sterile. The solvent may contain excipients that improve the stability of the powder or a reconstituted solution prepared from the powder, or other pharmacological components. Excipients that can be used include, but are not limited to, dextran, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agents. In one embodiment, the solvent may also contain a buffer at approximately a neutral pH, such as citrate, sodium phosphate, or potassium phosphate, or other such buffers known to those skilled in the art. The solution is then sterilely filtered and subsequently lyophilized under standard conditions known to those skilled in the art to obtain the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial will contain a single or multiple doses of the compound. The lyophilized powder can be stored under suitable conditions, such as at approximately 4°C to room temperature. Reconstitute the lyophilized powder with water for injection to provide a formulation for parenteral administration. To reconstitute, add the lyophilized powder to sterile water or other suitable carrier. The precise amount depends on the compound selected. This amount can be determined empirically.

The anti-TIM-3 (e.g., human TIM-3) antibodies described herein and other compositions provided herein can also be formulated to target specific tissues, receptors, or other regions of the body of a subject to be treated. Many such targeting methods are well known to those skilled in the art. All such targeting methods are contemplated herein for use in the compositions of the invention. For non-limiting examples of targeting methods, see, for example, U.S. Patent Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542, and 5,709,874, all of which are incorporated herein by reference in their entirety. In one specific embodiment, the antibody described herein targets a tumor.

The composition intended for in vivo administration can be sterile. This can be easily achieved through filtration, for example, using a sterile filter membrane.

6.4 Usage and Applications

On the other hand, this disclosure provides a method of treating a subject with an anti-TIM-3 (e.g., human TIM-3) antibody disclosed herein. Any disease or condition in the subject that would benefit from inhibition of TIM-3 (e.g., human TIM-3) function can be treated with the anti-TIM-3 (e.g., human TIM-3) antibody disclosed herein. The anti-TIM-3 (e.g., human TIM-3) antibody disclosed herein is particularly useful for suppressing immune system tolerance to tumors and is therefore suitable for use as an immunotherapy for cancer subjects. For example, in some embodiments, this disclosure provides a method of increasing T cell activation in response to a subject antigen, the method comprising administering to the subject an effective amount of an anti-TIM-3 (e.g., human TIM-3) antibody or a pharmaceutical composition thereof as disclosed herein. In some embodiments, this disclosure provides a method of treating a subject with cancer, the method comprising administering to the subject an effective amount of an antibody or a pharmaceutical composition thereof as disclosed herein. In some embodiments, this disclosure provides an antibody or pharmaceutical composition as disclosed herein in a method for treating cancer or an infectious disease. In some embodiments, this disclosure provides an antibody or pharmaceutical composition as disclosed herein for use as a medicament. In another embodiment, this disclosure provides the use of antibody or pharmaceutical compositions as disclosed herein for the preparation of medicaments for treating cancer or infectious diseases.

Cancers that can be treated with the anti-TIM-3 (e.g., human TIM-3) antibodies or pharmaceutical compositions disclosed herein include, but are not limited to, solid tumors, hematologic malignancies (e.g., leukemia, lymphoma, myeloma (e.g., multiple myeloma)), and metastatic lesions. In one embodiment, the 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, breasts, ovaries, lymph nodes, gastrointestinal tract (e.g., colon), anus, genitals and genitourinary tract (e.g., kidneys, urothelial cells, bladder cells, prostate), pharynx, CNS (e.g., brain, nerves, or glial cells), head and neck, skin (e.g., melanoma), and pancreas, as well as adenocarcinomas including malignancies such as colon cancer, rectal cancer, renal cell carcinoma, liver cancer, lung cancer (e.g., non-small cell lung cancer or small cell lung cancer), small bowel cancer, and esophageal cancer. The cancer may be in the early, intermediate, late, or metastatic stage. In some implementations, the cancer is associated with elevated PD-1 activity (e.g., elevated PD-1 expression).

In one embodiment, the cancer is selected from lung cancer (e.g., lung adenocarcinoma or non-small cell lung cancer (NSCLC) (e.g., NSCLC with squamous and/or non-squamous histological features, or NSCLC adenocarcinoma)), melanoma (e.g., advanced melanoma), kidney cancer (e.g., renal cell carcinoma), liver cancer (e.g., hepatocellular carcinoma), myeloma (e.g., multiple myeloma), prostate cancer, breast cancer (e.g., breast cancer that does not express one, two, or all of estrogen receptor, progesterone receptor, or Her2/neu, such as triple-negative breast cancer). Ovarian cancer, colorectal cancer, pancreatic cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC), anal cancer, gastroesophageal cancer (e.g., esophageal squamous cell carcinoma), mesothelioma, nasopharyngeal carcinoma, thyroid cancer, cervical cancer, epithelial cancer, peritoneal cancer, or lymphoproliferative disorders (e.g., post-transplant lymphoproliferative disorders). In one embodiment, the cancer is NSCLC. In one embodiment, the cancer is renal cell carcinoma. In one embodiment, the cancer is ovarian cancer. In a specific embodiment, the ovarian cancer is platinum-refractory ovarian cancer.

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

In another embodiment, the cancer is selected from carcinoma (e.g., advanced or metastatic cancer), melanoma, or lung cancer, such as non-small cell lung cancer.

In one implementation, the cancer is lung cancer, such as lung adenocarcinoma, non-small cell lung cancer, or small cell lung cancer.

In one embodiment, the cancer is melanoma, such as advanced melanoma. In one embodiment, the cancer is advanced or unresectable melanoma that is unresponsive to other therapies. In other embodiments, the cancer is melanoma with a BRAF mutation (e.g., BRAF V600 mutation). In still other embodiments, the anti-TIM-3 (e.g., human TIM-3) antibody or pharmaceutical composition disclosed herein is administered after treatment with an anti-CTLA-4 antibody (e.g., ipilimumab) with or without a BRAF inhibitor (e.g., vemurafenib or dabrafenib).

In another embodiment, the cancer is liver cancer, such as advanced liver cancer with or without viral infection, such as chronic viral hepatitis.

In another embodiment, the cancer is prostate cancer, such as advanced prostate cancer.

In another embodiment, the cancer is myeloma, such as multiple myeloma.

In another embodiment, the cancer is kidney cancer, such as renal cell carcinoma (RCC) (e.g., metastatic RCC, clear cell renal cell carcinoma (CCRCC), or papillary renal cell carcinoma).

In another embodiment, the cancer is selected from lung cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, leukemia, or metastatic lesions of cancer.

In some embodiments, this disclosure provides a method for preventing or treating an infectious disease in a subject, the method comprising administering to the subject an effective amount of an anti-TIM-3 (e.g., human TIM-3) antibody or a pharmaceutical composition thereof as disclosed herein. In one embodiment, this disclosure provides a method for preventing and/or treating an infection (e.g., a viral infection, bacterial infection, fungal infection, protozoan infection, or parasitic infection). The infection prevented and/or treated according to this method may be caused by an infectious agent identified herein. In one specific embodiment, the anti-TIM-3 (e.g., human TIM-3) antibody or a composition thereof described herein is the sole active agent administered to the subject. In some embodiments, the anti-TIM-3 (e.g., human TIM-3) antibody or a composition thereof described herein is used in combination with an anti-infective intervention (e.g., an antiviral drug, an antibacterial drug, an antifungal drug, or an anti-helmintic drug) for treating an infectious disease. Thus, in one embodiment, the present invention relates to the antibody and/or pharmaceutical composition of the present invention in a method for preventing and/or treating an infectious disease, optionally wherein said antibody or pharmaceutical composition is the sole active agent administered to the subject, or wherein said antibody or pharmaceutical composition is used in combination with an anti-infective intervention.

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

Preventable and/or treatable bacterial infections 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, but are not limited to, Mycobacteria rickettsia, Mycoplasma, Neisseria, Streptococcus pneumoniae, and Borrelia burgdorferi. Lyme disease, Bacillus anthracis (anthrax), tetanus, Streptococcus, Staphylococcus, Mycobacterium, pertussis, cholera, plague, diphtheria, chlamydia, Staphylococcus aureus, and Legionella.

The methods described herein can be used to prevent and/or treat protozoan diseases or infections caused by protozoa, including but not limited to diseases caused by Leishmaniasis, coccidiosis, trypanosomiasis, or malaria. The methods described herein can also be used to prevent and/or treat parasitic diseases or infections caused by parasites, including but not limited to diseases caused by Chlamydia and Rickettsia.

Fungal diseases or infections that can be prevented and/or treated according to the methods described herein include, but are not limited to, those caused by: Candida infection, zygomycosis, candidal mastitis, progressive disseminated trichosporidiosis with latent trichosporemia, disseminated candidiasis, pulmonary paracoccidioidomycosis, pulmonary aspergillosis, Pneumocystis carinii pneumonia, cryptococcal meningitis, coccidioidal meningoencephalitis and cerebrospinal vasculitis, Aspergillus niger infection, Fusarium keratitis. Keratitis, sinus fungal infections, Aspergillus fumigatus endocarditis, tibial chondropathy, Candida glabrata vaginitis, oropharyngeal candidiasis, X-linked chronic granulomatosis, tinea pedis, cutaneous candidiasis, fungal placenta inflammation, disseminated trichosporidiosis, allergic bronchopulmonary aspergillosis, fungal keratitis, Cryptococcus neoformans infection, fungal peritonitis, Curvularia geniculata infection, staphylococcal endophthalmitis, sporotrichosis, and dermatomycosis.

In some embodiments, these methods further include administering an additional therapeutic agent to the subject. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent, a radiotherapy agent, or a checkpoint target. In some embodiments, the chemotherapeutic agent is a demethylating agent (e.g., azacitidine). In some embodiments, the checkpoint target is selected from antagonist anti-CTLA-4 antibody, antagonist anti-PD-L1 antibody, antagonist anti-PD-L2 antibody, antagonist anti-PD-1 antibody, antagonist anti-TIM-3 antibody, antagonist anti-LAG-3 antibody, antagonist anti-CEACAM1 antibody, agonist anti-CD137 antibody, antagonist anti-TIGIT antibody, antagonist anti-VISTA antibody, agonist anti-GITR antibody, and agonist anti-OX40 antibody.

In one embodiment, the present invention relates to the antibody and/or pharmaceutical composition of the present invention used in a method of the present invention, wherein the method further includes administering an additional therapeutic agent to a subject. In one embodiment, the present invention relates to (a) the antibody and/or pharmaceutical composition of the present invention and (b) an additional therapeutic agent used as a medicament. In one embodiment, the present invention relates to (a) the antibody and/or pharmaceutical composition of the present invention and (b) an additional therapeutic agent used in a method of treating cancer. In another embodiment, the present invention relates to a pharmaceutical composition, cassette, or dispensing kit comprising (a) the antibody and/or pharmaceutical composition of the present invention and (b) an additional therapeutic agent. In one embodiment, the additional therapeutic agent is a chemotherapeutic agent, a radiotherapy agent, or a checkpoint targeting agent.

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

Further non-limiting examples of anti-PD-1 antibodies that can be used in the treatments disclosed herein are disclosed in the following patents and patent applications, all of which are incorporated herein by reference in their entirety for all purposes: U.S. Patent No. 6,808,710; U.S. Patent No. 7,332,582; U.S. Patent No. 7,488,802; U.S. Patent No. 8,008,449; U.S. Patent No. 8,114,845; U.S. Patent No. 8,168,757; U.S. Patent No. U.S. Patent No. 8,354,509; U.S. Patent No. 8,686,119; U.S. Patent No. 8,735,553; U.S. Patent No. 8,747,847; U.S. Patent No. 8,779,105; U.S. Patent No. 8,927,697; U.S. Patent No. 8,993,731; U.S. Patent No. 9,102,727; U.S. Patent No. 9,205,148; U.S. Publication No. US 2013/0202623 A1; U.S. Publication No. U US Publication No. S 2013/0291136 A1; US Publication No. US 2014/0044738 A1; US Publication No. US 2014/0356363 A1; US Publication No. US 2016/0075783 A1; and PCT Publication No. WO 2013/033091 A1; PCT Publication No. WO 2015/036394 A1; PCT Publication No. WO 2014/179664 A2; PC PCT published WO 2014/209804 A1; PCT published WO 2014/206107 A1; PCT published WO 2015/058573 A1; PCT published WO 2015/085847 A1; PCT published WO 2015/200119 A1; PCT published WO 2016/015685 A1; and PCT published WO 2016/020856 A1.

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

Non-limiting examples of anti-PD-L1 antibodies that can be used in the treatment methods disclosed herein are disclosed in the following patents and patent applications, all of which are incorporated herein by reference in their entirety for all purposes: U.S. Publication No. 7,943,743; U.S. Publication No. 8,168,179; U.S. Publication No. 8,217,149; U.S. Patent No. 8,552,154; U.S. Patent No. 8,779,108; U.S. Patent No. 8,981,063; U.S. Patent No. 9,175,082; U.S. Publication No. US2010/0203056A1; U.S. Publication No. US2003/0232323 A1; U.S. Publication No. US2013/0323249A1; U.S. Publication No. US2014/0341917 A1; U.S. Publication No. US2014/0044738 A1. 1; US Announcement No. US2015/0203580 A1; US Announcement No. US2015/0225483 A1; US Announcement No. US2015/0346208 A1; US Announcement No. US2015/0355184 A1; and PCT Announcement No. WO 2014/100079 A1; PCT Announcement No. WO2014/022758 A1; P CT published WO 2014/055897 A2; PCT published WO 2015/061668 A1; PCT published WO 2015/109124 A1; PCT published WO 2015/195163 A1; PCT published WO 2016/000619 A1; and PCT published WO 2016/030350 A1.

In some embodiments, the anti-TIM-3 (e.g., human TIM-3) antibody disclosed herein is administered to a subject in combination with a compound targeting immunomodulatory enzymes such as IDO (indoleamine-(2,3)-dioxygenase) and/or TDO (tryptophan 2,3-dioxygenase). Thus, in one embodiment, the additional therapeutic agent is a compound targeting an immunomodulatory enzyme, such as an inhibitor of indoleamine-(2,3)-dioxygenase (IDO). In some embodiments, such compounds are selected from epacadostat (IncyteCorp; see, e.g., WO 2010/005958, incorporated herein by reference in its entirety), F001287 (FlexusBiosciences/Bristol-Myers Squibb), indoximod (NewLink Genetics), and NLG919 (NewLink Genetics). In one embodiment, the compound is epacadostat. In another embodiment, the compound is F001287. In yet another embodiment, the compound is indoximod. In another embodiment, the compound is NLG919. In one specific embodiment, the anti-TIM-3 (e.g., human TIM-3) antibody disclosed herein is administered to a subject in combination with an IDO inhibitor for the treatment of cancer. The IDO inhibitor for the treatment of cancer as described herein is present in a solid dosage form of the pharmaceutical composition, such as tablets, pills, or capsules, wherein the pharmaceutical composition comprises an IDO inhibitor and a pharmaceutically acceptable excipient. Similarly, the antibody as described herein and the IDO inhibitor as described herein can be administered alone, sequentially, or simultaneously as independent dosage forms. In one embodiment, the antibody is administered parenterally, while the IDO inhibitor is administered orally. In a particular embodiment, the inhibitor is selected from epacadostat (Incyte Corporation), F001287 (Flexus Biosciences/Bristol-Myers Squibb), indoximod (NewLink Genetics), and NLG919 (NewLink Genetics). epacadostat is described in PCT Publication WO 2010/005958, which is incorporated herein by reference in its entirety for all purposes. In one embodiment, the inhibitor is Epacadostat. In another embodiment, the inhibitor is F001287. In yet another embodiment, the inhibitor is indoximod. In still another embodiment, the inhibitor is NLG919.

In some embodiments, the anti-TIM-3 (e.g., human TIM-3) antibody disclosed herein is administered in combination with a vaccine to a subject. The vaccine may be, for example, a peptide vaccine, a DNA vaccine, or an RNA vaccine. In some embodiments, the vaccine is a tumor vaccine based on heat shock proteins or a pathogen vaccine based on heat shock proteins. In some embodiments, the anti-TIM-3 (e.g., human TIM-3) antibody disclosed herein is administered in combination with a vaccine described in WO 2016/183486, which is incorporated herein by reference in its entirety (e.g., a vaccine comprising at least one synthetic peptide containing a cancer-specific mutation present in the subject's cancer), to a subject. In one specific embodiment, the anti-TIM-3 (e.g., human TIM-3) antibody disclosed herein is administered in combination with a heat shock protein-based tumor vaccine to a subject. Heat shock proteins (HSPs) are a highly conserved family of proteins that are ubiquitous in all species. Their expression can be efficiently induced to much higher levels by heat shock or other forms of stress, including exposure to toxins, oxidative stress, or glucose starvation. Based on molecular weight, they have been classified into five families: HSP-110, HSP-90, HSP-70, HSP-60, and HSP-28. HSPs deliver immunogenic peptides via cross-presentation pathways in antigen-presenting cells (APCs) such as macrophages and dendritic cells (DCs), thereby leading to T cell activation. HSPs act as chaperone protein carriers of tumor-associated antigenic peptides, forming complexes capable of inducing tumor-specific immunity. Upon release from dying tumor cells, the HSP-antigen complex is taken up by antigen-presenting cells (APCs), where the antigen is processed into peptides that bind MHC class I and II molecules, thereby leading to activation of anti-tumor CD8+ and CD4+ T cells. The immunity triggered by the HSP complex derived from tumor preparations is specifically targeted at a unique library of antigenic peptides expressed by the cancer of each subject. Therefore, in one embodiment, the present invention relates to (a) the antibody and/or pharmaceutical compositions of the present invention and (b) vaccines used as medicines, for example, in methods of treating cancer. In one embodiment, the present invention relates to a pharmaceutical composition, kit, or dispensing kit comprising (a) an antibody and/or pharmaceutical composition of the present invention and (b) a vaccine. In one embodiment, the vaccine is a tumor vaccine based on heat shock proteins. In one embodiment, the vaccine is a pathogen vaccine based on heat shock proteins.

The heat shock protein peptide complex (HSPPC) is a protein peptide complex composed of heat shock proteins non-covalently complexed with an antigenic peptide. HSPPCs elicit both innate and adaptive immune responses. In one specific embodiment, the antigenic peptide exhibits antigenicity against the targeted cancer. HSPPCs are efficiently captured by APCs via membrane receptors (primarily CD91) or by binding to Toll-like receptors. HSPPC internalization leads to APC functional maturation and the production of chemokines and cytokines, resulting in natural killer (NK) cell (NK) and monocyte activation, and Th1 and Th-2 mediated immune responses. In some embodiments, the HSPPCs used in the methods disclosed herein comprise one or more heat shock proteins from the hsp60, hsp70, or hsp90 family of stress proteins complexed with the antigenic peptide. In some embodiments, HSPPCs comprise hsc70, hsp70, hsp90, hsp110, gRP170, gp96, calreticulin, or a combination of two or more of these.

In one specific embodiment, the heat shock protein peptide complex (HSPPC) comprises a recombinant heat shock protein (e.g., hsp70 or hsc70) or its peptide-binding domain complexed with a recombinant antigenic peptide. The recombinant heat shock protein can be generated using recombinant DNA techniques, for example, using the human hsc70 sequence as described in Dworniczak and Mirault, Nucleic Acid Res. 15:5181-5197 (1987) and GenBank accession numbers P11142 and/or Y00371, each of which is incorporated herein by reference in its entirety. In some embodiments, the Hsp70 sequence is as described in Hunt and Morimoto, Proc. Natl. Acad. Sci. U.S.A. 82 (19), 6455-6459 (1985) and GenBank accession numbers P0DMV8 and/or M11717, each of which is incorporated herein by reference in its entirety. The antigenic peptide can also be prepared using recombinant DNA methods known in the art.

In some embodiments, the antigenic peptide comprises modified amino acids. In some embodiments, the modified amino acids comprise post-translational modifications. In some embodiments, the modified amino acids comprise post-translational modification mimics. In some embodiments, the modified amino acids are Tyr, Ser, Thr, Arg, Lys, or His amino acids that have been phosphorylated at a side-chain hydroxyl or amine. In some embodiments, the modified amino acids are mimics of Tyr, Ser, Thr, Arg, Lys, or His amino acids that have been phosphorylated at a side-chain hydroxyl or amine.

In one specific embodiment, the anti-TIM-3 (e.g., human TIM-3) antibody disclosed herein is combined with a heat shock protein peptide complex (HSPPC), such as heat shock protein peptide complex-96 (HSPPC-96), and administered to a subject to treat cancer. HSPPC-96 contains a 96 kDa heat shock protein (Hsp), i.e., gp96, complexed with an antigenic peptide. HSPPC-96 is a cancer immunotherapy agent derived from a subject's tumor and contains an antigenic "fingerprint" of the cancer. In some embodiments, this fingerprint contains a unique antigen present only in specific cancer cells of that particular subject, and the injection is intended to stimulate the subject's immune system to recognize and attack any cells with that specific cancer fingerprint. Therefore, in one embodiment, the present invention relates to antibody and/or pharmaceutical compositions of the present invention in combination with heat shock protein peptide complexes (HSPPC), used as a medicine and/or in methods of treating cancer.

In some embodiments, HSPPCs, such as HSPPC-96, are generated from the subject's tumor tissue. In one specific embodiment, HSPPCs (e.g., HSPPC-96) are generated from the cancer being treated or from a metastatic type of tumor. In another specific embodiment, HSPPCs (e.g., HSPPC-96) are autologous to the treated subject. In some embodiments, the tumor tissue is non-necrotic tumor tissue. In some embodiments, at least 1 gram (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 grams) of non-necrotic tumor tissue is used to generate the vaccine regimen. In some embodiments, the non-necrotic tumor tissue is frozen after surgical resection before use in vaccine preparation. In some embodiments, HSPPCs, such as HSPPC-96, are isolated from the tumor tissue using purification techniques, filtered, and prepared for use in an injectable vaccine. In some embodiments, 6-12 doses of HSPPCs, such as HSPPC-96, are administered to the subject. In such implementations, the first four doses of HSPPC (e.g., HSPPC-96) can be administered weekly, followed by two to eight additional doses every two weeks.

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

In some embodiments, the anti-TIM-3 antibody disclosed herein is administered to a subject in combination with an adjuvant. Various adjuvants may be used depending on the treatment context. Non-limiting examples of suitable adjuvants include, but are not limited to, complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), montanide ISA (incomplete Seppic adjuvant), Ribi adjuvant system (RAS), TiterMax, cell wall peptides, Syntex adjuvant formulations (SAF), alum (aluminum hydroxide and/or aluminum phosphate), aluminum salt adjuvants, adjuvants, nitrocellulose-adsorbed antigens, encapsulated or trapped antigens, 3De-O acylated monophospholipid A (3D-MPL), immunostimulatory oligonucleotides, toll-like receptor (TLR) ligands, mannan-binding lectin (MBL) ligands, STING agonists, and immunostimulatory complexes such as saponins, Quil A, QS-21, QS-7, ISCMATRIX, etc. Other adjuvants include CpG oligonucleotides and double-stranded RNA molecules such as poly(A) and poly(U). Combinations of the above adjuvants may also be used. See, for example, U.S. Patent Nos. 6,645,495, 7,029,678, and 7,858,589, all of which are incorporated herein by reference in their entirety. In one embodiment, the adjuvant used herein is QS-21STIMULON.

In some embodiments, the anti-TIM-3 antibody disclosed herein is administered to a subject in combination with an adjunct therapeutic agent containing a TCR. In some embodiments, the adjunct therapeutic agent is a soluble TCR. In some embodiments, the adjunct therapeutic agent is a cell expressing a TCR. Therefore, in one embodiment, the present invention relates to antibody and/or pharmaceutical compositions of the present invention in combination with an adjunct therapeutic agent containing a TCR, used as a medicament and/or in a method of treating cancer.

In some embodiments, the anti-TIM-3 antibody disclosed herein is administered to a subject in combination with cells expressing a chimeric antigen receptor (CAR). In some embodiments, the cells are T cells.

In some embodiments, the anti-TIM-3 antibody disclosed herein is administered in combination with a TCR mimic antibody to a subject. In some embodiments, the TCR mimic antibody is an antibody that specifically binds to a peptide-MHC complex. For non-limiting examples of TCR mimic antibodies, see, for example, U.S. Patent No. 9,074,000 and U.S. Publications Nos. US2009/0304679 A1 and US2014/0134191 A1, all of which are incorporated herein by reference in their entirety.

Anti-TIM-3 (e.g., human TIM-3) antibodies and adjunctive therapeutic agents (e.g., chemotherapeutic agents, radiotherapy agents, checkpoint targets, IDO inhibitors, vaccines, adjuvants, soluble TCRs, TCR-expressing cells, chimeric antigen receptor-expressing cells, and/or TCR mimic antibodies) can be administered alone, sequentially, or simultaneously as independent dosage forms. In one embodiment, the anti-TIM-3 (e.g., human TIM-3) antibody is administered parenterally, while the IDO inhibitor is administered orally.

The antibody or pharmaceutical compositions described herein can be delivered to subjects via a variety of routes. These include, but are not limited to, parenteral, intranasal, intratracheal, oral, intradermal, topical, intramuscular, intraperitoneal, transdermal, intravenous, intrathecal, intratumoral, intraconjunctival, intra-arterial, and subcutaneous routes. In some embodiments, the antibody or pharmaceutical composition is delivered intravenously. Lung administration may also be used, for example, by using an inhaler or nebulizer, and by formulation with a nebulizer for use as a spray. In some embodiments, the antibody or pharmaceutical compositions described herein are delivered subcutaneously or intravenously. In some embodiments, the antibody or pharmaceutical compositions described herein are delivered intra-arterially. In some embodiments, the antibody or pharmaceutical compositions described herein are delivered intratumorally. In some embodiments, the antibody or pharmaceutical compositions described herein are delivered to tumor-draining lymph nodes.

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

The precise dosage to be used in the composition will also depend on the route of administration and the severity of the infection or disease it causes, and should be determined based on the practitioner's judgment and the individual subject's condition. For example, the effective dosage can also vary depending on the route of administration, target site, the patient's physiological state (including age, weight, and health) (whether the patient is human or animal), and whether other medications administered or the treatment is prophylactic or therapeutic. Typically, the patient is human, but non-human mammals, including transgenic animals, can also be treated. Therapeutic dosages are optimally titrated to optimize safety and efficacy.

The anti-TIM-3 (e.g., human TIM-3) antibodies described herein can also be used to determine TIM-3 (e.g., human TIM-3) protein levels in biological samples using classical immunohistochemical methods known to those skilled in the art, including immunoassays such as enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, or Western blotting. Suitable antibody assay labels are known in the art and include enzyme labels such as glucose oxidase; radioactive isotopes such as iodine ( ^¹²⁵I , ^¹²¹I ), carbon ( ^¹⁴C ), sulfur ( ^³⁵S ), tritium ( ^³H ), indium ( ^¹²¹In ), and technetium ( ^⁹⁹Tc ); luminescent labels such as luminol; and fluorescent labels such as fluorescein and rhodamine, and biotin. Such labels can be used to label the antibodies described herein. Alternatively, a second antibody recognizing the anti-TIM-3 (e.g., human TIM-3) antibody described herein may be labeled and combined with the anti-TIM-3 (e.g., human TIM-3) antibody for detecting TIM-3 (e.g., human TIM-3) protein levels. Therefore, in one embodiment, the present invention relates to the use of the antibody of the present invention for in vitro detection of TIM-3 (e.g., human TIM-3) protein in biological samples. In another embodiment, the present invention relates to the use of the anti-TIM-3 (e.g., human TIM-3) antibody of the present invention for in vitro determination and/or detection of TIM-3 (e.g., human TIM-3) protein levels in biological samples, optionally wherein the anti-TIM-3 (e.g., human TIM-3) antibody is conjugated with a radionuclide or a detectable label and/or carries the label described herein, and/or is used using immunohistochemical methods.

The determination of TIM-3 (e.g., human TIM-3) protein expression levels is intended to include qualitatively or quantitatively measuring or estimating the TIM-3 (e.g., human TIM-3) protein level in a first biological sample, either directly (e.g., by measuring or estimating absolute protein levels) or relatively (e.g., by comparing it with disease-related protein levels in a second biological sample). The TIM-3 (e.g., human TIM-3) peptide expression level in the first biological sample can be measured or estimated and compared with a standard TIM-3 (e.g., human TIM-3) protein level, which is obtained from a second biological sample obtained from disease-free individuals or determined by averaging levels from a population of disease-free individuals. As is recognized in the art, once the “standard” TIM-3 (e.g., human TIM-3) peptide level is known, it can be repeatedly used as a comparative standard. Therefore, in another embodiment, the present invention relates to an in vitro method for determining and/or detecting the level of TIM-3 protein, such as human TIM-3 protein, in a biological sample, comprising qualitatively or quantitatively measuring or estimating the level of TIM-3 protein, such as human TIM-3 protein, in the biological sample by immunohistochemical methods.

As used herein, the term "biological sample" refers to any biological sample obtained from a subject, including cell lines, tissues, or other cell sources that may express TIM-3 (e.g., human TIM-3). Methods for obtaining tissue biopsy samples and body fluids from animals (e.g., humans) are well known in the art. Biological samples include peripheral blood mononuclear cells.

The anti-TIM-3 (e.g., human TIM-3) antibodies described herein can be used for prognostic, diagnostic, surveillance, and screening applications, including in vitro and in vivo applications known and standard to those skilled in the art and based on this specification. Prognostic, diagnostic, surveillance, and screening assays and kits for in vitro assessment and evaluation of immune system status and/or immune response can be used to predict, diagnose, and monitor patient samples, including those known to have or suspected of having immune system dysfunction, or to predict, diagnose, and monitor expected or desired immune system responses, antigen responses, or vaccine responses. Assessment and evaluation of immune system status and/or immune response can also be used to determine a patient's suitability for a drug clinical trial or for administration of a specific chemotherapeutic agent, radiotherapy agent, or antibody (including combinations thereof), relative to different agents or antibodies. This type of prognostic and diagnostic surveillance and assessment has already utilized antibodies against the HER2 protein in breast cancer (HercepTest ^™ , Dako), and said assays are also used to evaluate patients in response to the antibody therapy used. In vivo applications include targeted cell therapy and radiographic imaging of the immune system modulation and immune response. Therefore, in one embodiment, the present invention relates to anti-TIM-3 antibodies and/or pharmaceutical compositions of the present invention, used as diagnostic agents. In one embodiment, the present invention relates to anti-TIM-3 antibodies and/or pharmaceutical compositions of the present invention, used in methods for predicting, diagnosing, and monitoring patients with or suspected of having immune system dysfunction and/or predicting, diagnosing, and monitoring immune system responses, antigen responses, or vaccine responses relative to expected or desired responses. In another embodiment, the present invention relates to the use of the anti-TIM-3 antibody of the present invention for predicting, diagnosing, and monitoring patients with or suspected of having immune system dysfunction, or for predicting, diagnosing, and monitoring immune system responses, antigen responses, or vaccine responses, by in vitro determination and/or detection of human TIM-3 protein levels in biological samples of a subject.

In one embodiment, an anti-TIM-3 (e.g., human TIM-3) antibody can be used in the immunohistochemistry of a biopsy sample. In one embodiment, the method is an in vitro method. In another embodiment, an anti-TIM-3 (e.g., human TIM-3) antibody can be used to detect the level of TIM-3 (e.g., human TIM-3) or the level of cells containing TIM-3 (e.g., human TIM-3) on their membrane surface, and that level can then be correlated with certain disease symptoms. The anti-TIM-3 (e.g., human TIM-3) antibodies described herein can carry a detectable or functional label and/or can be conjugated to a radionuclide or a detectable label. When fluorescent labeling is used, currently available microscopy and fluorescence activated cell sorting analysis (FACS) or a combination of two methods known in the art can be used to identify and quantify specific binding members. The anti-TIM-3 (e.g., human TIM-3) antibodies described herein can carry a fluorescent label or be conjugated to a fluorescent label. Exemplary fluorescent labels include, for example, reactive and conjugated probes such as aminocoumarin, fluorescein, and Texas red, Alexa Fluor dye, Cy dye, and DyLight dye. Anti-TIM-3 (e.g., human TIM-3) antibodies may carry or be conjugated to radiolabels or radionuclides, such as isotopes ^3H , ^14C , ^32P , 35S, ^36Cl , ^51Cr , ^57Co , ^58Co , ^59Fe , ^67Cu , 90Y, ^99Tc , ^111In , ^117Lu , ^121I , ^124I , ^125I , ^131I , ^{198Au, 211At , 213Bi} , ^{225Ac , and 186Re .} When using radiolabeling, currently available counting procedures known in the art can be used to identify and quantify the specific binding of anti-TIM-3 (e.g., human TIM-3) antibodies to TIM-3 (e.g., human TIM-3). In cases where the label is an enzyme, detection can be performed using any currently employed colorimetric, spectrophotometric, fluorescence spectrophotometric, galvanometry, or gas quantification techniques known in the art. This can be achieved by contacting a sample or control sample with an anti-TIM-3 (e.g., human TIM-3) antibody under conditions that allow for the formation of a complex between the antibody and TIM-3 (e.g., human TIM-3). Any complexes formed between the antibody and TIM-3 (e.g., human TIM-3) are detected and compared in the sample or control sample. Given the specific binding of the antibodies described herein to TIM-3 (e.g., human TIM-3), the antibodies can be used to specifically detect TIM-3 (e.g., human TIM-3) expression on cell surfaces. The antibodies described herein can also be used to purify TIM-3 (e.g., human TIM-3) via immunoaffinity purification. This document also includes assay systems for the quantitative analysis of the presence levels of, for example, TIM-3 (e.g., human TIM-3) or TIM-3 (e.g., human TIM-3)/TIM-3 (e.g., human TIM-3) ligand complexes, which can be prepared in the form of test kits, cartridges, or dispensing kits. The system, test kit, cartridge, or dispensing kit may contain labeled components, such as labeled antibodies, and one or more additional immunochemical reagents.

6.5 Polynucleotides, vectors, and methods for generating anti-TIM-3 antibodies

On the other hand, this document provides polynucleotides comprising nucleotide sequences encoding antibodies described herein or fragments thereof (e.g., light chain variable regions and/or heavy chain variable regions) that specifically bind to TIM-3 (e.g., human TIM-3) antigens, and vectors, such as vectors comprising such polynucleotides for recombinant expression in host cells (e.g., *E. coli* and mammalian cells). This document provides polynucleotides comprising nucleotide sequences encoding the heavy chain and/or light chain of any antibody provided herein, and vectors comprising such polynucleotide sequences, such as expression vectors for efficient expression in host cells, such as mammalian cells.

As used herein, an "isolated" polynucleotide or nucleic acid molecule is a polynucleotide or nucleic acid molecule separate from other nucleic acid molecules present in the natural source of that nucleic acid molecule (e.g., mouse or human). Furthermore, "isolated" nucleic acid molecules, such as cDNA molecules, may be substantially free of other cellular material, or, when produced by recombinant technology, substantially free of culture medium, or, when chemically synthesized, substantially free of chemical precursors or other chemicals. For example, the language "substantially free" includes polynucleotide or nucleic acid molecule formulations having less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (especially less than about 10%) of other substances (e.g., cellular material, culture medium, other nucleic acid molecules, chemical precursors, and/or other chemicals). In one specific embodiment, the nucleic acid molecule encoding the antibody described herein is isolated or purified.

In certain aspects, this document provides antibodies comprising an antibody encoding a TIM-3 (e.g., human TIM-3) peptide that specifically binds to it and comprising an amino acid sequence as described herein, as well as polynucleotide sequences of nucleotide sequences that compete with such antibodies for binding to the TIM-3 (e.g., human TIM-3) peptide (e.g., in a dose-dependent manner) or with antibodies that bind to the same epitope as such antibodies.

In some respects, this document provides polynucleotides comprising nucleotide sequences encoding a light chain or a heavy chain of an antibody described herein. The polynucleotide may comprise a nucleotide sequence encoding a light chain (see, for example, Table 1) of a VL FR and CDR containing an antibody described herein, or a nucleotide sequence encoding a heavy chain (see, for example, Table 1) of a VH FR and CDR containing an antibody described herein.

This document also provides polynucleotides encoding anti-TIM-3 (e.g., human TIM-3) antibodies, optimized, for example, through codon/RNA optimization, heterologous signal sequence substitution, and elimination of mRNA instability elements. Methods for generating optimized nucleic acids encoding anti-TIM-3 (e.g., human TIM-3) antibodies or fragments thereof (e.g., light chain, heavy chain, VH domain, or VL domain) for recombinant expression by introducing codon changes and/or eliminating repressive regions in mRNA can be performed by correspondingly employing optimization methods described, for example, in U.S. Patents 5,965,726, 6,174,666, 6,291,664, 6,414,132, and 6,794,498, all of which are incorporated herein by reference in their entirety. For example, potential splicing sites and instability elements within the RNA (e.g., A/T-rich or A/U-rich elements) can be mutated without altering the amino acids encoded by the nucleic acid sequence to improve the stability of the RNA for recombinant expression. The alteration utilizes the degeneracy of the genetic code, for example, by using alternative codons for the same amino acid. In some embodiments, it may be desirable to alter one or more codons to encode a conserved mutation, such as a similar amino acid having a similar chemical structure and properties and/or function to the original amino acid. Such methods can increase the expression of anti-TIM-3 (e.g., human TIM-3) antibodies or fragments thereof by at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times or more relative to the expression of anti-TIM-3 (e.g., human TIM-3) antibodies encoded by unoptimized polynucleotides.

In some embodiments, an optimized polynucleotide sequence encoding an anti-TIM-3 (e.g., human TIM-3) antibody or a fragment thereof (e.g., VL domain and/or VH domain) described herein may hybridize with an antisense (e.g., complementary) polynucleotide sequence encoding an unoptimized polynucleotide sequence encoding an anti-TIM-3 (e.g., human TIM-3) antibody or a fragment thereof (e.g., VL domain and/or VH domain). In a specific embodiment, an optimized nucleotide sequence encoding an anti-TIM-3 (e.g., human TIM-3) antibody or a fragment thereof described herein hybridizes with an antisense polynucleotide sequence encoding an unoptimized polynucleotide sequence encoding an anti-TIM-3 (e.g., human TIM-3) antibody or a fragment thereof under high-toughness hybridization conditions. In one specific embodiment, an optimized nucleotide sequence encoding an anti-TIM-3 (e.g., human TIM-3) antibody or a fragment thereof described herein hybridizes with an antisense polynucleotide sequence encoding an unoptimized polynucleotide sequence encoding an anti-TIM-3 (e.g., human TIM-3) antibody or a fragment thereof described herein under high-toughness, medium-toughness, or low-toughness hybridization conditions. Information regarding hybridization conditions has been described, see, for example, U.S. Patent Application Publication No. US2005/0048549 (e.g., paragraphs 72-73), which is incorporated herein by reference in its entirety.

Polynucleotides can be obtained by any method known in the art, and their nucleotide sequences can be determined. The nucleotide sequences encoding antibodies described herein (e.g., antibodies described in Table 1) and modified forms of these antibodies can be determined using methods known in the art, i.e., assembling in a manner where nucleotide codons encoding specific amino acids are known to produce nucleic acids encoding that antibody. Such antibody-encoding polynucleotides can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al. (1994), BioTechniques 17:242-6, which is incorporated herein by reference in its entirety), which, in brief, involves synthesizing overlapping oligonucleotides containing portions of the antibody-encoding sequence, annealing and ligating those oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

Alternatively, the polynucleotide encoding the antibody described herein can be produced from nucleic acids from a suitable source (e.g., hybridoma) using methods known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers that hybridize to the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells that produce the target antibody. Such PCR amplification methods can be used to obtain nucleic acids containing sequences encoding the antibody light chain and/or heavy chain. Such PCR amplification methods can be used to obtain nucleic acids containing sequences encoding variable light chain regions and/or variable heavy chain regions of the antibody. The amplified nucleic acids can be cloned into vectors for expression in host cells and for further cloning, for example, to produce chimeric and humanized antibodies.

If a clone containing a nucleic acid encoding a specific antibody is unavailable, but the sequence of the antibody molecule is known, the nucleic acid encoding the immunoglobulin can be chemically synthesized or obtained by PCR amplification using synthetic primers that hybridize to the 3' and 5' ends of that sequence, or by cloning using oligonucleotide probes specific to a particular gene sequence to identify, for example, cDNA clones from a cDNA library encoding an antibody, from a suitable source (e.g., an antibody cDNA library or a cDNA library derived from nucleic acids (preferably polyA+RNA) isolated from any tissue or cell expressing an antibody (such as hybridoma cells selected for expressing the antibodies described herein). The amplified nucleic acid generated by PCR can then be cloned into a reproducible cloning vector using any method known in the art.

DNA encoding the anti-TIM-3 (e.g., human TIM-3) antibody described herein can be readily isolated and sequenced using conventional procedures, such as by using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of the anti-TIM-3 (e.g., human TIM-3) antibody. Hybridoma cells can be used as a source of such DNA. Once isolated, the DNA can be placed in an expression vector, which is then transfected into host cells such as *E. coli* cells, ape COS cells, Chinese hamster ovary (CHO) cells (e.g., CHO cells from CHO GS System ^™ (Lonza)), or myeloma cells (which do not additionally produce immunoglobulins) to obtain the synthesis of anti-TIM-3 (e.g., human TIM-3) antibodies in recombinant host cells.

To generate full-length antibodies, the VH or VL sequence in an scFv clone can be amplified using PCR primers comprising the VH or VL nucleotide sequence, a restriction site, and flanking sequences protecting the restriction site. Using cloning techniques known to those skilled in the art, the PCR-amplified VH domain can be cloned into a vector expressing a heavy chain constant region (e.g., the human γ4 constant region), and the PCR-amplified VL domain can be cloned into a vector expressing a light chain constant region (e.g., the human κ or λ constant region). In some embodiments, the vector for expressing the VH or VL domain comprises an EF-1α promoter, a secretion signal, a variable region cloning site, a constant domain, and a selection marker such as neomycin. The VH and VL domains can also be cloned into a vector expressing the necessary constant region. The heavy chain conversion vector and the light chain conversion vector are then co-transfected into cell lines using techniques known to those skilled in the art to generate stable or transient cell lines expressing full-length antibodies (e.g., IgG).

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

Also provided are polynucleotides that hybridize with polynucleotides encoding the antibodies described herein under high-strictness, moderate-strictness, or low-strictness hybridization conditions. In a specific embodiment, the polynucleotides described herein hybridize with polynucleotides encoding the VH domain and/or VL domain provided herein under high-strictness, moderate-strictness, or low-strictness hybridization conditions.

Hybridization conditions have been described in the art and are known to those skilled in the art. For example, hybridization under stringent conditions may involve hybridization of DNA bound to a filter in 6x sodium chloride/sodium citrate (SSC) at about 45°C, followed by washing once or more in 0.2x SSC/0.1% SDS at about 50-65°C; hybridization under highly stringent conditions may involve hybridization of nucleic acids bound to a filter in 6x SSC at about 45°C, followed by washing once or more in 0.1x SSC/0.2% SDS at about 68°C. Hybridization under other stringent hybridization conditions is known to those skilled in the art and has been described, see, for example, Ausubel FM et al. (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 and 2.10.3, which are incorporated herein by reference in their entirety.

In some aspects, this document provides cells (e.g., host cells) that express (e.g., recombinantly express) antibodies described herein that specifically bind to TIM-3 (e.g., human TIM-3), and associated polynucleotides and expression vectors. This document provides vectors (e.g., expression vectors) containing polynucleotide sequences encoding anti-TIM-3 (e.g., human TIM-3) antibodies or fragments for recombinant expression in host cells, preferably in mammalian cells. This document also provides host cells containing such vectors for recombinant expression of anti-TIM-3 (e.g., human TIM-3) antibodies (e.g., human or humanized antibodies) described herein. In one particular aspect, this document provides a method for generating the antibodies described herein, comprising expressing such antibodies from host cells.

Recombinant expression of antibodies described herein that specifically bind to TIM-3 (e.g., human TIM-3) (e.g., full-length antibodies, heavy chains and/or light chains of antibodies, or single-chain antibodies) involves constructing an expression vector containing a polynucleotide encoding the antibody. Once a polynucleotide encoding the antibody molecule described herein, the heavy chain and/or light chain of the antibody, or a fragment thereof (e.g., the variable region of the heavy chain and/or light chain) is obtained, the vector for generating the antibody molecule can be produced using recombinant DNA techniques known in the art. Therefore, methods for preparing proteins by expressing polynucleotides containing nucleotide sequences encoding antibodies or antibody fragments (e.g., light chains or heavy chains) are described herein. Methods known to those skilled in the art can be used to construct expression vectors containing the coding sequence of an antibody or antibody fragment (e.g., light chain or heavy chain) and appropriate transcription and translation control signal sequences. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are reproducible vectors comprising, optionally linked to a promoter, nucleotide sequences encoding the antibody molecule described herein, the heavy or light chain of an antibody, a variable region of the heavy or light chain of an antibody or a fragment thereof, or a CDR of the heavy or light chain. Such vectors may, for example, comprise nucleotide sequences encoding constant regions of an antibody molecule (see, for example, International Publications WO 86/05807 and WO 89/01036; and U.S. Patent No. 5,122,464, which are incorporated herein by reference in their entirety), and antibody variable regions may be cloned into such vectors for expression of the entire heavy chain, the entire light chain, or both the entire heavy and light chains.

Expression vectors can be transferred into cells (e.g., host cells) using conventional techniques, and the resulting cells can then be cultured using conventional techniques to produce the antibodies or fragments thereof described herein. Therefore, this document provides host cells containing polynucleotides encoding the antibodies or fragments thereof described herein, or their heavy or light chains or fragments thereof, or the single-chain antibodies described herein, said polynucleotides being operatively linked to a promoter for expressing such sequences in the host cells. In some embodiments, for the expression of double-chain antibodies, vectors encoding both the heavy and light chains separately can be co-expressed in host cells to express the entire immunoglobulin molecule, as detailed below. In some embodiments, the host cell contains polynucleotides comprising both the heavy and light chains encoding the antibodies or fragments described herein. In a specific embodiment, the host cell contains two distinct vectors, a first vector containing a polynucleotide encoding the heavy chain or a heavy chain variable region of the antibody or fragment described herein, and a second vector containing a polynucleotide encoding the light chain or a light chain variable region of the antibody or fragment described herein. In other embodiments, the first host cell comprises a first vector containing a polynucleotide encoding a heavy chain or a heavy chain variable region encoding an antibody or a fragment thereof described herein, while the second host cell comprises a second vector containing a light chain or a light chain variable region encoding an antibody or a fragment thereof described herein. In a specific embodiment, the heavy chain/heavy chain variable region expressed by the first cell associates with the light chain/light chain variable region of the second cell to form the anti-TIM-3 (e.g., human TIM-3) antibody described herein. In some embodiments, a host cell population comprising such first host cells and such second host cells is provided herein.

In one particular embodiment, a family of vectors is provided herein comprising a first vector containing a polynucleotide encoding a light chain/light chain variable region of an antibody against TIM-3 (e.g., human TIM-3) as described herein, and a second vector containing a polynucleotide encoding a heavy chain/heavy chain variable region of an antibody against TIM-3 (e.g., human TIM-3) as described herein.

A variety of host-expression vector systems can be used to express the antibody molecules described herein (see, for example, U.S. Patent No. 5,807,715, which is incorporated herein by reference in its entirety). Such host-expression systems represent media through which a target coding sequence can be generated and subsequently purified, but also represent cells that can express the antibody molecules described herein in situ upon transformation or transfection with an appropriate nucleotide coding sequence. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant phage DNA, plasmid DNA, or copious DNA expression vectors containing antibody-encoding sequences (e.g., *Escherichia coli* and *Bacillus subtilis*); yeast transformed with recombinant yeast expression vectors containing antibody-encoding sequences (e.g., *Saccharomyces pichia*); insect cell systems infected with recombinant viral expression vectors containing antibody-encoding sequences (e.g., baculoviruses); and plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors containing antibody-encoding sequences (e.g., Ti plasmids) (e.g., green algae such as *Chlamydomonas*). reinhardtii); or mammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NSO, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa and NIH3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB/20 and BMT10 cells) carrying recombinant expression constructs containing promoters derived from mammalian cell genomes (e.g., metallothionein promoters) or promoters derived from mammalian viruses (e.g., adenovirus late promoters; vaccinia virus 7.5K promoters). In one specific embodiment, the cells used to express the antibodies described herein are CHO cells, such as CHO cells from the CHO GS System ^™ (Lonza). In one specific embodiment, the cells used to express the antibodies described herein are human cells, such as human cell lines. In one specific embodiment, the mammalian expression vector is pOptiVEC ^™ or pcDNA3.3. In one specific embodiment, bacterial cells such as *Escherichia coli* or eukaryotic cells (e.g., mammalian cells) are used to express the recombinant antibody molecules, particularly for expressing the complete recombinant antibody molecules. For example, mammalian cells such as Chinese hamster ovary (CHO) cells, which bind to vectors such as major mid-early gene promoter elements from human cytomegalovirus, are efficient antibody expression systems (Foecking MK & Hofstetter H (1986) Gene 45:101-5; and Cockett MI et al. (1990) Biotechnology 8(7):662-7, each incorporated herein by reference in its entirety). In some embodiments, the antibodies described herein are produced via CHO cells or NSO cells. In one specific embodiment, the expression of the nucleotide sequence encoding the antibody described herein that specifically binds to TIM-3 (e.g., human TIM-3) is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

In bacterial systems, a variety of expression vectors can be advantageously selected based on the intended use of the expressed antibody molecule. For example, when a large quantity of such antibodies is to be produced for the creation of pharmaceutical compositions containing antibody molecules, a vector that directs the high-level expression of an easily purified fusion protein product may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983) EMBO J 2:1791-1794), in which the antibody coding sequence can be separately coupled to the lac Z coding region in the vector to produce the fusion protein; the pIN vector (Inouye S & Inouye M (1985) Nuc Acids Res 13:3101-3109; Van Heeke G & Schuster SM (1989) J Biol Chem 24:5503-5509); and so on, all of which are incorporated herein by reference in their entirety. For example, the pGEX vector can also be used to express exogenous peptides as fusion proteins with glutathione 5-transferase (GST). Generally, such fusion proteins are soluble and can be readily purified from lysed cells by adsorption and binding to a matrix of glutathione agarose beads, followed by elution in the presence of free glutathione. The pGEX vector is designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In insect systems, for example, the alfalfa silver-striped armyworm (Autographa californica) nucleopolyhedrovirus (AcNPV) can be used as a vector for expressing exogenous genes. The virus grows in Spodoptera frugiperda cells. Antibody-coding sequences can be cloned separately into non-essential regions of the virus (e.g., polyhedrosis protein genes) and placed under the control of AcNPV promoters (e.g., polyhedrosis protein promoters).

In mammalian host cells, a variety of virus-based expression systems can be utilized. In cases where adenovirus is used as the expression vector, the target antibody-coding sequence can be linked to an adenoviral transcription/translation control complex, such as a late promoter and a triplet leader sequence. This chimeric gene can then be inserted into the adenoviral genome via in vitro or in vivo recombination. Insertion into non-essential regions of the viral genome (e.g., E1 or E3 regions) will produce a viable recombinant virus capable of expressing antibody molecules in an infected host (e.g., see Logan J & Shenk T (1984) PNAS 81(12):3655-9, which is incorporated herein by reference in its entirety). Specific initiation signal sequences can also be required for efficient translation of the inserted antibody-coding sequence. These signal sequences include the ATG start codon and adjacent sequences. Furthermore, the start codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire inserted fragment. These exogenous translation control signal sequences and start codons can have a variety of origins, both natural and synthetic. Expression efficiency can be enhanced by including appropriate transcriptional enhancer elements, transcription terminators, etc. (see, for example, Bitter G et al. (1987) Methods Enzymol. 153:516-544, which is incorporated herein by reference in its entirety).

Additionally, host cell lines can be selected to regulate the expression of the inserted sequence or to modify and process the gene product in a desired specific manner. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for protein function. Different host cells possess unique and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure proper modification and processing of the expressed exogenous protein. For this purpose, eukaryotic host cells with appropriate cellular mechanisms for processing primary transcripts, glycosylation, and phosphorylation of gene products can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, MDCK, HEK293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NSO (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB/20, BMT10 and HsS78Bst cells. In some embodiments, the anti-TIM-3 (e.g., human TIM-3) antibody described herein is produced in mammalian cells, such as CHO cells.

In one specific embodiment, the antibody described herein has a reduced fucose content or no fucose content. Such antibodies can be produced using techniques known to those skilled in the art. For example, the antibody can be expressed in cells with defective or absent fucosylation capacity. In one specific example, a cell line with two alleles knocked out of α1,6-fucosyltransferase can be used to produce antibodies with reduced fucose content. The Lonza system is an example of such a system that can be used to produce antibodies with reduced fucose content.

To achieve long-term, high-yield production of recombinant proteins, stable expression cells can be generated. For example, cell lines stably expressing the anti-TIM-3 (e.g., human TIM-3) antibody described herein can be generated. In a specific embodiment, the cells provided herein stably express light chain/light chain variable regions and heavy chain/heavy chain variable regions, which associate to form the antibody described herein.

In some respects, without using expression vectors containing viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selection markers. After the introduction of exogenous DNA/polynucleotides, engineered cells can be allowed to grow in enrichment media for 1–2 days, followed by conversion to selection media. The selection markers in the recombinant plasmid confer resistance to selection and allow cells to stably integrate the plasmid into their chromosome and grow to form loci that can then be cloned and amplified into cell lines. This approach can be advantageously used to engineer cell lines expressing the anti-TIM-3 (e.g., human TIM-3) antibody or fragments thereof described herein. Such engineered cell lines can be particularly useful for screening and evaluating compositions that interact directly or indirectly with antibody molecules.

A variety of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler M et al. (1977) Cell 11(1):223-32), hypoxanthine-guanine phosphoribosyltransferase (Szybalska EH & Szybalski W (1962) PNAS 48(12):2026-2034) and adenine phosphoribosyltransferase (Lowy I et al. (1980) Cell 22(3):817-23) genes in tk-, hgprt-, or aprt- cells, all of which are incorporated herein by reference in their entirety. Similarly, antimetabolite resistance can be used as a basis for selection on the following genes: dhfr, which confers resistance to methotrexate (Wigler M et al. (1980) PNAS 77(6):3567-70; O’Hare K et al. (1981) PNAS 78:1527-31); gpt, which confers resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4):2072-6); neo, which confers resistance to aminoglycoside G-418 (Wu GY & Wu CH (1991) Biotherapy 3:87-95; Tolstoshev P (1993) AnnRe v Pharmacol Toxicol 32:573-596; Mulligan RC (1993) Science 260:926-932; and Morgan RA & Anderson WF (1993) Ann Rev Biochem 62:191-217; Nabel GJ & Felgner PL (1993) Trends Biotechnol 11(5):211-5); and hygro, which confers resistance to hygromycin (Santerre RF et al. (1984) Gene 30(1-3):147-56), all of which are incorporated herein by reference in their entirety. Methods commonly known in the field of recombinant DNA technology can be routinely applied to select desired recombinant clones, and such methods are described, for example, in Ausubel FM et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler M, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Dracopoli NC et al. (eds.), Current Protocols in Human Genetics, Chapters 12 and 13, John Wiley & Sons, NY (1994); Colbère-Garapin F et al. (1981) J Mol Biol 150:1-144, which are incorporated herein by reference in their entirety.

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

Host cells can be co-transfected with two or more expression vectors as described herein, with the first vector encoding a polypeptide derived from the heavy chain and the second vector encoding a polypeptide derived from the light chain. Both vectors may contain the same selection marker, enabling the heavy and light chain polypeptides to be expressed equivalently. Host cells can be co-transfected with different amounts of two or more expression vectors. For example, host cells can be transfected with a first expression vector and a second expression vector in any of the following ratios: 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 encoding and capable of expressing both the heavy and light chain polypeptides can be used. In such cases, the light chain should be placed before the heavy chain to avoid excessive toxicity of the free heavy chain (Proudfoot NJ (1986) Nature 322:562-565; and G (1980) PNAS 77:2197-2199, each incorporated herein by reference in its entirety). The coding sequences for the heavy and light chains can comprise cDNA or genomic DNA. The expression vector can be monocistronic or polycistronic. Polycistronic nucleic acid constructs can encode 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, or in the range of 2-5, 5-10, or 10-20 genes/nucleotide sequences. For example, a bicistronic nucleic acid construct can contain a promoter, a first gene (e.g., the heavy chain of the antibody described herein), and a second gene (e.g., the light chain of the antibody described herein) in the following order. In such expression vectors, transcription of both genes can be driven by this promoter, and translation of mRNA from the first gene can be performed via a cap-dependent scanning mechanism while translation of mRNA from the second gene can be performed via a cap-independent mechanism, such as via IRES.

Once the antibody molecules described herein have been generated through recombinant expression, they can be purified using any method known in the art for the purification of immunoglobulin molecules, such as chromatography (e.g., ion exchange chromatography, affinity chromatography, and for specific antigens following protein A, particularly affinity chromatography and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. Furthermore, the antibodies described herein can be fused with heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

In specific embodiments, the antibodies described herein are isolated or purified. Typically, isolated antibodies are antibodies substantially free of other antibodies having antigen specificity different from that of the isolated antibody. For example, in one particular embodiment, the formulation of the antibody described herein is substantially free of cellular material and/or chemical precursors. The language “substantially free of cellular material” includes antibody formulations in which the antibody is separated from cellular components of the cells from which it is isolated or recombined. Thus, antibodies substantially free of cellular material include formulations having less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of heterologous protein (also referred to herein as “contaminating protein”), and/or antibody variants, such as different post-translational modifications of the antibody or other different forms of the antibody (e.g., antibody fragments). When the antibody is recombined, it is also generally substantially free of culture medium, i.e., the culture medium constitutes less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the protein formulation. When antibodies are produced through chemical synthesis, they are typically substantially free of chemical precursors or other chemicals; that is, they are separate from the chemical precursors or other chemicals involved in protein synthesis. Therefore, such antibody formulations contain less than about 30%, 20%, 10%, or 5% (on a dry weight) of chemical precursors or compounds other than the target antibody. In one specific embodiment, the antibody described herein is isolated or purified.

Antibodies or fragments thereof that specifically bind to TIM-3 (e.g., human TIM-3) can be produced by any method known in the art for antibody synthesis, such as by chemical synthesis or by recombinant expression techniques. Unless otherwise stated, the methods described herein employ conventional techniques from the fields of molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and other relevant prior art. These techniques are described and fully illustrated in, for example, in the references cited herein. 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, 2nd ed., Cold Spring Harbor Laboratory Press; Sambrook J et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al., Current Protocols in Molecular Cloning Biology, John Wiley & Sons (1987 and annual update); Current Protocols in Immunology, John Wiley & Sons (1987 and annual update); 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. (ed.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press, all of which are incorporated herein by reference in their entirety.

In one specific embodiment, the antibody described herein is an antibody (e.g., a recombinant antibody) prepared, expressed, generated, or isolated by any means involving the generation of DNA sequences (e.g., via synthesis), genetic engineering. In some embodiments, such antibodies comprise sequences (e.g., DNA sequences or amino acid sequences) within an antibody germline library that are not naturally present in animals or mammals (e.g., humans).

On one hand, this document provides methods for preparing antibodies that specifically bind to TIM-3 (e.g., human TIM-3), comprising culturing the cells or host cells described herein. In one embodiment, the method is performed in vitro. In some aspects, this document provides methods for generating antibodies that specifically bind to TIM-3 (e.g., human TIM-3), comprising expressing (e.g., recombinantly expressing) the antibody using the cells or host cells described herein (e.g., cells or host cells containing polynucleotides encoding the antibodies described herein). In one particular embodiment, the cells are isolated cells. In one particular embodiment, exogenous polynucleotides have been introduced into the cells. In one particular embodiment, the method further includes the step of purifying the antibody obtained from the cells or host cells.

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

Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including hybridoma, recombinant, and phage display techniques, or combinations thereof. For example, monoclonal antibodies can be produced using hybridoma techniques, including those known in the art and, for example, those taught in Harlow E & Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed., 1988); and Hammerling GJ et al., Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, N.Y., 1981) (each incorporated herein by reference in its entirety). The term “monoclonal antibody” as used herein is not limited to antibodies produced by hybridoma techniques. For example, monoclonal antibodies can be produced from exogenous expression of the antibodies described herein or fragments thereof, for example, through recombinant host cells of the light and/or heavy chains of such antibodies.

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

Methods for generating and screening specific antibodies using hybridoma technology are routine and well-known in the art. For example, in hybridoma methods, mice or other suitable host animals such as sheep, goats, rabbits, rats, hamsters, or macaques are immunized to induce the production or ability to produce antibodies that will specifically bind to proteins used for immunization (e.g., TIM-3 (e.g., human TIM-3)). 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 hybridoma cells (Goding JW (ed.), Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986), which is incorporated herein by reference in its entirety). Additionally, RIMMS (Multi-site Repeat Immunization) technology can be used to immunize animals (Kilpatrick KE et al. (1997) Hybridoma 16:381-9, which is incorporated herein by reference in its entirety).

In some embodiments, mice (or other animals such as rats, monkeys, donkeys, pigs, sheep, hamsters, or dogs) are immunized with an antigen (e.g., TIM-3 (e.g., human TIM-3)) and, once an immune response is detected, for example, by detecting antibodies specific to the antigen in mouse serum, the mouse spleen is harvested and spleen cells are isolated. The spleen cells are then fused with any suitable myeloma cells, such as cells from the SP20 cell line available from the U.S. Type Culture Collection (Manassas, VA), using known techniques to form hybridomas. The hybridomas are selected and cloned using limiting dilutions. In some embodiments, lymph nodes from immunized mice are harvested and fused with NSO myeloma cells.

Hybridoma cells prepared in this way are inoculated and grown in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if the parental myeloma cells are deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma culture medium will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells.

The specific implementation scheme employs efficient fusion to support stable, high-level antibody production in selected antibody-producing cells and myeloma cells sensitive to culture media such as HAT medium. These myeloma cell lines include mouse myeloma lines, such as the NS0 cell line or those derived from MOPC-21 and MPC-11 mouse tumors, available from the Salk Institute Cell Distribution Center, San Diego, CA, USA, and SP-2 or X63-Ag8.653 cells, available from the U.S. Type Culture Collection (Rockville, MD, USA). Human myeloma and mouse-human hybrid myeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor D (1984) J Immunol 133:3001-5; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987), each of which is incorporated herein by reference in its entirety).

The production of monoclonal antibodies against TIM-3 (e.g., human TIM-3) in the culture medium for hybridoma cell growth is measured. The binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by methods known in the art (e.g., immunoprecipitation) or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

After identifying hybridoma cells that produce antibodies with the desired specificity, affinity, and/or activity, clones can be subcloned via a limiting dilution process and cultured using standard methods (Goding JW, Monoclonal Antibodies: Principles and Practice, ibid.). Suitable media for this purpose include, for example, D-MEM or RPMI 1640. Alternatively, hybridoma cells can be grown in vivo as ascites tumors in animals.

Monoclonal antibodies secreted by subclones are appropriately separated from culture medium, ascites, or serum using routine immunoglobulin purification processes, such as protein A agarose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

The antibodies described herein comprise antibody fragments that recognize specific TIM-3 (e.g., human TIM-3) and can be generated by any technique known to those skilled in the art. For example, the Fab and F(ab') ₂ fragments described herein can be generated by proteolytic cleavage of an immunoglobulin molecule using enzymes such as papain (for generating the Fab fragment) or trypsin (for generating the F(ab') ₂ fragment). The Fab fragment corresponds to one of the 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 in the hinge region.

Furthermore, the antibodies described herein can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of a phage particle carrying a polynucleotide sequence encoding them. Specifically, DNA sequences encoding the VH and VL domains are amplified from an animal cDNA library (e.g., a human or mouse cDNA library of the affected tissue). The DNA encoding the VH and VL domains is recombined with an scFv adapter by PCR and cloned into a phage particle vector. The vector is electroporated into *E. coli*, and the *E. coli* is infected with a helper phage. The phages used in these methods are typically filamentous phages, including fd and M13, and the VH and VL domains are typically recombinantly fused with phage gene III or gene VIII. Phages expressing antigen-binding domains that bind to specific antigens can be selected or identified using antigens, such as labeled antigens or antigens that bind to or capture solid surfaces or beads. Examples of phage display methods that can be used to prepare the antibodies described herein include Brinkman U et al. (1995) J Immunol Methods 182:41-50; Ames RS et al. (1995) J Immunol Methods 184:177-186; Kettleborough CA et al. (1994) Eur J Immunol 24:952-958; Persic L et al. (1997) Gene 187:9-18; Burton DR & Barbas CF (1994) Advan Immunol 57:191-280; PCT application number PCT/GB91/001134; International Publications Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, WO 95/20401 and WO 97/13844; and U.S. Patent Nos. 5,698,426, 5,223,409, and 5,403, Those disclosed in Nos. 484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108 are all incorporated herein by reference in their entirety.

As described in the above literature, after phage selection, antibody coding regions derived from phages can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen-binding fragments, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, for example, as described below. Techniques for recombinantly generating antibody fragments such as Fab, Fab', and F(ab') ₂ fragments can also be utilized using methods known in the art, such as those disclosed 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, all of which are incorporated herein by reference in their entirety.

In some embodiments, to generate full-length antibodies, PCR primers comprising the VH or VL nucleotide sequence, restriction sites, and flanking sequences protecting the restriction sites can be used to amplify the VH or VL sequence from a template (e.g., an scFv clone). Using cloning techniques known to those skilled in the art, the PCR-amplified VH domain can be cloned into a vector expressing the VH constant region, and the PCR-amplified VL domain can be cloned into a vector expressing the VL constant region (e.g., the human κ or λ constant region). The VH and VL domains can also be cloned into a vector expressing the necessary constant region. The heavy chain transformation vector and the light chain transformation vector are then co-transfected into cell lines using techniques known to those skilled in the art to generate stable or transient cell lines expressing full-length antibodies (e.g., IgG).

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

Humanized antibodies are capable of binding to a predetermined antigen and comprise a framework region having an amino acid sequence substantially that of a human immunoglobulin and a core region (CDR) having an amino acid sequence substantially that of a non-human immunoglobulin (e.g., mouse immunoglobulin). In a particular embodiment, the humanized antibody further comprises at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin. The antibody may also comprise CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. Humanized antibodies may be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotypes, including _IgG1 , _IgG2 , _IgG3 , and _IgG4 . Humanized antibodies can be produced using a variety of techniques known in the art, including but not limited to, CDR transplantation (European Patent No. EP 239400; International Publication No. WO 91/09967; and US Patent Nos. 5,225,539, 5,530,101 and 5,585,089), veneering, or resurfacing (European Patent Nos. EP 592106 and EP 519596; Padlan EA (1991) Mol Immunol 28(4/5):489-498; Studnicka GM et al. (1994) Prot Engineering 7(6):805-814; and Roguska MA et al. (1994) PNAS). 91:969-973), chain remodeling (US Patent No. 5,565,332), and, for example, US Patent No. 6,407,213, US Patent No. 5,766,886, International Publication No. WO 93/17105; Tan P et al. (2002) J Immunol 169:1119-25; Caldas C et al. (2000) Protein Eng. 13(5):353-60; Morea V et al. (2000) Methods 20(3):267-79; Baca M et al. (1997) J Biol Chem 272(16):10678-84; Roguska MA et al. (1996) Protein Eng 9(10):895 904; Couto JR et al. (1995) Cancer The techniques disclosed in Res. 55(23 Supplement): 5973s-5977s; Couto JR et al. (1995) Cancer Res 55(8): 1717-22; Sandhu JS (1994) Gene 150(2): 409-10; and Pedersen JT et al. (1994) J Mol Biol 235(3): 959-73 (all of which are incorporated herein by reference in their entirety). See also U.S. Application Publication No. US2005/0042664 A1 (February 24, 2005), which is incorporated herein by reference in its entirety.

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

Single-domain antibodies, such as those lacking a light chain, can be produced by methods known in the art. See Riechmann L & Muyldermans S (1999) J Immunol 231:25-38; Nuttall SD et al. (2000) Curr Pharm Biotechnol 1(3):253-263; Muyldermans S, (2001) J Biotechnol 74(4):277-302; U.S. Patent No. 6,005,079; and International Publications Nos. WO 94/04678, WO 94/25591 and WO 01/44301, all of which are incorporated herein by reference in their entirety.

Furthermore, antibodies that specifically bind to TIM-3 (e.g., human TIM-3) antigens can instead be used to generate anti-idiotype antibodies against “mimicking” antigens using techniques known to those skilled in the art. See, for example, Greenspan NS & Bona CA (1989) FASEB J 7(5):437-444; and Nissinoff A (1991) J Immunol 147(8):2429-2438, each of which is incorporated herein by reference in its entirety.

In certain embodiments, the antibody described herein that binds to the same TIM-3 (e.g., human TIM-3) epitope as the anti-TIM-3 (e.g., human TIM-3) antibody described herein is a human antibody. In certain embodiments, the antibody described herein that competitively blocks (e.g., in a dose-dependent manner) the binding of any of the antibodies described herein to TIM-3 (e.g., human TIM-3) is a human antibody. Human antibodies can be generated using any method known in the art. For example, transgenic mice that cannot express functional endogenous immunoglobulins but can express human immunoglobulin genes can be used. Specifically, the human heavy chain and light chain immunoglobulin gene complex can be introduced into mouse embryonic stem cells randomly or via homologous recombination. Alternatively, in addition to the human heavy chain and light chain genes, human variable, constant, and polymorphic regions can be introduced into mouse embryonic stem cells. Independently or simultaneously with the introduction of human immunoglobulin gene loci via homologous recombination, the mouse heavy chain and light chain immunoglobulin genes can be rendered nonfunctional. Specifically, 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. These chimeric mice can then breed to produce homozygous offspring expressing human antibodies. The transgenic mice are immunized in their normal form with a selected antigen, such as a whole or a portion of an antigen (e.g., TIM-3 (e.g., human TIM-3)). Monoclonal antibodies against the antigen can be obtained from the immunized transgenic mice using conventional hybridoma techniques. The human immunoglobulin transgenes carried by the transgenic mice undergo rearrangement during B-cell differentiation, followed by class switching and somatic mutations. Therefore, using such techniques, it is possible to generate therapeutically useful IgG, IgA, IgM, and IgE antibodies. For a review of this technique for generating human antibodies, see Lonberg N & Huszar D (1995) Int Rev Immunol 13:65-93, which is incorporated herein by reference in its entirety. For a detailed discussion of the techniques and protocols used to generate human antibodies and human monoclonal antibodies, see, for example, International Publications WO 98/24893, WO 96/34096 and WO96/33735; and U.S. Patents 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318 and 5,939,598, all of which are incorporated herein by reference in their entirety. Examples of mice capable of producing human antibodies include Xenomouse ^™ (Abgenix, Inc.; U.S. Patent Nos. 6,075,181 and 6,150,184), HuAb-Mouse ^™ (Mederex, Inc./Gen Pharm; U.S. Patent Nos. 5,545,806 and 5,569,825), Trans ChromoMouse ^™ (Kirin), and KM Mouse ^™ (Medarex/Kirin), all of which are incorporated herein by reference in their entirety.

Human antibodies that specifically bind to TIM-3 (e.g., human TIM-3) can be prepared by a variety of methods known in the art, including the phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also U.S. Patents 4,444,887, 4,716,111, and 5,885,793; and International Publications WO 98/46645, WO 98/50433, WO98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741, all of which are incorporated herein by reference in their entirety.

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

6.6 medicine box

Kits containing one or more antibodies or pharmaceutical compositions or conjugates described herein are also provided. In one specific embodiment, this document provides pharmaceutical packaging or kits containing one or more containers filled with one or more components of a pharmaceutical composition described herein, such as one or more antibodies provided herein. In some embodiments, the kit contains a pharmaceutical composition described herein and any preventative or therapeutic agent, such as those described herein. In some embodiments, the kit may contain T-cell mitogens, such as phytohemagglutinin (PHA) and/or phorbol myristate (PMA), or TCR complex-stimulating antibodies, such as anti-CD3 antibodies and anti-CD28 antibodies. Optionally associated with such containers may be a notification in the form prescribed by a government agency regulating the production, use, and sale of a pharmaceutical or biological product, reflecting the agency's approval for its production, use, and sale for human administration.

Kits for use in the methods described above are also provided. In one embodiment, the kit contains the antibody described herein, preferably a purified antibody, in one or more containers. In one specific embodiment, the kit described herein contains substantially isolated TIM-3 (e.g., human TIM-3) antigen as a control. In another specific embodiment, the kit described herein further contains a control antibody that does not respond to the TIM-3 (e.g., human TIM-3) antigen. In yet another specific embodiment, the kit described herein contains one or more elements for detecting the binding of the antibody to the TIM-3 (e.g., human TIM-3) antigen (e.g., the antibody may be conjugated to a detectable matrix such as a fluorescent compound, enzyme substrate, radioactive compound, or luminescent compound, or a second antibody recognizing the first antibody may be conjugated to a detectable matrix). In specific embodiments, the kits provided herein may include recombinantly generated or chemically synthesized TIM-3 (e.g., human TIM-3) antigen. The TIM-3 (e.g., human TIM-3) antigen provided in the kit may also be attached to a solid support. In a more specific embodiment, the detection method of the above-described kit includes a solid support to which the TIM-3 (e.g., human TIM-3) antigen is attached. Such kits may also include non-attached reporter factor-labeled anti-human antibodies or anti-mouse/rat antibodies. In this embodiment, the binding of the antibody to the TIM-3 (e.g., human TIM-3) antigen can be detected by the binding of the reporter factor-labeled antibody. In one embodiment, the present invention relates to the use of the kit for the in vitro determination and/or detection of the TIM-3 antigen (e.g., human TIM-3) in biological samples.

7. Example

The embodiments in this section (i.e., Section 7) are provided by way of illustration rather than by way of limitation.

7.1 Example 1: Generation and characterization of a novel antibody against human TIM-3

This embodiment describes the generation and characterization of antibodies that bind to human T-cell immunoglobulin and mucin domain-3 (TIM-3). Specifically, this embodiment describes the generation of human antibodies that specifically bind to human TIM-3 and inhibit human TIM-3 function.

In some studies described below, the activity of the anti-TIM-3 antibody of the present invention is compared with the activity of reference anti-TIM-3 antibodies pab1944w or Hum11. Antibody pab1944w was generated based on the variable region of antibody 8213HV0LV0 provided in U.S. Patent No. 8,552,156 (which is incorporated herein by reference in its entirety). The sequence of pab1944w is shown in Table 7. Antibody pab1944w expresses an IgG ₁ antibody containing the N297A mutation in the Fc region according to the EU numbering system. Antibody Hum11 was generated based on the variable region of antibody ABTIM3-hum11 provided in U.S. Patent Publication No. US2015/0218274 (which is incorporated herein by reference in its entirety). The sequence of Hum11 is shown in Table 7. Antibody Hum11 expresses an IgG ₄ antibody containing the S228P mutation in the Fc region according to the EU numbering system.

Table 7. Sequence of reference anti-TIM-3 antibody.

7.1.1 Using Retrocyte Display ^™ technology to generate anti-TIM-3 antibodies

This article describes the generation of Retrocyte Display ^™ libraries. To generate library inserts, total RNA was extracted from FACS-sorted CD19-positive human B lymphocytes using phenol/chloroform. The total RNA was used for first-strand cDNA synthesis using the RevertAid First-Strand cDNA Synthesis Kit from Fermentas (Cat# K1621 and K1622). The antibody variable region was amplified from the cDNA by PCR and cloned into a retroviral expression vector (pCMA). These constructs were then used to transduce mouse preB (preB) cells to express the antibody on their surface using Retrocyte Display ^™ technology.

Screening of the Retrocyte Display ^™ library generated as described above for recombinant human TIM-3 and recombinant cynomolgus monkey TIM-3 resulted in the identification of two antibodies, named pab2085 and pab2088. Table 4 summarizes the sequence information of the variable regions of pab2085 and pab2088. Antibodies pab2085 and pab2088 express _IgG1 antibodies and were analyzed in the assays described below.

7.1.2 Binding of anti-TIM-3 antibody to TIM-3 expressing cells

The binding of antibodies pab2085 and pab2088 to TIM-3-expressing cells was tested 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 (BDPharmingen, Cat#553142) to reduce non-specific binding. After washing, cells were stained with anti-TIM-3 antibody or an allotype control antibody and analyzed using 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 (Figure 1).

7.1.3 Stability determination of anti-TIM-3 antibody

The selectivity of pab2085 and pab2088 for TIM-3 relative to family members TIM-1 and TIM-4 was evaluated using a suspension array technique. The microspheres were coupled to recombinant human TIM-3Fc (R&D Systems, Cat#2365-TM), recombinant human TIM-3His (Sino Biological, Cat#10390-H08H), recombinant cynomolgus monkey TIM-3Fc (R&D Systems, Cat#7914-TM), recombinant human TIM-1His (R&D Systems, Cat#1750-TM), or recombinant human TIM-4His (R&D, Cat#2929-TM) via amine coupling to the surface of the COOH beads. Purified pab2085, pab2088, and IgG ₁ isotype control antibodies were diluted to 10 ng/ml, 100 ng/ml, and 1000 ng/ml in assay buffer (Roche, Cat#11112589001). Each dilution (25 μl) was incubated with 1500 microspheres in 5 μl of assay buffer for 1 hour in the dark (20°C, 650 rpm). Standard curves were generated using two 25 μl aliquots of human IgG _1κ standard (Sigma, Cat#I5154) at a 1:3 dilution series (0.08–540 ng/ml). The assay was performed using 60 μl of R-PE-labeled goat anti-human IgG F(ab) ₂ (2.5 μg/ml; Jackson ImmunoResearch, Cat#109-116-097) followed by incubation for 1 hour (20°C, 650 rpm). The plates were analyzed using a Millipore 200 system. A total of 100 beads were counted per well in a 48 μl sample volume. PE MFI values were used to determine whether the binding to the aforementioned recombinant protein was specific or non-specific.

Both pab2085 (Figure 2A) and pab2088 (Figure 2B) showed specific binding to TIM-3 in humans and cynomolgus monkeys, while no significant binding to TIM-1 or TIM-4 was observed at the tested concentrations.

7.1.4 Optimization of Anti-TIM-3 Antibodies Using Retrocyte Display ^™ Technology

Antibodies pab2085 and pab2088 share the same heavy chain. To obtain additional anti-TIM-3 antibodies, a heavy chain Retrocyte Display ^™ sublibrary was generated based on the heavy chains of pab2085 and pab2088 and combined with a more diverse light chain library. Further screening of this novel Retrocyte Display ^™ library targeting recombinant human TIM-3 and recombinant cynomolgus monkey TIM-3 led to the identification of 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. The sequence information of the variable regions of these light chain optimized variants is listed in Table 4. These light chain optimized variants express antibodies containing either the wild-type IgG1 _Fc region or the _IgG1 variant Fc region. This _IgG1 variant Fc region does not affect the effector function of the Fc region.

The light chain optimized antibody pab2188 is an antibody containing a T109S substitution (i.e., a threonine-serine substitution at position 109 relative to the wild-type sequence) in the light chain constant domain. This substitution facilitates co-cloning of the variable region into the constant region. This mutation is a conserved modification that does not affect antibody binding or function. A wild-type counterpart, named pab2188w, was also generated, containing a threonine residue at position 109 of the light chain, according to the Kabat number. Antibody pab2188w is expressed as an antibody containing the _IgG1 N297A Fc region.

7.1.5 Binding of anti-TIM-3 antibody to TIM-3 expressing cells

The binding of the light chain optimized variants to cells expressing human or cynomolgus monkey TIM-3 was evaluated in flow cytometry assays similar to those described above. All variants showed binding to mouse 1624-5 cells engineered to express human TIM-3 (Fig. 3A and 3B) or cynomolgus monkey TIM-3 (Fig. 3C and 3D), but not to wild-type mouse 1624-5 cells (data not shown).

The binding of light chain-optimized variants to primary human T cells was compared with the binding of the parental antibody pab2085. In short, magnetic separation (Miltenyi Biotec) was used to enrich unreached whole T cells from peripheral blood mononuclear cells (PBMCs) isolated from healthy donor erythrocyte sedimentation rate (ESR) amber layer (Research Blood Components, LLC) via a Ficoll gradient. The enriched T lymphocyte population was then activated for _{3 days} at 37°C and 5% CO2 in RPMI medium supplemented with 10% heat-inactivated FBS using plate-bound anti-CD3 antibody (SP34, 3 μg/ml) and soluble anti-CD28 antibody (CD28.1, 2 μg/ml). After activation, cells were incubated with a human Fc receptor blocker for 15 minutes at room temperature to reduce non-specific binding (FcR block, Biolegend). Add anti-TIM-3 or IgG isotype control antibody (12-point titration, 10,000 ng/ml to 0.06 ng/ml) to individual samples and incubate at 4°C for 30 min. Wash samples twice and add a mixture of antibodies containing FITC-conjugated anti-κ antibody, as well as anti-CD3 (BV711, OKT3), anti-CD4 (BV605, OKT4), and anti-CD8a (PE, RPA-T8, all 2.5 μg/ml) diluted in buffer (PBS, 2 mM EDTA, 0.5% BSA, pH 7.2) to each sample and incubate at 4°C for 30 min. Wash samples twice and analyze using an LSR Tortessa flow cytometer (BD Biosciences). Analyze flow cytometry plots using a combination of FACSDIVA and WEHI Weasel software.

As shown in Figure 4, all light chain optimized variants tested in this study showed stronger binding to activated human CD8+ T cells than the parental antibody pab2085.

Next, the binding of the anti-TIM-3 antibody pab2188 to primary cynomolgus monkey cells was detected. Cryopreserved PBMCs isolated from cynomolgus monkeys (Worldwide Primates, Inc.) were thawed, washed, and then analyzed by flow cytometry. Cells were treated with 10% cynomolgus monkey serum (Abcam) at room temperature for 15 minutes before antibody incubation to reduce non-specific binding. Anti-TIM-3 or IgG isotype control antibodies (10-point titration, 20,000 ng/ml to 0.6 ng/ml) were added to individual samples and incubated at 4°C for 30 minutes. Samples were washed twice, and a mixture of FITC-conjugated anti-κ antibody and anti-CD11b (BV785, M1/70) antibodies diluted 2.5 μg/ml in buffer (PBS, 2 mM EDTA, 0.5% BSA, pH 7.2) was added to each sample and incubated at 4°C for 30 minutes. Samples were washed twice and analyzed using an LSR Tortessa flow cytometer (BDBiosciences). Flow cytometry plots were analyzed using a combination of FACSDIVA and WEHI Weasel software.

As shown in Figure 5, the anti-TIM-3 antibody pab2188 binds to primary cynomolgus monkey bone marrow cells in a dose-dependent manner.

7.1.6 Ligand blocking activity of anti-TIM-3 antibody

The ability of anti-TIM-3 antibodies to block the binding of recombinant human or cynomolgus TIM-3 to phosphatidylserine expressed by irradiated WR19L mouse lymphoma cells was tested. Recombinant human TIM-3Fc (R&D Systems, #2365-TM) or recombinant cynomolgus TIM-3Fc (R&D Systems, #7914-TM) (10,000 ng/ml) prepared in 1X Annexin-V binding buffer (adjusted to pH 7.4 with 10 mM Hepes, 140 mM NaCl, and 2.5 mM _CaCl2 ) was incubated together at room temperature for 30 minutes. WR19L cells, irradiated with 20 Gy and resuspended in 1X Annexin-V binding buffer, were added to an anti-TIM-3:TIM-3-Fc mixture to a final density of 1 x ^10⁶ cells/ml and incubated at room temperature for 45 min. Samples were washed once, and an antibody mixture containing PE-conjugated anti-Fc antibody (1:100 dilution) and viability stain (Biolegend, NIR channel; 1:1000 dilution), diluted in 1X Annexin-V binding buffer, was added to each sample and incubated at room temperature for 20 min. 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 using an LSR Tortessa 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 (Figure 6A) and recombinant cynomolgus monkey TIM-3 (Figure 6B) to phosphatidylserine.

7.1.7 Effect of anti-TIM-3 antibody on human PBMCs after stimulation with staphylococcal enterotoxin A (SEA)

The functional activity of a light chain optimized variant on primary human PBMCs following staphylococcal enterotoxin A (SEA) stimulation was evaluated. Briefly, cryopreserved human PBMCs (Research Blood Components) were plated at 1 x ^10⁵ cells/well in 96-well NUNCLON delta plates (NUNC ^™ ) supplemented with Normocin ^™ (Invivogen #ant-nr) and 10% heat-inactivated FBS (Gibco, Invitrogen Corporation) in RPMI 1640. Cells were cultured for 6 days at 37°C and 5% CO₂ in the presence of 5 μg/ml anti-PD-1 antibody pembrolizumab (batch 7002688300, Myoderm), anti-TIM-3 antibody (10 μg/ml), and SEA superantigen (100 ng/ml, Toxin Technologies). Cell-free _supernatant was collected and stored at -80°C until analysis. IFNγ levels were determined using AlphaLISA (Perkin Elmer).

When combined with the anti-PD-1 antibody pembrolizumab, the light chain-optimized variant enhanced IFNγ production in this primary human PBMC assay (Figure 7).

The functional activity of pab2188w was analyzed using a modified protocol in a SEA stimulation assay. Cryopreserved human PBMCs (Research Blood Components) were plated at 1 x ^10⁵ cells/well in 96-well NUNCLON delta surface plates (NUNC ^™ ) supplemented with Normocin ^™ (Invivogen #ant-nr) and 10% heat-inactivated FBS (Gibco, Invitrogen Corporation) in RPMI 1640. Cells were cultured for 9 days at 37°C and 5% CO₂ in the presence of 5 μg/ml anti-PD-1 antibody pembrolizumab (batch 7002688300, Myoderm), anti-TIM-3 antibody (10 μg/ml), and SEA superantigen (100 ng/ml, _Toxin Technologies). Cells were then washed once and restimulated for 2 days with fresh SEA and antibody. Cell-free supernatant was collected and stored at -80°C until analysis. IFNγ levels were determined using AlphaLISA (Perkin Elmer).

As shown in Figures 8A-8F, in this SEA stimulation assay, the anti-TIM-3 antibody pab2188w (IgG ₁ N297A), alone or in combination with the anti-PD-1 antibody pembrolizumab, enhanced IFNγ production in human PBMCs from multiple donors.

7.2 Example 2: Optimization of anti-TIM-3 antibody using CDR mutagenesis

To improve binding affinity, the anti-TIM-3 antibody pab2188w was modified using targeted mutagenesis of the CDR residues in the variable regions of both the heavy and light chains. In short, six Fab phage display libraries were generated based on the parental antibody pab2188w, each containing a CDRH or CDRL region modified using NNK degenerate codon randomization. The Fab phage libraries underwent affinity-driven selection against the recombinant human and cynomolgus monkey TIM-3 antigen. Nine clones, named 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. The sequence information of the variable regions for these nine clones is summarized in Table 4. All these variants shared the light chain of pab2188w but contained a mutation in the heavy chain CDR1. AM-1 through AM-9 were expressed as full-length antibodies containing the _IgG1 N297A Fc region and analyzed in the experiments described below.

7.2.1 Binding of anti-TIM-3 antibody to TIM-3 expressing cells

In flow cytometry analysis, the binding of antibodies AM-1 to AM-9 to Jurkat cells ectopically expressing human TIM-3 was compared with that of the parental antibody pab2188w. As shown in Figure 9A, all variants of Jurkat cells expressing TIM-3, as well as AM-2 and AM-6, showed stronger binding than the parental antibody pab2188w. The binding of AM-2 and AM-6 was further analyzed by flow cytometry using Kasumi-3 (CRL-2725 ^™ ), endogenously TIM-3-expressing human acute myeloid leukemia cell lines (Figure 9B), and human CD8+ T cells stimulated with staphylococcal enterotoxin A (SEA) (Figure 9C) and SEA-stimulated cynomolgus monkey CD8+ T cells (Figure 9D). For binding to human CD8+ T cells, human PBMCs isolated from healthy donor erythrocyte sedimentation rate (ESR) amber layer (Research Blood Components, LLC) via a Ficoll gradient were activated for 8 days with _SEA (100 ng/ml) in RPMI medium supplemented with 10% heat-inactivated FBS at 37°C and 5% CO2. After activation, cells were incubated at room temperature for 15 minutes with a human Fc receptor blocker (FcR blocker, Biolegend) to reduce nonspecific binding. Individual samples were added with anti-TIM-3 or IgG isotype control antibody (12-point titration, 10,000 ng/ml to 0.06 ng/ml) and incubated at 4°C for 30 minutes. Similarly, for binding to cynomolgus monkey CD8+ T cells, cynomolgus monkey PBMCs isolated from cryopreservation stock (Worldwide Primates Inc.) were activated for 5 days at 37°C and 5% _CO2 in RPMI medium supplemented with 10% heat-inactivated FBS using SEA (100 ng/ml). The activated cynomolgus monkey PBMCs were incubated at room temperature for 15 minutes with a combination of human Fc receptor blocker (FcR blocker, Biolegend) and cynomolgus monkey serum (Abcam) to reduce nonspecific binding. A mixture of phycoerythrin-conjugated AM-2 antibody or isotype control antibody (Biolegend PE conjugated, 6-point titration, 10,000 ng/ml to 41 ng/ml) and 2.5 μg/ml anti-CD4 antibody (BV605, OKT4) and anti-CD8a antibody (PE, SK1) was diluted in buffer (PBS, 2 mM EDTA, 0.5% BSA, pH 7.2) and added to each sample, then incubated at 4°C for 30 minutes. The samples were washed twice, and a mixture containing FITC-conjugated anti-κ antibody (all at 2.5 μg/ml) and antibodies against CD3 (BV711, OKT3), CD4 (BV605, OKT4), and CD8a (PE, RPA-T8) was diluted in buffer (PBS, 2 mM EDTA, 0.5% BSA, pH 7.2) and added to each sample, then incubated at 4°C for 30 minutes. Samples were washed twice and analyzed using LSR Fretessa flow cytometry (BD Biosciences). Flow cytometry plots were analyzed using a combination of FACSDIVA 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).

Next, in a similar assay, the binding of phycoerythrin (PE)-conjugated pab2188w, AM-2, or isotype control antibodies to primary human and cynomolgus monkey CD14+ bone marrow cells was analyzed by flow cytometry. In short, frozen PBMCs isolated from humans or cynomolgus monkeys (Worldwide Primates, Inc.) were thawed, washed, and then analyzed by flow cytometry. Cells were treated with 10% cynomolgus monkey serum (Abcam, Cat#ab155109) at room temperature for 15 minutes before antibody incubation to reduce nonspecific binding. PE-conjugated anti-TIM-3 or IgG isotype control antibodies (12-point titration, 10,000 ng/ml to 0.05 ng/ml for human PBMCs and 100,000 ng/ml to 0.5 ng/ml for cynomolgus monkey PBMCs) were added to individual samples containing a mixture of antibodies with anti-CD14 antibodies (APC, M5E2) and Zombie Green ^™ immobilizable activity markers, and then incubated at 4°C for 30 min. Additional samples were reserved for single-staining compensation controls (CD45-FITC, CD45-PE, and CD45-APC; clone MB4-6D6, Miltenyi). Samples were washed twice in buffer and analyzed using an LSR Tortessa flow cytometer (BD Biosciences). Flow cytometry plots were analyzed using a combination of FACSDIVA and WEHI Weasel software. AM-2 showed stronger binding to human (Fig. 9E) and cynomolgus monkey (Fig. 9F) CD14+ bone marrow cells than the parental antibody pab2188w.

7.2.2 Selectivity assay of anti-TIM-3 antibody

The selectivity of AM-2 and AM-6 for TIM-3 was evaluated using a suspension array technique. Microspheres were coupled to recombinant human TIM-3His (Sino Biological, #10390-H08H), recombinant cynomolgus monkey TIM-3Fc (Sino Biological, #90312-C02H), recombinant mouse TIM-3Fc (R&D Systems, #1529-TM), recombinant human TIM-1His (R&D Systems, #1750-TM), recombinant human TIM-4His (R&D, #2929-TM), recombinant human OX40His (Sino Biological, #10481-H08H), recombinant human GITR Fc (R&D Systems, #689-GR), recombinant human DR3Fc (R&D Systems, #943-D3), and recombinant human CD137 Fc (internal production material) via amine coupling to the surface of COOH beads. Purified pab2188w (IgG ₁ N297A), AM-2 (IgG ₁ N297A), AM-6 (IgG ₁ N297A), and IgG ₁ N297A isotype control antibodies were diluted to titers ranging from 10,000 ng/mL to 0.1 ng/mL in assay buffer (Roche 11112589001). Each dilution (25 μl) was incubated with 1500 microspheres in 5 μl of assay buffer for 1 hour in the dark (20°C, 650 rpm). Detection was performed using 60 μl of R-PE-labeled goat anti-human IgG F(ab) ₂ (2.5 μg/mL; JIR 109-116-097) followed by a further incubation for 1 hour (20°C, 650 rpm). The plates were analyzed using a Millipore 200 system. In a 48 μl sample volume, a total of 100 beads were counted per well. PE MFI values were used to determine whether the binding to the recombinant protein was specific or nonspecific.

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

7.2.3 Ligand blocking activity of anti-TIM-3 antibody

Further analysis was conducted on the ability of anti-TIM-3 antibodies AM-2 and AM-6 to block the binding of phosphatidylserine to human or cynomolgus TIM-3. In brief, anti-TIM-3 or IgG isotype control antibodies (10-point titration, 40,000 ng/ml to 1,000 ng/ml) were incubated with recombinant human TIM-3Fc (R&D Systems, #2365-TM) or recombinant cynomolgus TIM-3Fc (R&D Systems, #7914-TM) (10,000 ng/ml) prepared in 1X Annexin-V binding buffer (adjusted to pH 7.4 with 10 mM Hepes, ₁₄₀ mM NaCl, and 2.5 mM CaCl2) at room temperature for 30 minutes. WR19L cells, irradiated with 20 Gy and resuspended in 1X Annexin-V binding buffer, were added to an anti-TIM-3:TIM-3-Fc mixture to a final density of 1 x ^10⁶ cells/ml and incubated at room temperature for 45 min. Samples were washed once, and an antibody mixture containing PE-conjugated anti-Fc antibody (1:100 dilution) and viability stain (Biolegend, NIR channel; 1:1000 dilution) diluted in 1X Annexin-V binding buffer was added to each sample and incubated at room temperature for 20 min. Samples were then washed once in 1X Annexin-V binding buffer and analyzed using an LSR Tortessa flow cytometer (BD Biosciences). Flow cytometry plots were analyzed using FACSDIVA.

As shown in Figures 11A and 11B, the anti-TIM-3 antibodies pab2188w, AM-2, and AM-6 effectively blocked the binding of human or cynomolgus monkey TIM-3 to phosphatidylserine-expressing cells.

7.2.4 Effect of anti-TIM-3 antibody on human PBMCs after stimulation with staphylococcal enterotoxin A (SEA)

Functional activity of the pab2188w variant was analyzed using primary human PBMCs stimulated with staphylococcal enterotoxin A (SEA). Briefly, cryopreserved human PBMCs (Research Blood Components) were plated at 1 x ^10⁵ cells/well in 96-well NUNCLON delta plates (NUNC ^™ ) supplemented with Normocin ^™ (Invivogen #ant-nr) and 10% heat-inactivated FBS (Gibco, Invitrogen Corporation) in RPMI 1640. Cells were cultured for 9 days at 37°C and 5% CO₂ in the presence of 5 μg/ml anti-PD-1 antibody pembrolizumab (batch 7002688300, Myoderm), anti-TIM-3 antibody (10 μg/ml), and _SEA superantigen (100 ng/ml, Toxin Technologies). Cells were then washed once and restimulated for 2 days with fresh SEA and antibody. Collect cell-free supernatant and store at -80°C until analysis. IFNγ levels were measured using AlphaLISA (PerkinElmer).

As shown in Figures 12A and 12B, many pab2188w variants, alone or in combination with the anti-PD-1 antibody pembrolizumab, exhibit enhancements derived from IFNγ production in human PBMCs from two different donors.

7.2.5 Effects of anti-TIM-3 antibody on cytokine production in tumor-infiltrating lymphocytes

Further evaluation was conducted on the ability of anti-TIM-3 antibodies, alone or in combination with anti-PD-1 antibodies, to stimulate cytokine production in activated primary tumor-infiltrating lymphocytes (TILs). Single-cell suspensions from fresh non-small cell lung cancer (NSCLC) (stage II), gallbladder adenocarcinoma (stage IV), or breast cancer (stage II) tumors (UMass Medical School, Worcester, MA) were isolated by mechanical microdissection. In some cases, enzymatic digestion was required depending on the level of fibrosis (Liberase and DNAseI, Roche). Cells were paced at 5 x ^10⁴ cells/well in 96-well NUNCLON delta surface plates (NUNC ^™ ) supplemented with Normocin ^™ (Invivogen #ant-nr), recombinant human IL-2 (20 U/ml, R&D Systems), and 10% heat-inactivated FBS (Gibco, Invitrogen Corporation) in RPMI 1640 for 1 day. The next day, the samples were centrifuged and added to fresh medium containing the target antibodies (20 μg/ml anti-TIM-3 antibody and 5 μg/ml anti-PD-1 antibody pembrolizumab) and anti-CD3/CD28 microbeads (1:1 bead:cell ratio) to a final volume of 100 μl, and allowed to incubate at 37°C and 5% _CO2 for 3 days. The cell-free supernatant was collected and stored at -80°C until analysis. IFNγ and TNFα levels were measured using AlphaLISA (Perkin Elmer).

As shown in Figures 13A-13F, anti-TIM-3 antibodies enhanced the production of IFNγ and TNFα in activated primary TILs from NSCLC, gallbladder adenocarcinoma, or breast cancer tumors.

7.2.6 Internalization of anti-TIM-3 antibody after binding

In this embodiment, the internalization of anti-TIM-3 antibodies into cells was analyzed. In the first set of experiments, the internalization of anti-TIM-3 antibodies was assessed using αHFc-NC-DM1 (an anti-human IgG Fc antibody conjugated to maytansinoid DM1 via a non-cleavable linker, Moradec LLC). This secondary antibody conjugate αHFc-NC-DM1 binds to the test antibody (e.g., anti-TIM-3 antibody) and, upon internalization, induces the release of the cytotoxic payload DM1 into the cytoplasm of the cell. In the second set of experiments, the internalization was evaluated using anti-TIM-3 antibodies pab2188w (IgG ₁ N297A) and Hum11 (IgG ₄ S228P), which are directly conjugated to monomethylaurestatin E (MMAE). Each antibody exhibited a similar drug-antibody ratio (DAR; isotype control = 3.5, pab2188w = 4.0, Hum11 = 3.0), supporting the delivery of equivalent levels of antibody-drug conjugates (ADCs) after internalization. In the third set of experiments, internalization was assessed by subcellular localization of TIM-3 protein labeled with a cell-impermeable fluorescent dye.

In short, Kasumi-3 (CRL-2725 ^™ ), endogenously TIM-3-expressing acute myeloid leukemia cell lines, and engineered Jurkat cell lines overexpressing TIM-3 were plated into white-background tissue culture plates at a density of 2 x ^10⁴ cells per well. For the first set of experiments using the secondary antibody drug conjugate αHFc-NC-DM1, anti-TIM-3 antibody or IgG isotype control antibody was added to the cells at 8-point titrations (3,333 ng/ml to 1 ng/ml) along with αHFc-NC-DM1 (1:1 with the primary antibody), resulting in a final volume of 100 μl/well. Cells were incubated with the primary and secondary antibody drug conjugates at 37°C and 5% _CO₂ for 72 hours.

As demonstrated by reduced cell survival across a wide range of antibody concentrations, in the αHFc-NC-DM1 assay, anti-TIM-3 antibodies pab2188w (IgG ₁ N297A), AM-2 (IgG ₁ N297A), and AM-6 (IgG ₁ N297A) were more effectively internalized in Jurkat cells (Fig. 14A) and Kasumi-3 cells (Fig. 14B) expressing TIM-3 than the reference anti-TIM-3 antibodies Hum11 (IgG ₄ S228P) and pab1944w (IgG ₁ N297A).

For the second set of experiments, the direct conjugation of antibodies pab2188w (IgG1 N297A) and Hum11 (reference, IgG4S228P) to similar concentrations of MMAE is the reason for the potential difference in the binding tendency of the secondary drug conjugate (αHFc-NC-DM1) to different Fc regions of the antibody. Nine dose titrations (6,666 ng/ml to 1 ng/ml) of MMAE-conjugated anti-TIM-3 antibody or MMAE-conjugated IgG isotype control antibody were added to the cells, resulting in a final volume of 100 μl/well. Cells were incubated with the conjugated antibody at 37°C and 5% _CO2 for 72 hours. After incubation, 90 μl of reconstituted Cell Titer Glo (Promega) was added to each well, and the cells were incubated at room temperature for 5 minutes. The resulting fluorescence was recorded using an Envision instrument (Perkin Elmer).

As shown in Figure 14C, antibody pab2188w (IgG1 N297A) induced a greater reduction in cell survival than antibody Hum11 (reference, IgG4S228P), suggesting that the effects observed with secondary antibody conjugates (e.g., as shown in Figure 14A) can be attributed to the internalization potential of each TIM-3 antibody.

In the third set of experiments, the internalization of anti-TIM-3 antibodies was analyzed by confocal fluorescence microscopy of live cells. Jurkat cells expressing the HaloTag-TIM-3 fusion protein were first incubated for 30 minutes at 37°C and 5% _CO2 with 1 μM purple proliferation dye 450 (BD). After incubation, the cells were washed in PBS and resuspended in culture medium. To detect the extracellular domain of TIM-3, Jurkat HaloTag-TIM-3 cells were stained for 15 minutes at 37°C and 5% _CO2 with membrane-impermeable HaloTag Alexa Fluor 488 ligand (Promega, 1 μM). The cells were then resuspended in fresh culture medium and plated in 384-well microplates (15,000 cells/well) containing anti-TIM-3 antibody AM-2 (IgG ₁ N297A) or an allotype control (10 μg/ml for each antibody). Active images were acquired using an ImageXpress Micro Confocal High-Content microscope (Molecular Devices) under controlled environmental conditions (37°C and 5% _CO2 ), with images acquired 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 (purple proliferation dye 450) and the internalized TIM-3 signaling level in each cell was quantified by the FITC channel (HaloTag Alexa Fluor 488).

As shown in Figure 15, compared to cells incubated with the isotype control antibody, cells incubated with the anti-TIM-3 antibody AM-2 showed increased TIM-3 internalization over time. Specifically, at 3.5 hours, AM-2 antibody treatment resulted in twice the percentage of TIM-3-positive cells exhibiting TIM-3 internalization compared to TIM-3-positive cells treated with the isotype control antibody (i.e., 15.1% vs. 7.2%). Furthermore, the internalization signal observed in AM-2 antibody-treated cells at 3.5 hours was significantly stronger than that in cells treated with the isotype control antibody (p = 0.00027, one-tailed T-test). There was no statistically significant difference at the 0-hour time point (p = 0.91, one-tailed T-test).

7.3 Example 3: Epitope mapping of anti-TIM-3 antibody

In this embodiment, epitopes of anti-TIM-3 antibodies pab2188 (IgG ₁ variant), pab2187 (IgG ₁ variant), and AM-2 (IgG ₁ N297A) are characterized.

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

The binding characteristics of the anti-TIM-3 antibodies pab2188 (IgG ₁ variant) and pab2187 (IgG ₁ variant) were evaluated using alanine scanning. In short, human TIM-3 mutants with alanine substitutions in the extracellular domains were generated using the QuikChange HT protein engineering system (Cat#G5901A) from Agilent Technologies (Cat#G5901A). Retroviral transduction was used to express the human TIM-3 mutants on the surface of mouse 1624-5 pre-B cells. The transduction efficiency or percentage of cells expressing human TIM-3 was kept below 5% to ensure that most cells did not express two or more different TIM-3 mutants.

As demonstrated in flow cytometry using binding to a polyclonal anti-TIM-3 antibody (R&D Systems, Cat#AF2365), further selection was made of cells expressing correctly folded human TIM-3 mutants to obtain a subpopulation of human TIM-3 mutants expressing either the monoclonal anti-TIM-3 antibody pab2188 (IgG ₁ variant) or pab2187 (IgG ₁ variant). Cells exhibiting specific antibody binding were separated from the non-binding cell populations using preparative, high-speed FACS (FACSAriaII, BD Biosciences). The antibody-reactive or non-reactive cell libraries were then re-amplified in tissue cultures, and antibody-guided cell sorting and tissue culture amplification cycles were repeated until a definitively detectable non-reactive cell population of anti-TIM-3 antibodies (pab2188 (IgG ₁ variant) or pab2187 (IgG ₁ variant)) was obtained. This population of cells unresponsive to the anti-TIM-3 antibody (pab2188 (IgG ₁ variant) or pab2187 (IgG ₁ variant)) undergoes a final, single-cell or batch sorting step. Several days after cell expansion, the single-cell or batch-sorted cells are retested using flow cytometry to determine binding to the polyclonal anti-TIM-3 antibody and non-binding to the monoclonal antibody pab2188 (IgG ₁ variant) or pab2187 (IgG ₁ variant).

To link phenotype with genotype, bulk-sorted cells expressing human TIM-3 mutants were subjected to NGS sequencing. Sequence analysis revealed that cells responsive to polyclonal anti-TIM-3 antibodies but unresponsive to monoclonal anti-TIM-3 antibodies pab2188 (IgG ₁ variant) or pab2187 (IgG ₁ variant) expressed a human TIM-3 mutant at position 40, where Phe was mutated to Ala (according to SEQ ID NO: 79).

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

In the first study, hydrogen-deuterium exchange (HDX) mass spectrometry was used to investigate the interaction between pab2188 (IgG ₁ variant) and human TIM-3.

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

For pepsin digestion, 6.9 μg of native or deglycosylated human TIM-3/Fc chimera was denatured for 3 min for native or deglycosylated human TIM-3/Fc chimera in 115 μl of control buffer (50 mM phosphate, 100 mM sodium chloride, pH 7.4) by adding 115 μl of 4 M guanidine hydrochloride and 0.85 M TCEP buffer (final pH 2.5) and incubating the mixture at 10 °C for 3 min. The mixture was then subjected to on-column pepsin digestion using an internally packed peptide column, and the resulting peptides were analyzed using a UPLC-MS system coupled with a Waters Acquity UPLC and a Q Exactive ^™ Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo). Peptides were separated from 2-32% solvent B (0.1% formic acid in acetonitrile) at a gradient of 20.5 min on a 50 mm x 1 mm C8 column. Peptide identification was performed by searching MS/MS data for human TIM-3 sequences using Mascot software. The mass tolerances for the precursor and product ions were 20 ppm and 0.05 Da, respectively.

Incubate 10 μl of natural or deglycosylated human TIM-3/Fc chimera (6.9 μg), 10 μl of natural human TIM-3/Fc chimera and antibody mixture (6.9 μg:12.9 μg), or 10 μl of deglycosylated human TIM-3/Fc chimera and antibody mixture (6.9 μg:12.9 μg) with 105 μl of deuterium-labeled buffer (50 mM phosphate, 100 mM sodium chloride, pD 7.4) for 0 s, 60 s, 300 s, 1800 s, 7200 s, 14400 s, and 28800 s. For natural human TIM-3/Fc chimera and its antibody complex, deuterium exchange is performed at 10 °C, or for deglycosylated human TIM-3/Fc chimera and its antibody complex, at 4 °C. Deuterium exchange was quenched by adding 115 μl of 4 M guanidine hydrochloride and 0.85 M TCEP buffer (final pH 2.5). The quenched sample was then subjected to on-column pepsin digestion and LC-MS analysis as described above. Mass spectra were recorded in MS-only mode. To calculate deuterium incorporation, the mass spectra of a given peptide at the extracted ion chromatographic peaks were combined and a weighted average m/z was calculated. The mass increment from the mass of the native peptide (0 min) to the weighted average mass corresponds to the deuterium incorporation level.

The sequence coverage achieved by native and deglycosylated human TIM-3 was 71.6% and 98.4%, respectively. While most TIM-3 peptides exhibited similar or identical deuterium levels with and without anti-TIM-3 antibodies, several peptide fragments were found to show significantly reduced deuterium incorporation upon antibody binding. Native and deglycosylated human TIM-3 showed significantly reduced deuterium uptake upon binding to the anti-human TIM-3 antibody pab2188 (IgG ₁ variant) in the regions consisting of the amino acid sequences of SEQ ID NO:94 (VCWGKGACPVFECGNVVL) and SEQ ID NO:95 (RIQIPGIMND). The strongest reduction in deuterium uptake was observed in the region consisting of the amino acid sequence of SEQ ID NO:93 (PVFECGN).

Next, the interaction between AM-2 (IgG ₁ N297A) and human TIM-3 was investigated in a similar HDX mass spectrometry study. Briefly, deglycosylated human TIM-3/Fc chimeras were incubated in deuterium oxide alone or in combination with the anti-human TIM-3 antibody AM-2 (IgG ₁ N297A). Deuterium exchange was performed at 10 °C for 0, 60, 300, 1800, 7200, and 14400 seconds. The exchange reaction was quenched with low pH, and the quenched samples were subjected to on-column pepsin/protease XIII or protease XVIII digestion and LC-MS analysis as described above. Raw MS data were processed using HDX WorkBench software for analyzing H/D exchange MS data (J. Am. Soc. Mass Spectrom. 2012, 23(9), 1512-1521, which is incorporated herein by reference in its entirety). Deuterium levels were calculated using the average mass difference between the deuterated peptide and its native form ( _t0 ).

For deglycosylated human TIM-3, 100% sequence coverage was achieved. The anti-TIM-3 antibody AM-2 (IgG ₁ N297A) exhibited a binding pattern similar to that shown by pab2188 (IgG ₁ variant). When deglycosylated human TIM-3 binds to the anti-TIM-3 antibody AM-2 (IgG ₁ N297A), two regions, one consisting of the amino acid sequence of SEQ ID NO:94 (VCWGKGACPVFECGNVVL) and the other consisting of the amino acid sequence of SEQ ID NO:96 (RIQIPGIMNDEKFNLKL), undergo strong deuterium protection. The strongest reduction was observed in the region consisting of the amino acid sequence of SEQ ID NO:93 (PVFECGN).

7.3.3 Epitope mapping of anti-TIM-3 antibody using Pepscan analysis

The binding of the synthesized TIM-3-related peptide fragments to the anti-TIM-3 antibody pab2188 (IgG ₁ variant) was measured for a peptide array fabricated into a chip. Analysis was performed using Pepscan Presto BV, Lelystad, and Netherlands. In short, a peptide library was synthesized to reconstruct the human TIM-3 epitope. An amino-functionalized polypropylene support was obtained by grafting with a proprietary hydrophilic polymer formulation, followed by a reaction with dicyclohexylcarbodiimide (DCC) and N-hydroxybenzotriazole (HOBt) with tert-butyloxycarbonyl-hexamethylenediamine (BocHMDA), and subsequent cleavage of the Boc group using trifluoroacetic acid (TFA). Peptides were synthesized on the amino-functionalized solid support using a custom-modified JANUS liquid processing station (Perkin Elmer) via the standard Fmoc-peptide synthesis method. Structural mimics were synthesized using Pepscan's proprietary chemically linked peptide on scaffold (CLIPS) technology. CLIPS technology allows peptide structures to be constructed in monocyclic, bicyclic, tricyclic, sheet-like, helical, and combinations thereof. The binding of antibodies to each synthetic peptide was tested in a PEPSCAN-based ELISA. The peptide array was incubated overnight at 4°C with the primary antibody solution. After washing, the peptide array was incubated at 25°C for one hour with a goat anti-human HRP conjugate (Southern Biotech, Cat#2010-05). After washing, the peroxidase substrate 2,2'-azido-di-3-ethylbenzothiazoline sulfonate (ABTS) and 2 μl/ml of 3% _H₂O₂ were added _. After one hour, color development was measured and quantified using a charge-coupled device (CCD) camera and image processing system.

Pepscan studies have demonstrated that the anti-TIM-3 antibody pab2188 (IgG ₁ variant) recognizes the human TIM-3 chain segment, including the region consisting of the amino acid sequence of SEQ ID NO:99 (GKGACPVFE) and the region consisting of the amino acid sequence of SEQ ID NO:100 (DFTAAFPR).

***

This invention is not limited in scope to the specific embodiments described herein. In fact, various modifications to the invention, other than those described, will become apparent to those skilled in the art from the foregoing description and drawings. Such modifications are intended to fall within the scope of the appended claims.

All references cited herein (e.g., publications, patents, or patent applications) are incorporated herein by reference in their entirety and for all purposes, to the same extent as if each individual reference (e.g., publications, patents, or patent applications) were explicitly stated separately to be incorporated herein by reference in their entirety and for all purposes.

Other embodiments are described in the following claims.

This application also involves the following items.

1. An isolated antibody that specifically binds to human TIM-3, said antibody comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising complementarity-determining regions CDRH1, CDRH2, and CDRH3, said light chain variable region comprising complementarity-determining regions CDRL1, CDRL2, and CDRL3, wherein:

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

_X1 is R, S, A, G, K, M, or T.

X ₂ can be Q, S, A, G, R, or T.

X ₃ can be N, Y, G, or Q.

X ₄ is either A or Q, and

X ₅ can be 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 X ₁ ASQSVX ₂ SSYLA (SEQ ID NO:52), in which

_X1 is R or G, and

_X2 either does not exist or is S;

(e) CDRL2 contains the amino acid sequence X ₁ ASX ₂ RAT (SEQ ID NO:53).

in

_X1 is either D or G, and

_X2 is N, S, or T; and

(f) CDRL3 contains the amino acid sequence QQYGSSPX ₁ T (SEQ ID NO:54), where X ₁ is L or I.

2. An isolated antibody that specifically binds to human TIM-3, said antibody comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region having complementarity-determining regions CDRH1, CDRH2 and CDRH3, said light chain variable region having complementarity-determining regions CDRL1, CDRL2 and CDRL3, wherein said antibody is internalized upon binding to cells expressing human TIM-3, and wherein said CDRH3 contains the amino acid sequence AKGGDYGGNYFD (SEQ ID NO:3).

3. The antibodies isolated according to Project 2, wherein:

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

_X1 is R, S, A, G, K, M, or T.

X ₂ can be Q, S, A, G, R, or T.

X ₃ can be N, Y, G, or Q.

X ₄ is either A or Q, and

X ₅ can be 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 X ₁ ASQSVX ₂ SSYLA (SEQ ID NO:52), in which

_X1 is R or G, and

_X2 either does not exist or is S;

(d) CDRL2 contains the amino acid sequence X ₁ ASX ₂ RAT (SEQ ID NO:53).

in

_X1 is either D or G, and

_X2 is N, S, or T; and

(e)CDRL3 contains the amino acid sequence QQYGSSPX ₁ T (SEQ ID NO:54), where X ₁ is L or I.

4. The antibody isolated according to any one of items 1-3, wherein CDRH1 comprises the amino acid sequence of _{X1 X2} NAWS (SEQ ID NO:49), wherein: _X1 is R or A; and _X2 is Q or R.

5. The antibody isolated according to any one of items 1-3, wherein CDRH1 comprises the amino acid sequence _{X1 X2 GQX3} S (SEQ ID NO: 50), wherein: _X1 is K, M or G; _X2 is A or S; and _X3 is S or T.

6. The antibody isolated according to any one of items 1-3, wherein CDRH1 comprises the amino acid sequence _{X1 X2} QQAS (SEQ ID NO:51), wherein: _X1 is S, R, T or G; and _X2 is A, S, T or G.

7. The antibody isolated according to any one of items 1-6, wherein CDRH1 comprises an amino acid sequence selected from SEQ ID NO:1 and 4-12.

8. The antibody isolated according to any one of items 1-7, wherein CDRL1 comprises an amino acid sequence selected from SEQ ID NO:13-16.

9. The antibody isolated according to any one of items 1-8, wherein CDRL2 comprises an amino acid sequence selected from SEQ ID NO:17-21.

10. The antibody isolated according to any one of items 1-9, wherein CDRL3 comprises an amino acid sequence selected from SEQ ID NO: 22 and 23.

11. The antibody isolated according to any one of items 1-10, wherein CDRH1, CDRH2 and CDRH3 respectively contain the amino acid sequences of CDRH1, CDRH2 and CDRH3 listed in 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.

12. The antibody isolated according to any one of items 1-11, wherein CDRL1, CDRL2 and CDRL3 respectively comprise the amino acid sequences of CDRL1, CDRL2 and CDRL3 listed in 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.

13. The antibody isolated according to any one of items 1-12, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 respectively comprise the amino acid sequences listed in SEQ ID NO: 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.

14. An isolated antibody that specifically binds to human TIM-3, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions CDRH1, CDRH2, and CDRH3, and the light chain variable region comprises complementarity-determining regions CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 respectively comprise the amino acid sequences listed in SEQ ID NO: 1, 2, 3, 14, 21, and 22.

15. An isolated antibody that specifically binds to human TIM-3, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions CDRH1, CDRH2, and CDRH3, and the light chain variable region comprises complementarity-determining regions CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 respectively comprise the amino acid sequences listed in SEQ ID NO: 5, 2, 3, 14, 21, and 22.

16. An isolated antibody that specifically binds to human TIM-3, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions CDRH1, CDRH2, and CDRH3, and the light chain variable region comprises complementarity-determining regions CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 respectively comprise the amino acid sequences listed in SEQ ID NO: 9, 2, 3, 14, 21, and 22.

17. An isolated antibody that specifically binds to human TIM-3, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions CDRH1, CDRH2, and CDRH3, and the light chain variable region comprises complementarity-determining regions CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 respectively comprise the amino acid sequences listed in SEQ ID NO: 1, 2, 3, 15, 18, and 22.

18. The isolated antibody according to any one of items 1-17, wherein the antibody is internalized after binding to cells expressing human TIM-3.

19. An isolated antibody that specifically binds to human TIM-3, wherein the antibody is internalized after binding to cells expressing human TIM-3.

20. The antibody isolated according to item 18 or 19, wherein, in an assay comprising the following steps, the percentage of said human TIM-3-expressing cells surviving in the presence of said antibody is lower than that in the presence of 1944w (IgG ₁ N297A):

(a) The cells expressing human TIM-3 were seeded in tissue culture plates at a density of 2 x ^10⁴ cells per well;

(b) Add 1111 ng/ml of αHFc-NC-DM1 and 1111 ng/ml of the antibody or pab1944w (IgG ₁ N297A) to a final volume of 100 μl/well;

(c) _Incubate at 37°C and 5% CO2 for 72 hours;

(d) Measure the survival of the cells expressing human TIM-3; and

(e) Calculate the percentage of cell survival relative to untreated cells expressing human TIM-3.

21. The antibody isolated according to Item 20, wherein 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 (IgG ₁ N297A).

22. The antibody isolated according to item 18 or 19, wherein, in an assay comprising the following steps, the percentage of said human TIM-3-expressing cells surviving in the presence of said antibody is lower than in the presence of Hum11 (IgG ₄ S228P):

(a) The cells expressing human TIM-3 were seeded in tissue culture plates at a density of 2 x ^10⁴ cells per well;

(b) Add 1111 ng/ml of αHFc-NC-DM1 and 1111 ng/ml of the antibody or Hum11 (IgG ₄ S228P) to a final volume of 100 μl/well;

(c) _Incubate at 37°C and 5% CO2 for 72 hours;

(d) Measure the survival of the cells expressing human TIM-3; and

(e) Calculate the percentage of cell survival relative to untreated cells expressing human TIM-3.

23. The antibody isolated according to item 22, wherein 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).

24. The antibody isolated according to any one of items 2-23, wherein the cell expressing human TIM-3 is a Kasumi-3 cell.

25. The antibody isolated according to any one of items 2-23, wherein the cell expressing human TIM-3 is a Jurkat cell engineered to express human TIM-3.

26. The antibody isolated according to any one of items 1-25, wherein the antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO:55.

27. An antibody isolated according to any one of items 1-25, wherein the antibody comprises a heavy chain variable region comprising at least 75%, 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as selected from SEQ ID NO:24-35.

28. The antibody isolated according to item 27, wherein the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO:24-35.

29. The antibody isolated according to item 28, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:25.

30. The antibody isolated according to item 28, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:28.

31. The antibody isolated according to item 28, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:32.

32. The antibody isolated according to any one of items 1-31, wherein the antibody comprises a light chain variable region containing the amino acid sequence of SEQ ID NO:56.

33. An antibody isolated according to any one of items 1-31, wherein the antibody comprises a light chain variable region comprising at least 75%, 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as selected from SEQ ID NO:36-47.

34. The antibody isolated according to item 33, wherein the light chain variable region comprises an amino acid sequence selected from SEQ ID NO:36-47.

35. The antibody isolated according to item 34, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:46.

36. An isolated antibody that specifically binds to human TIM-3, said antibody comprising a heavy chain variable region containing an amino acid sequence selected from SEQ ID NO:24-35.

37. The antibody isolated according to item 36, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:25.

38. The antibody isolated according to item 36, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:28.

39. The antibody isolated according to item 36, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:32.

40. The antibody isolated according to item 37, wherein the antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO:58.

41. The antibody isolated according to item 38, wherein the antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO:61.

42. The antibody isolated according to item 39, wherein the antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO:65.

43. An isolated antibody that specifically binds to human TIM-3, said antibody comprising a light chain variable region containing an amino acid sequence selected from SEQ ID NO:36-47.

44. The antibody isolated according to item 43, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:46.

45. The antibody isolated according to item 44, wherein the antibody comprises a light chain containing the amino acid sequence of SEQ ID NO:69.

46. An isolated antibody that specifically binds to human TIM-3, said antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region each comprise the amino acid sequence listed in 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.

47. The antibody isolated according to item 46, wherein the heavy chain variable region and the light chain variable region comprise the amino acid sequences listed in SEQ ID NO:25 and 46, respectively.

48. The antibody isolated according to item 46, wherein the heavy chain variable region and the light chain variable region comprise the amino acid sequences listed in SEQ ID NO:28 and 46, respectively.

49. The antibody isolated according to item 46, wherein the heavy chain variable region and the light chain variable region comprise the amino acid sequences listed in SEQ ID NO:32 and 46, respectively.

50. An isolated antibody that specifically binds to human TIM-3, said antibody 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.

51. An isolated antibody that specifically binds to human TIM-3, said antibody 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.

52. An isolated antibody that specifically binds to human TIM-3, said antibody 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.

53. The isolated antibody according to any one of the preceding items, wherein the antibody comprises a heavy chain variable region having an amino acid sequence derived from the human IGHV3-23 germline sequence.

54. The isolated antibody according to any one of the preceding items, wherein the antibody comprises a light chain variable region having an amino acid sequence derived from a human ancestral sequence selected from IGKV1-27, IGKV3-11, IGKV3-20 and IGKV3D-20.

55. An isolated antibody that specifically binds to human TIM-3, said antibody comprising a heavy chain variable region having an amino acid sequence derived from a human IGHV3-23 germline sequence and a light chain variable region having an amino acid sequence derived from a human germline sequence selected from IGKV1-27, IGKV3-11, IGKV3-20 and IGKV3D-20.

56. An antibody isolated according to any one of items 1-39, 43-49 and 53-55, wherein the antibody comprises a heavy chain constant region selected from human _IgG1 , _IgG2 , _IgG3 , _IgG4 , _IgA1 and _IgA2 .

57. The antibody isolated according to any one of items 1-39, 43-49 and 53-55, wherein the heavy chain constant region is _IgG1 .

58. The antibody isolated according to item 57, wherein the amino acid sequence of _IgG1 contains the N297A mutation numbered according to the EU numbering system.

59. The antibody isolated according to item 58, wherein the antibody comprises a heavy chain constant region containing the amino acid sequence of SEQ ID NO:72.

60. The antibody isolated according to item 57, wherein the amino acid sequence of _IgG1 contains the N297Q mutation numbered according to the EU numbering system.

61. The antibody isolated according to item 57, wherein the _IgG1 is non-fucosylated _IgG1 .

62. The antibody isolated according to any one of items 1-39, 43-49 and 53-55, wherein the heavy chain constant region is _IgG4 .

63. The antibody isolated according to item 62, wherein the amino acid sequence of _IgG4 contains the S228P mutation numbered according to the EU numbering system.

64. The antibody isolated according to item 63, wherein the antibody comprises a heavy chain constant region containing the amino acid sequence of SEQ ID NO:74.

65. An antibody isolated according to any one of items 1-44, 46-49 and 53-64, wherein the antibody comprises a light chain constant region selected from human IgGκ and IgGλ.

66. The antibody isolated according to any one of items 1-44, 46-49 and 53-64, wherein the light chain constant region is IgGκ.

67. The antibody isolated according to item 66, wherein the antibody comprises a light chain constant region containing the amino acid sequence of SEQ ID NO:76.

68. An isolated antibody that cross-competes with any of the preceding items for binding to human TIM-3.

69. An isolated antibody that binds to the same epitope of human TIM-3 as any of the antibodies described in any of the preceding items.

70. The isolated antibody according to any one of the preceding items, wherein the antibody specifically binds to a variant TIM-3 protein having the amino acid sequence of SEQ ID NO: 101 with a lower affinity than that of wild-type TIM-3 protein having the amino acid sequence of SEQ ID NO: 79.

71. The isolated antibody according to any one of the preceding items, wherein the antibody does not specifically bind to the variant TIM-3 protein having the amino acid sequence of SEQ ID NO: 101.

72. The isolated antibody according to any one of the preceding items, wherein the antibody binds to residue 40 of SEQ ID NO:79.

73. The isolated antibody according to any one of the preceding items, wherein the antibody binds to an epitope located in the human TIM-3 region consisting of the amino acid sequence of SEQ ID NO: 93.

74. The isolated antibody according to any one of the preceding items, wherein the antibody binds to an epitope located within the human TIM-3 region consisting of the amino acid sequence of SEQ ID NO: 94.

75. The isolated antibody according to any one of the preceding items, wherein the antibody binds to an epitope located within the human TIM-3 region consisting of the amino acid sequence of SEQ ID NO: 95.

76. The isolated antibody according to any one of the preceding items, wherein the antibody binds to an epitope located within the human TIM-3 region consisting of the amino acid sequence of SEQ ID NO: 96.

77. The isolated antibody according to any one of the preceding items, wherein the antibody binds to an epitope located in the human TIM-3 region consisting of the amino acid sequence of SEQ ID NO: 97.

78. The isolated antibody according to any one of the preceding items, wherein the antibody binds to an epitope located within the human TIM-3 region consisting of the amino acid sequence of SEQ ID NO: 98.

79. The isolated antibody according to any one of the preceding items, wherein the antibody binds to an epitope located within the human TIM-3 region consisting of the amino acid sequence of SEQ ID NO: 99.

80. The isolated antibody according to any one of the preceding items, wherein the antibody binds to an epitope located within the human TIM-3 region consisting of the amino acid sequence of SEQ ID NO: 100.

81. The isolated antibody according to any one of items 68-80, wherein the antibody comprises a human IgG heavy chain constant region variant of a wild-type human IgG heavy chain constant region, wherein the variant human IgG heavy chain constant region has a lower affinity for binding to a human Fcγ receptor than the wild-type human IgG heavy chain constant region has for binding to the human Fcγ receptor.

82. The antibody isolated according to item 81, wherein the human Fcγ receptor is selected from FcγRI, FcγRII and FcγRIII.

83. The antibody isolated according to item 81, wherein the variant human IgG heavy chain constant region is an IgG ₁ constant region containing the N297A mutation numbered according to the EU numbering system.

84. The isolated antibody according to any one of the preceding items, wherein the antibody is a human antibody.

85. The isolated antibody according to any one of the preceding items, wherein the antibody antagonizes human TIM-3.

86. The antibody isolated according to item 85, wherein the antibody inactivates, reduces or inhibits human TIM-3 activity.

87. The antibody isolated according to item 85, wherein the antibody inhibits the binding of human TIM-3 to phosphatidylserine.

88. The antibody isolated according to item 85, wherein the antibody induces the production of IFNγ by peripheral blood mononuclear cells (PBMCs) stimulated by staphylococcal enterotoxin A (SEA).

89. The antibody isolated according to item 85, wherein the antibody induces tumor-infiltrating lymphocytes (TILs) stimulated with anti-CD3 and anti-CD28 antibodies to produce IFNγ or TNFα.

90. The isolated antibody according to any one of the preceding items, wherein the antibody is internalized after binding to cells expressing human TIM-3.

91. The isolated antibody according to any one of the preceding items, which is conjugated to a cytotoxic agent, a cell inhibitor, a toxin, a radionuclide, or a detectable label.

92. A pharmaceutical composition comprising the isolated antibody as described in any one of the preceding items and a pharmaceutically acceptable carrier or excipient.

93. An isolated polynucleotide encoding the heavy and/or light chain of the antibody described in any one of items 1-91.

94. A vector comprising the polynucleotide described in item 93.

95. A recombinant host cell comprising the polynucleotide described in item 93 or the vector described in item 94.

96. A method for generating an antibody that binds to human TIM-3, the method comprising culturing the host cells described in item 95 to express the polynucleotide and generate the antibody.

97. A method for increasing T-cell activation in response to a subject antigen, the method comprising administering to the subject an effective amount of any one of items 1-92, an antibody or pharmaceutical composition.

98. A method of treating a subject with cancer, the method comprising administering to the subject an effective amount of any one of items 1-92, an antibody or pharmaceutical composition.

99. The method according to item 97 or 98, wherein the antibody or pharmaceutical composition is administered subcutaneously or intravenously.

100. The method according to item 97 or 98, wherein the antibody or pharmaceutical composition is administered intratumorally.

101. The method according to any one of items 97-100, further comprising administering an additional therapeutic agent to the subject.

102. The method according to item 101, wherein the additional therapeutic agent is selected from chemotherapeutic agents, radiotherapy agents, or checkpoint targeting agents.

103. The method according to Item 102, wherein the checkpoint target is selected from antagonist anti-PD-1 antibody, antagonist anti-PD-L1 antibody, antagonist anti-PD-L2 antibody, antagonist anti-CTLA-4 antibody, antagonist anti-TIM-3 antibody, antagonist anti-LAG-3 antibody, antagonist anti-CEACAM1 antibody, agonist anti-GITR antibody and agonist anti-OX40 antibody.

104. The method according to item 101, wherein the additional therapeutic agent is an anti-PD-1 antibody, optionally wherein the anti-PD-1 antibody is pembrolizumab or nivolumab.

105. The method according to item 101, wherein the additional therapeutic agent is an indoleamine-2,3-dioxygenase (IDO) inhibitor.

106. The method according to item 105, wherein the inhibitor is selected from epacadostat, F001287, indoximod and NLG919.

107. The method according to item 106, wherein the inhibitor is epacadostat.

108. The method according to item 101, wherein the additional therapeutic agent is a vaccine.

109. The method according to item 108, wherein the vaccine comprises a heat shock protein peptide complex (HSPPC) containing a heat shock protein complexed with an antigenic peptide.

110. The method according to item 109, wherein the heat shock protein is hsc70 and is complexed with a tumor-associated antigen peptide.

111. The method according to item 109, wherein the heat shock protein is gp96 and is complexed with a tumor-associated antigen peptide, wherein the HSPPC is derived from a tumor obtained from a subject.

112. An isolated antibody or pharmaceutical composition according to any one of items 1-92, for the treatment of cancer or infectious diseases.

113. Use of the isolated antibody or pharmaceutical composition according to any one of items 1-92 for the preparation of a medicament for treating cancer or infectious diseases.

Claims (30)

1. An isolated antibody that specifically binds to human TIM-3, said antibody comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising complementarity-determining regions CDRH1, CDRH2, and CDRH3, said light chain variable region comprising complementarity-determining regions CDRL1, CDRL2, and CDRL3, wherein: (a) CDRH1 contains the amino acid sequence X ₁ X ₂ X ₃ X ₄ X ₅ S (SEQ ID NO:48), in which _X1 is R, S, A, G, K, M, or T. X ₂ can be Q, S, A, G, R, or T. X ₃ can be N, Y, G, or Q. X ₄ is either A or Q, and X ₅ can be 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 X ₁ ASQSVX ₂ SSYLA (SEQ ID NO:52), in which _X1 is R or G, and _X2 either does not exist or is S; (e) CDRL2 contains the amino acid sequence X ₁ ASX ₂ RAT (SEQ ID NO:53). in _X1 is either D or G, and _X2 is N, S, or T; and (f) CDRL3 contains the amino acid sequence QQYGSSPX ₁ T (SEQ ID NO:54), where X ₁ is L or I; and The antibodies described herein do not contain the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 as listed in SEQ ID NO: 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. 2. The isolated antibody according to claim 1, wherein CDRH1 comprises the amino acid sequence _{X1 X2} NAWS (SEQ ID NO:49), wherein: _X1 is R or A; and _X2 is Q or R; the amino acid sequence _{X1 X2 GQX3} S (SEQ ID NO:50), wherein: _X1 is K, M or G; _X2 is A or S; and _X3 is S or T; or the amino acid sequence _{X1 X2} QQAS (SEQ ID NO:51), wherein: _X1 is S, R, T or G; and _X2 is A, S, T or G. 3. The isolated antibody according to claim 1 or 2, wherein CDRH1 comprises an amino acid sequence selected from SEQ ID NO:1 and 4-12. 4. The isolated antibody according to any one of claims 1-3, wherein CDRL1 comprises an amino acid sequence selected from SEQ ID NO:13-16. 5. The isolated antibody according to any one of claims 1-4, wherein CDRL2 comprises an amino acid sequence selected from SEQ ID NO:17-21. 6. The isolated antibody according to any one of claims 1-5, wherein CDRL3 comprises an amino acid sequence selected from SEQ ID NO: 22 and 23. 7. The isolated antibody according to any one of claims 1-6, wherein CDRH1, CDRH2 and CDRH3 respectively comprise the amino acid sequences of CDRH1, CDRH2 and CDRH3 listed in 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. 8. The isolated antibody according to any one of claims 1-7, wherein CDRL1, CDRL2 and CDRL3 respectively comprise the amino acid sequences of CDRL1, CDRL2 and CDRL3 listed in 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. 9. The isolated antibody according to any one of claims 1-8, wherein the antibody is internalized after binding to cells expressing human TIM-3. 10. The isolated antibody according to claim 9, wherein in an assay comprising the following steps, the percentage of said human TIM-3-expressing cells surviving in the presence of said antibody is lower than in the presence of pab1944w (IgG ₁ N297A): (a) The cells expressing human TIM-3 were seeded in tissue culture plates at a density of 2 x ^10⁴ cells per well; (b) Add 1111 ng/ml of αHFc-NC-DM1 and 1111 ng/ml of the antibody or pab1944w (IgG ₁ N297A) to a final volume of 100 μl/well; (c) _Incubate at 37°C and 5% CO2 for 72 hours; (d) Measure the survival of the cells expressing human TIM-3; and (e) Calculate the percentage of cell survival relative to untreated cells expressing human TIM-3; or In the assay including the following steps, the percentage of cells expressing human TIM-3 that survived in the presence of the antibody was lower than that in the presence of Hum11 (IgG ₄ S228P): (a) The cells expressing human TIM-3 were seeded in tissue culture plates at a density of 2 x ^10⁴ cells per well; (b) Add 1111 ng/ml of αHFc-NC-DM1 and 1111 ng/ml of the antibody or Hum11 (IgG ₄ S228P) to a final volume of 100 μl/well; (c) _Incubate at 37°C and 5% CO2 for 72 hours; (d) Measure the survival of the cells expressing human TIM-3; and (e) Calculate the percentage of cell survival relative to untreated cells expressing human TIM-3. 11. The isolated antibody according to claim 10, wherein 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 (IgG ₁ N297A) or Hum11 (IgG ₄ S228P). 12. The isolated antibody according to any one of claims 9-11, wherein the cell expressing human TIM-3 is a Kasumi-3 cell or a Jurkat cell engineered to express human TIM-3. 13. An isolated antibody that specifically binds to human TIM-3, said antibody comprising a heavy chain variable region having an amino acid sequence derived from a human IGHV3-23 germline sequence and a light chain variable region having an amino acid sequence derived from a human germline sequence selected from IGKV1-27, IGKV3-11, IGKV3-20 and IGKV3D-20. 14. The isolated antibody according to any one of claims 1-13, wherein the antibody comprises a heavy chain constant region, wherein the heavy chain constant region: (a) Selected from human IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ and IgA ₂ ; (b) is the constant region of the IgG ₁ heavy chain; (c) is the constant region of the IgG ₁ heavy chain, which contains the N297A mutation numbered according to the EU numbering system; (d) Contains the amino acid sequence of SEQ ID NO:72; (e) is the constant region of the IgG ₁ heavy chain, which contains the N297Q mutation numbered according to the EU numbering system; (f) represents non-fucosylated _IgG1 ; (g) represents the constant region of the IgG ₄ heavy chain; (h) is the constant region of the IgG ₄ heavy chain, which contains the S228P mutation numbered according to the EU numbering system; or (i) Contains the amino acid sequence of SEQ ID NO:74; (j) is a variant of the wild-type human IgG heavy chain constant region, wherein the affinity of the variant human IgG heavy chain constant region for binding to the human Fcγ receptor is lower than that of the wild-type human IgG heavy chain constant region for binding to the human Fcγ receptor. 15. The isolated antibody according to any one of claims 1-14, wherein the antibody comprises a light chain constant region, wherein the light chain constant region: (a) Selected from human IgGκ and IgGλ; (b) is the constant region of the human IgGκ light chain; or (c) Contains the amino acid sequence of SEQ ID NO:76. 16. An isolated antibody that cross-competitively binds to human TIM-3 with the antibody of any one of claims 1-15. 17. An isolated antibody that binds to the same epitope of human TIM-3 as the antibody of any one of claims 1-15. 18. The isolated antibody according to any one of claims 1-17, wherein the antibody specifically binds to a variant TIM-3 protein having the amino acid sequence SEQ ID NO: 101 with a lower affinity than that of wild-type TIM-3 protein having the amino acid sequence SEQ ID NO: 79, or wherein the antibody does not specifically bind to a variant TIM-3 protein having the amino acid sequence SEQ ID NO: 101. 19. The isolated antibody according to any one of claims 1-18, wherein the antibody binds to residue 40 of SEQ ID NO:79. 20. The isolated antibody according to any one of claims 1-19, wherein the antibody binds to an epitope located in the human TIM-3 region consisting of any one of the amino acid sequences in SEQ ID NOs: 93-100. 21. The isolated antibody according to any one of claims 1-20, wherein the antibody: (a) Human antibodies; (b) Antagonism of human TIM-3; (c) Inactivation, reduction or inhibition of human TIM-3 activity; (d) Inhibits the binding of human TIM-3 to phosphatidylserine; (e) Inducing the production of IFNγ in peripheral blood mononuclear cells (PBMCs) stimulated by staphylococcal enterotoxin A (SEA); (f) Inducing the production of IFNγ or TNFα from tumor-infiltrating lymphocytes (TILs) stimulated with anti-CD3 and anti-CD28 antibodies; and/or (g) It is internalized after binding to cells expressing human TIM-3. 22. The isolated antibody according to any one of claims 1-21, wherein it is conjugated with a cytotoxic agent, a cell inhibitor, a toxin, a radionuclide, or a detectable label. 23. A pharmaceutical composition comprising the isolated antibody of any one of claims 1-22 and a pharmaceutically acceptable carrier or excipient. 24. An isolated polynucleotide encoding the heavy and/or light chain of the antibody according to any one of claims 1-22. 25. A vector comprising the polynucleotide of claim 24. 26. A recombinant host cell comprising the polynucleotide of claim 24 or the vector of claim 25. 27. A method for generating an antibody that binds to human TIM-3, the method comprising culturing the host cell of claim 26 to express the polynucleotide and generate the antibody. 28. The antibody or pharmaceutical composition according to any one of claims 1-23, used in a method of increasing T cell activation in response to a subject antigen. 29. The antibody or pharmaceutical composition according to any one of claims 1-23, for use in treating cancer or infectious diseases in subjects in need. 30. The use according to claim 28 or 29, wherein: (a) The antibody or pharmaceutical composition is administered subcutaneously, intravenously, or intratumorally; (b) The use also includes the administration of an additional therapeutic agent, wherein the additional therapeutic agent: (i) is a chemotherapy agent, a radiotherapy agent, or a checkpoint targeting agent; (ii) is a checkpoint targeting agent, wherein the checkpoint targeting agent is selected from antagonist anti-PD-1 antibody, antagonist anti-PD-L1 antibody, antagonist anti-PD-L2 antibody, antagonist anti-CTLA-4 antibody, antagonist anti-TIM-3 antibody, antagonist anti-LAG-3 antibody, antagonist anti-CEACAM1 antibody, agonist anti-GITR antibody and agonist anti-OX40 antibody; (iii) is an anti-PD-1 antibody, wherein the anti-PD-1 antibody is selected from pembrolizumab or nivolumab; (iv) is an inhibitor of indoleamine-2,3-dioxygenase (IDO); (v) is an IDO inhibitor, wherein the IDO inhibitor is selected from epacadostat, F001287, indoximod and NLG919; (vi) refers to vaccines; (vii) is a vaccine comprising a heat shock protein peptide complex (HSPPC) containing a heat shock protein complexed with an antigenic peptide; (viii) is a vaccine containing HSPPC, which contains hsc70 complexed with a tumor-associated antigen peptide; (ix) is a vaccine comprising HSPPC containing gp96 complexed with a tumor-associated antigen peptide, wherein the HSPPC is derived from a tumor obtained from a subject.