Antibodies against tim3 and uses thereof

Abstract

To provide a medicine targeting TIM3 and a method for using the medicine for new cancer immunotherapy design and traditional cancer immunotherapy improvement.SOLUTION: Provided herein are antibodies or antigen-binding portions thereof that bind to T-cell immunoglobulin and mucin domain-containing 3 (T1MM3) protein. Also provided are uses of these antibodies or antigen-binding portions thereof in therapeutic applications such as cancer treatment. Also provided are cells that produce the antibodies or antigen-binding portions thereof, polynucleotides encoding the heavy and / or light chain regions of the antibodies or antigen-binding portions thereof, and vectors comprising polynucleotides encoding the heavy and / or light chain regions of the antibodies or antigen-binding portions thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Citation of sequence listings submitted electronically via EFS-WEB The contents of the Sequence Listing, an ASCII text file provided electronically with this application (Name: 3338_098PC01_SequenceListing.txt; Size: 912,981 bytes; Created: January 11, 2019), are incorporated herein by reference in their entirety. [Background technology]

[0002] Background of the Invention T-cell immunoglobulin and mucin domain-containing 3 (TIM3), also known as hepatitis A virus cellular receptor 2 (HAVCR2), is a type I transmembrane protein that functions as a key regulator of immune responses. TIM3 mediates activation of activated IFN-γ-producing T cells (e.g., type 1 helper CD4 + T cells and cytotoxic CD8 + TIM3 has been identified in T cells and shown to induce T cell death or exhaustion after binding to galectin-9. Recent studies have also identified that TIM3 expression is important in regulating the activity of many innate immune cells (e.g., macrophages, monocytes, dendritic cells, mast cells, and natural killer cells). See Han G et al., Front Immunol. 4: 449 (2013).

[0003] Like many inhibitory receptors (e.g., PD-1 and CTLA-4), TIM3 expression is associated with many types of chronic diseases, including cancer. + T cells have been detected in patients with advanced melanoma, non-small cell lung cancer, or follicular B-cell non-Hodgkin's lymphoma. + The presence of regulatory T cells has been described as a useful indicator of lung cancer progression. See Anderson AC. Cancer Immunol Res. 2: 393-8 (2014).

[0004] Several potential ligands for TIM3 have been identified: galectin-9, HMGB1, semaphorin-4A, CEACAM-1, ILT-4, and phosphatidylserine (PtdSer or PS). PS is an important plasma membrane component and is normally localized to the inner leaflet of the plasma membrane. However, when cells undergo apoptosis, PS is redistributed and exposed to the outer membrane. This redistribution is also observed in many tumor cell lines. See Riedl S et al., Biochim Biophys Acta. 1808: 2638-2645 (2011). Binding of TIM3 to PS is important for phagocytosis and cross-presentation. See Nakayama M et al., Blood. 113: 3821-30 (2009).

[0005] Studies have shown a close relationship between TIM3 and the inhibitory receptor PD-1. For example, many tumor-specific T cells express both PD-1 and TIM3, and these T cells are more dysfunctional than T cells expressing only PD-1 or TIM3. See Fourcade J et al., J Exp Med. 207: 2175-2186 (2010). Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, agents that target TIM3 and methods for using such agents are highly desirable for designing new cancer immunotherapies and improving traditional cancer immunotherapies. [Means for solving the problem]

[0007] Summary of the Invention Provided herein are isolated antibodies, such as monoclonal antibodies or antigen-binding portions thereof, particularly human (e.g., monoclonal) antibodies, that specifically bind to human T-cell immunoglobulin and mucin domain-containing 3 (TIM3) ("anti-TIM3 antibodies") and comprise heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3. In certain embodiments, the heavy chain CDR3 comprises SEQ ID NO:414 or SEQ ID NO:416. In certain embodiments, the heavy chain CDR1 comprises SEQ ID NO:45. In certain embodiments, the heavy chain CDR2 comprises SEQ ID NO:413 or SEQ ID NO:415. In certain embodiments, the light chain CDR1 comprises SEQ ID NO:64. In certain embodiments, the light chain CDR2 comprises SEQ ID NO:66. In certain embodiments, the light chain CDR3 comprises SEQ ID NO:69, SEQ ID NO:68, or SEQ ID NO:419.

[0008] In certain embodiments, the anti-TIM3 antibody, or antigen-binding portion thereof, comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3, wherein (a) heavy chain CDR1 comprises SEQ ID NO: 45; (b) the heavy chain CDR2 comprises SEQ ID NO: 413 or SEQ ID NO: 415; (c) the heavy chain CDR3 comprises SEQ ID NO: 414 or SEQ ID NO: 416; (d) light chain CDR1 comprises SEQ ID NO: 64; (e) the light chain CDR2 comprises SEQ ID NO: 66; and (f) the light chain CDR3 comprises SEQ ID NO: 69, SEQ ID NO: 68, or SEQ ID NO: 419.

[0009] In some embodiments, (a) heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 45, 413 and 414, respectively, and light chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 64, 66 and 69, respectively; (b) heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 45, 415 and 416, respectively, and light chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 64, 66 and 68, respectively; or (c) heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 45, 415 and 416, respectively, and light chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 64, 66 and 419, respectively.

[0010] In certain embodiments, heavy chain CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 45, 413, and 414, respectively, and light chain CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 69, respectively. In certain embodiments, heavy chain CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 45, 415, and 416, respectively, and light chain CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 68, respectively. In certain embodiments, heavy chain CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 45, 415, and 416, respectively, and light chain CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 419, respectively.

[0011] In certain embodiments, the anti-TIM3 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO:410.

[0012] In certain embodiments, the anti-TIM3 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VL comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO:411.

[0013] In certain embodiments, the anti-TIM3 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VL comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO:412.

[0014] In certain embodiments, the anti-TIM3 antibody, or antigen-binding portion thereof, (a) a heavy chain variable region (VH) comprising SEQ ID NO: 410 and a light chain variable region (VL) comprising SEQ ID NO: 417; (b) a heavy chain variable region (VH) comprising SEQ ID NO: 411 and a light chain variable region (VL) comprising SEQ ID NO: 60; (c) a heavy chain variable region (VH) comprising SEQ ID NO: 411 and a light chain variable region (VL) comprising SEQ ID NO: 418; or (d) a heavy chain variable region (VH) comprising SEQ ID NO: 412 and a light chain variable region (VL) comprising SEQ ID NO: 60 The antibody cross-competes for binding to TIM3 with a control antibody comprising:

[0015] In certain embodiments, the anti-TIM3 antibody or antigen-binding portion thereof (a) heavy chain CDR1, CDR2, and CDR3 of a heavy chain variable region (VH) comprising SEQ ID NO: 410 and light chain CDR1, CDR2, and CDR3 of a light chain variable region (VL) comprising SEQ ID NO: 417; (b) heavy chain CDR1, CDR2, and CDR3 of a heavy chain variable region (VH) comprising SEQ ID NO: 411 and light chain CDR1, CDR2, and CDR3 of a light chain variable region (VL) comprising SEQ ID NO: 60; (c) a heavy chain CDR1, CDR2, and CDR3 of a heavy chain variable region (VH) comprising SEQ ID NO: 411 and a light chain CDR1, CDR2, and CDR3 of a light chain variable region (VL) comprising SEQ ID NO: 418; or (d) heavy chain CDR1, CDR2, and CDR3 of a heavy chain variable region (VH) comprising SEQ ID NO: 412 and light chain CDR1, CDR2, and CDR3 of a light chain variable region (VL) comprising SEQ ID NO: 60 Includes:

[0016] In certain embodiments, the anti-TIM3 antibody, or antigen-binding portion thereof, has the following activity as determined by HDX: (a) a heavy chain variable region (VH) comprising SEQ ID NO: 410 and a light chain variable region (VL) comprising SEQ ID NO: 417; (b) a heavy chain variable region (VH) comprising SEQ ID NO: 411 and a light chain variable region (VL) comprising SEQ ID NO: 60; (c) a heavy chain variable region (VH) comprising SEQ ID NO: 411 and a light chain variable region (VL) comprising SEQ ID NO: 418; or (d) a heavy chain variable region (VH) comprising SEQ ID NO: 412 and a light chain variable region (VL) comprising SEQ ID NO: 60 binds to the same epitope as the control antibody, including

[0017] In certain embodiments, the anti-TIM3 antibody or antigen-binding portion thereof (a) heavy chain CDR1, CDR2, and CDR3 of a heavy chain variable region (VH) comprising SEQ ID NO: 410 and light chain CDR1, CDR2, and CDR3 of a light chain variable region (VL) comprising SEQ ID NO: 417; (b) heavy chain CDR1, CDR2, and CDR3 of a heavy chain variable region (VH) comprising SEQ ID NO: 411 and light chain CDR1, CDR2, and CDR3 of a light chain variable region (VL) comprising SEQ ID NO: 60; (c) a heavy chain CDR1, CDR2, and CDR3 of a heavy chain variable region (VH) comprising SEQ ID NO: 411 and a light chain CDR1, CDR2, and CDR3 of a light chain variable region (VL) comprising SEQ ID NO: 418; or (d) heavy chain CDR1, CDR2, and CDR3 of a heavy chain variable region (VH) comprising SEQ ID NO: 412 and light chain CDR1, CDR2, and CDR3 of a light chain variable region (VL) comprising SEQ ID NO: 60 Includes:

[0018] In certain embodiments, the anti-TIM3 antibody, or antigen-binding portion thereof, comprises a VH and a VL, wherein the VH comprises SEQ ID NO: 410 and the VL comprises SEQ ID NO: 417. In certain embodiments, the VH comprises SEQ ID NO: 411 and the VL comprises SEQ ID NO: 60. In certain embodiments, the VH comprises SEQ ID NO: 411 and the VL comprises SEQ ID NO: 418. In certain embodiments, the VH comprises SEQ ID NO: 412 and the VL comprises SEQ ID NO: 60.

[0019] In some embodiments, an anti-TIM3 antibody disclosed herein comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3, wherein (a) heavy chain CDR1 comprises SEQ ID NO: 469; (b) heavy chain CDR2 comprises SEQ ID NO: 470; and (c) heavy chain CDR3 comprises SEQ ID NO: 471. In some embodiments, (a) light chain CDR1 comprises SEQ ID NO: 472; (b) light chain CDR2 comprises SEQ ID NO: 473; and (c) light chain CDR3 comprises SEQ ID NO: 474.

[0020] In certain embodiments, the anti-TIM3 antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein (a) the VH comprises SEQ ID NO: 449 or SEQ ID NO: 456; and (b) the VL comprises SEQ ID NO: 450.

[0021] In some embodiments, the anti-TIM3 antibody or antigen-binding portion thereof is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, and variants thereof. In some embodiments, the anti-TIM3 antibody or antigen-binding portion thereof is an IgG1 antibody. In some embodiments, the anti-TIM3 antibody or antigen-binding portion thereof comprises an effector-less IgG1 Fc.

[0022] In certain embodiments, the anti-TIM3 antibody, or antigen-binding portion thereof, (a1) heavy and light chain sequences comprising SEQ ID NOs: 386 (or 387) and 408, respectively; (a2) heavy and light chain sequences comprising SEQ ID NOs: 388 (or 389) and 408, respectively; (a3) heavy and light chain sequences comprising SEQ ID NOs: 390 (or 391) and 408, respectively; (a4) heavy and light chain sequences comprising SEQ ID NOs: 392 (or 393) and 408, respectively; (b1) heavy and light chain sequences comprising SEQ ID NOs: 394 (or 395) and 29, respectively; (b2) heavy and light chain sequences comprising SEQ ID NOs: 394 (or 395) and 409, respectively; (b3) heavy and light chain sequences comprising SEQ ID NOs: 396 (or 397) and 29, respectively; (b4) heavy and light chain sequences comprising SEQ ID NOs: 398 (or 399) and 29, respectively; (b5) heavy and light chain sequences comprising SEQ ID NOs: 400 (or 401) and 29, respectively; (b6) heavy and light chain sequences comprising SEQ ID NOs: 402 (or 403) and 29, respectively; (b7) heavy and light chain sequences comprising SEQ ID NOs: 404 (or 405) and 29, respectively; (b8) heavy and light chain sequences comprising SEQ ID NOs: 406 (or 407) and 29, respectively; (c1) heavy and light chain sequences comprising SEQ ID NOs: 451 (or 461) and 453, respectively; (c2) heavy and light chain sequences comprising SEQ ID NOs: 452 (or 462) and 453, respectively; (d1) heavy and light chain sequences comprising SEQ ID NOs: 457 (or 465) and 453, respectively; or (d2) heavy and light chain sequences comprising SEQ ID NOs: 458 (or 466) and 453, respectively Includes:

[0023] In certain embodiments, the anti-TIM3 antibody or antigen-binding portion thereof has one or more of the following properties: (1) For example, an EC of 1 μg / mL or less (e.g., 0.01 μg / mL to 0.05 μg / mL) for membrane-bound human TIM3, as measured by flow cytometry. 50 Combine with; (2) binds to membrane-bound cynomolgus monkey TIM3, e.g., as measured by flow cytometry; (3) 5 × 10 hTIM3-IgV was detected by the method described in Example 22. -8 M, 2 x 10 -8 M, 10 -8 M or 5x10 -9 K below M D Combine with; (4) hTIM3-IgV was measured by the method described in Example 22. -7 M, 2 x 10 -8 M or 10 -8 K below M D Combine with; (5) 5 × 10 TIM3-ECD cells were measured using the method described in Example 22. -7 M, 2 x 10 -8 M or 10 -8 K below M D Combine with; (6) 8 × 10 hTIM3-ECD was detected by the method described in Example 22. -8 M, 5 x 10 -8 M or 10 -8 K below M D Combine with; (7) induce or enhance T cell activation (e.g., by blocking or reducing the inhibitory effect of TIM3), as evidenced by increased proliferation of TIM3-expressing T cells (e.g., Th1 cells or TILs); and / or (8) Competes in either or both directions for binding to human TIM3 with an antibody comprising the VH and VL of any of 14H7, 23B3, 13A3, TIM3.7, TIM3.10, TIM3.11, TIM3.12, TIM3.13, TIM3.14, TIM3.15, TIM3.16, TIM3.17, TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24, TIM3.25, 17C3, 9F6, TIM3.7, 3G4, and 17C8.

[0024] In certain embodiments, the anti-TIM3 antibody comprises (i) a VH CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 45, 415, and 416, respectively, and a VL CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 64, 66, and 68, respectively; or (ii) a VH comprising SEQ ID NO: 412 and a VL comprising SEQ ID NO: 60, wherein the antibody binds to human TIM3-IgV at 5×10 -9 K below M D 10 to cynomolgus monkey TIM3-IgV -7 K below M D and / or 5 x 10 to cynomolgus TIM3-ECD -6 K below M D Combine with.

[0025] In certain embodiments, the anti-TIM3 antibody comprises (i) a VH CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 45, 413, and 414, respectively, and a VL CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 64, 66, and 69, respectively; or (ii) a VH comprising SEQ ID NO: 410 and a VL comprising SEQ ID NO: 417, wherein the antibody binds to human TIM3-IgV at 5×10 -8 K below M D 5 × 10 to cynomolgus monkey TIM3-IgV -9 K below M D and / or 5 x 10 to cynomolgus TIM3-ECD -9 K below M D Combine with.

[0026] In some embodiments, the heavy chain of an anti-TIM3 antibody disclosed herein comprises a C-terminal lysine. In other embodiments, the heavy chain of an anti-TIM3 antibody does not comprise a C-terminal lysine.

[0027] Provided herein are bispecific molecules comprising an anti-TIM3 antibody, or antigen-binding portion thereof, herein conjugated to a molecule with a second binding specificity.

[0028] Provided herein are nucleic acids encoding the VH and / or VL of an anti-TIM3 antibody, or antigen-binding portions thereof, expression vectors containing the nucleic acids, and cells transformed with the expression vectors.

[0029] Provided herein are immunoconjugates comprising an anti-TIM3 antibody, or antigen-binding portion thereof, described herein, linked to an agent.

[0030] Provided herein are compositions comprising an anti-TIM3 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate described herein and a carrier. Also provided herein are kits comprising an anti-TIM3 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate described herein and instructions for use.

[0031] Provided herein is a method for producing an anti-TIM3 antibody or antigen-binding portion thereof, comprising expressing the anti-TIM3 antibody or antigen-binding portion thereof in a cell and isolating the antibody or antigen-binding portion thereof from the cell.

[0032] Provided herein is a method of stimulating an antigen-specific T cell response, comprising contacting a T cell with an anti-TIM3 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate described herein, such that the antigen-specific T cell response is stimulated (e.g., by inhibiting the negative effect of TIM3 on a cell, e.g., a T cell).

[0033] Provided herein are methods of activating or costimulating T cells, e.g., effector T cells (e.g., Th1 cells), comprising contacting a cell, e.g., an effector T cell, with an anti-TIM3 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate described herein and CD3, wherein the effector T cell is activated or costimulated (e.g., by inhibiting the negative effect of TIM3 on the cell, e.g., T cell).

[0034] Provided herein are methods for increasing IFN-γ production in and / or proliferation of T cells, e.g., Th1 cells or TILs, comprising contacting the T cells with an effective amount of an anti-TIM3 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate described herein.

[0035] Provided herein is a method for increasing IFN-γ production in T cells in a subject, comprising administering to the subject an effective amount of an anti-TIM3 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate described herein, such that IFN-γ production from the T cells is increased.

[0036] Provided herein is a method of stimulating TIL activity in a subject, comprising administering to the subject a therapeutically effective amount of an anti-TIM3 antibody or antigen-binding portion thereof described herein, such that TILs proliferate or secrete cytokines, e.g., IFN-γ.

[0037] Provided herein are methods for stimulating NK cells (e.g., by increasing the cytotoxic activity of NK cells) and / or macrophages or other antigen-presenting cells in a subject, comprising administering to the subject an effective amount of an anti-TIM3 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate described herein. For example, the anti-TIM3 antibodies described herein can increase IL-12 secretion by antigen-presenting cells that contact the TIM3 antibody.

[0038] Provided herein is a method of stimulating an immune response in a subject, comprising administering to the subject an anti-TIM3 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate described herein, such that an immune response is stimulated in the subject. In some embodiments, the subject has a tumor, and an immune response against the tumor is stimulated.

[0039] Provided herein are methods for inhibiting tumor growth or reducing tumor size in a subject, comprising administering to the subject an anti-TIM3 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate described herein, such that tumor growth in the subject is inhibited.

[0040] Provided herein are methods for treating cancer, e.g., by immunotherapy, comprising administering a therapeutically effective amount of an anti-TIM3 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate described herein to a subject in need thereof to treat the cancer. In some embodiments, the cancer is selected from the group consisting of bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, colon cancer, kidney cancer, head and neck cancer, lung cancer, gastric cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system neoplasms, lymphoma, leukemia, myeloma, sarcoma, virus-associated cancer, and any combination thereof. In some embodiments, the cancer is metastatic cancer, refractory cancer, or recurrent cancer. In some embodiments, the cancer is a cold tumor.

[0041] In certain embodiments, the methods described herein further comprise administering one or more additional therapeutic agents, e.g., an anti-PD-1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, an anti-GITR antibody, and / or an anti-PD-L1 antibody and an anti-TIM3 antibody.

[0042] Provided herein is a method for detecting the presence of TIM3 protein in a sample, comprising contacting the sample with an anti-TIM3 antibody or antigen-binding portion thereof under conditions that allow the formation of a complex between the antibody or antigen-binding portion thereof and TIM3, and detecting the formation of the complex.

[0043] Implementation Embodiment 1. Binds to human T-cell immunoglobulin and mucin domain containing 3 (TIM3) and (a) Binds to soluble human TIM3; (b) binds to membrane-bound human TIM3; (c) inducing or enhancing T cell activation; and optionally: (d) binds to soluble cynomolgus monkey TIM3; and (e) Binds to membrane cynomolgus monkey TIM3 An isolated antibody (e.g., a human antibody) or antigen-binding portion thereof, exhibiting the properties of

[0044] Embodiment 2. The antibody or antigen-binding portion thereof of embodiment 1, wherein the antibody stimulates an anti-tumor immune response.

[0045] Embodiment 3. The antibody or antigen-binding portion thereof of embodiment 1 or 2, wherein the antibody stimulates an antigen-specific T cell response.

[0046] Embodiment 4. The antibody or antigen-binding portion thereof of any of the preceding embodiments, wherein the antibody increases IFN-γ production in TIM3-expressing T cells.

[0047] Embodiment 5. The antibody or antigen-binding portion thereof of any of the preceding embodiments, wherein the antibody increases T cell proliferation.

[0048] Embodiment 6. The antibody or antigen-binding portion thereof of any of the preceding embodiments, wherein the antibody does not bind to an Fc receptor or the antibody lacks effector function.

[0049] Embodiment 7. The antibody has a K of 10 nM or less for soluble human TIM3 as measured by Biacore. D10. The antibody or antigen-binding portion thereof of any of the preceding embodiments, wherein the antibody or antigen-binding portion binds to

[0050] Embodiment 8. The antibody has a K of 100 nM or less for soluble cynomolgus TIM3 as measured by Biacore. D 10. The antibody or antigen-binding portion thereof of any of the preceding embodiments, wherein the antibody or antigen-binding portion binds to

[0051] Embodiment 9. The antibody or antigen-binding fragment thereof of any of the preceding embodiments, wherein the antibody is an antagonist antibody that inhibits negative cell (e.g., T cell) signaling by TIM3.

[0052] Embodiment 10. The antibody has an EC of 0.1 or 1 μg / mL or less as measured by flow cytometry. 50 10. The antibody or antigen-binding portion thereof of any of the preceding embodiments, which binds to membrane-bound human TIM3 at

[0053] Embodiment 11. The antibody has a K of 1 nM or less as measured by Scatchard analysis. D 10. The antibody or antigen-binding portion thereof of any of the preceding embodiments, which binds to membrane-bound human TIM3 at

[0054] Embodiment 12. The antibody has an EC of 1 μg / mL or less as measured by flow cytometry. 50 or the antibody binds to membrane-bound cynomolgus monkey TIM3 with a K of 1 nM or less as measured by Scatchard analysis. D 2. The antibody or antigen-binding portion thereof of any preceding embodiment, which binds to membrane-bound cynomolgus TIM3 at 100 ng / mL.

[0055] Embodiment 13. The antibody, or antigen-binding portion thereof, of any of the preceding embodiments, wherein the antibody, or antigen-binding portion thereof, comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3, wherein the heavy chain CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, or SEQ ID NO:129.

[0056] Embodiment 14. The antibody or antigen-binding portion thereof of embodiment 13, wherein the heavy chain CDR1 comprises X1, X2, X3, X4, Y, X5, and X6, wherein X1 is S or absent, X2 is R or absent, X3 is S, R, or D, X4 is Y or H, X5 is W or M, and X6 is G, N, S, or H.

[0057] Embodiment 15. The antibody or antigen-binding portion thereof of embodiment 13 or 14, wherein the heavy chain CDR1 comprises X1, Y, Y, M and X2, wherein X1 is S or D and X2 is H or S.

[0058] Embodiment 16. The antibody or antigen-binding portion thereof of embodiment 13 or 14, wherein the heavy chain CDR1 comprises R, X1, Y, W and X2, where X1 is H or Y and X2 is N or S.

[0059] Embodiment 17. Heavy chain CDR2 is selected from the group consisting of X1, I, X2, X3, X4, G, X5, X6, X7, X8, Y, X9, X 10 , X 11 , X 12 , X 13 and X 14 wherein X1 is S, Y, I or F, X2 is Y, H, N or S, X3 is Y, P, G, T or S, X4 is S, T, R or G, X5 is F, S or D, X6 is S, T or I, X7 is I or absent, X8 is Y, N or I, X9 is N, Q, S or A, and X 10 is P, S, Q or D, and X 11 is S or K, and X 12 is L, F or V, and X 13 is K or Q and X 14 is S or G.

[0060] Embodiment 18. An antibody or antigen-binding portion thereof of any of embodiments 13 to 17, wherein the heavy chain CDR2 comprises Y, I, H, Y, X1, G, S, T, N, Y, N, X2, S, L, K and S, where X1 is S or T and X2 is S or P.

[0061] Embodiment 19. An antibody or antigen-binding portion thereof of any of embodiments 13 to 17, wherein the heavy chain CDR2 comprises F, I, S, X1, X2, G, S, X3, I, Y, Y, A, D, S, V, K and G, where X1 is G, T or S, X2 is G or S, and X3 is T or I.

[0062] Embodiment 20. An antibody or antigen-binding portion thereof of any of embodiments 13 to 17, wherein the heavy chain CDR2 comprises I, I, N, P, R, G, D, S, I, I, Y, A, Q, K, F, Q and G.

[0063] Embodiment 21. The antibody or antigen-binding portion thereof of any of embodiments 13 to 20, wherein the light chain CDR1 comprises SEQ ID NO: 64 or SEQ ID NO: 65.

[0064] Embodiment 22. The antibody or antigen-binding portion thereof of any of embodiments 13 to 21, wherein the light chain CDR2 comprises SEQ ID NO: 66 or SEQ ID NO: 67.

[0065] Embodiment 23. The antibody or antigen-binding portion thereof of any of embodiments 13 to 22, wherein the light chain CDR3 comprises SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71.

[0066] Embodiment 24. An antibody, or antigen-binding portion thereof, comprising heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3, wherein (a) the heavy chain CDR1 is selected from the group consisting of SEQ ID NO:41, SEQ ID NO:42; SEQ ID NO:43; SEQ ID NO:44; and SEQ ID NO:45; (b) the heavy chain CDR2 is selected from the group consisting of SEQ ID NO:46, SEQ ID NO:47; SEQ ID NO:48; SEQ ID NO:49; SEQ ID NO:50; SEQ ID NO:51; SEQ ID NO:52; SEQ ID NO:122; SEQ ID NO:123; SEQ ID NO:124 and SEQ ID NO:125; (c) the heavy chain CDR3 is selected from the group consisting of SEQ ID NO:53, SEQ ID NO:54; SEQ ID NO:55; SEQ ID NO:56; SEQ ID NO:57; SEQ ID NO:58; SEQ ID NO:59; SEQ ID NO:126; SEQ ID NO:127; SEQ ID NO:128 and SEQ ID NO:129; (d) the light chain CDR1 comprises SEQ ID NO: 64 or SEQ ID NO: 65; (e) the light chain CDR2 comprises SEQ ID NO: 66 or SEQ ID NO: 67; and (f) the light chain CDR3 comprises SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, or SEQ ID NO: 71; 10. The antibody or antigen-binding portion thereof of any of the preceding embodiments.

[0067] Embodiment 25. An isolated antibody, or antigen-binding portion thereof, that binds to human TIM3, (a1) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 41, 46, and 53, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 68, respectively; (a2) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 41, 122, and 53, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 68, respectively; (a3) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 41, 123, and 53, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 68, respectively; (a4) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 41, 124, and 53, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 68, respectively; (a5) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 41, 46, and 126, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 68, respectively; (a6) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 41, 46, and 127, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 68, respectively; (a7) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 41, 46, and 128, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 68, respectively; (a8) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 41, 46, and 129, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 68, respectively; (a9) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 41, 122, and 128, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 68, respectively; (a10) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 41, 122, and 126, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 68, respectively; (b1) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 42, 47, and 54, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 69, respectively; (b2) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 42, 125, and 54, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 69, respectively; (c) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 43, 48, and 55, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 64, 66, and 69, respectively; (d) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 44, 49, and 56, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 64, 66, and 68, respectively; (e) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 45, 50, and 57, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 64, 66, and 69, respectively; (f) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 45, 50, and 57, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 64, 66, and 71, respectively; (g1) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 45, 50, and 57, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 65, 67, and 70, respectively; (g2) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 45, 50, and 57, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 71, respectively; (g3) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 45, 50, and 57, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 69, respectively; (h) heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 45, 51 and 58, respectively, and / or light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 64, 66 and 68, respectively; (i) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 45, 52, and 59, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 64, 66, and 69, respectively; An antibody or antigen-binding portion thereof comprising:

[0068] Embodiment 26. The antibody or antigen-binding portion thereof of embodiment 25, wherein the antibody comprises heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 41, 46 and 53, respectively, and / or light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 64, 66 and 68, respectively.

[0069] Embodiment 27. The antibody or antigen-binding portion thereof of embodiment 25, wherein the antibody comprises heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 42, 47 and 54, respectively, and / or light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 64, 66 and 69, respectively.

[0070] Embodiment 28. The antibody or antigen-binding portion thereof of embodiment 25, wherein the antibody comprises heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 43, 48 and 55, respectively, and / or light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 64, 66 and 69, respectively.

[0071] Embodiment 29. The antibody or antigen-binding portion thereof of embodiment 25, wherein the antibody comprises heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 44, 49 and 56, respectively, and / or light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 64, 66 and 68, respectively.

[0072] Embodiment 30. The antibody or antigen-binding portion thereof of embodiment 25, wherein the antibody comprises heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 45, 50 and 57, respectively, and / or light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 64, 66 and 69, respectively.

[0073] Embodiment 31. The antibody or antigen-binding portion thereof of embodiment 25, wherein the antibody comprises heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 45, 50 and 57, respectively, and / or light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 64, 66 and 71, respectively.

[0074] Embodiment 32. The antibody or antigen-binding portion thereof of embodiment 25, wherein the antibody comprises heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 45, 50 and 57, respectively, and / or light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 65, 67 and 70, respectively.

[0075] Embodiment 33. The antibody or antigen-binding portion thereof of embodiment 25, wherein the antibody comprises heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 45, 51 and 58, respectively, and / or light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 64, 66 and 68, respectively.

[0076] Embodiment 34. The antibody or antigen-binding portion thereof of embodiment 25, wherein the antibody comprises heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 45, 52 and 59, respectively, and / or light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 64, 66 and 69, respectively.

[0077] Embodiment 35. An isolated antibody or antigen-binding portion thereof that binds to human TIM3 and comprises heavy and light chain variable regions, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 35, 36, 37, 38, 39, 40, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, and 364.

[0078] Embodiment 36. An isolated antibody or antigen-binding portion thereof that binds to human TIM3 and comprises a heavy chain and a light chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 60, 61, 62, and 63.

[0079] Embodiment 37. An isolated antibody, or antigen-binding portion thereof, that binds to human TIM3 and cross-competes for binding to human TIM3 with a control antibody comprising a VH and a VL, wherein the VH and VL are (a) a VH comprising the amino acid sequence set forth in SEQ ID NO: 34 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 60; (b) a VH comprising the amino acid sequence set forth in SEQ ID NO: 35 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 61; (c) a VH comprising the amino acid sequence set forth in SEQ ID NO: 36 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 61; (d) a VH comprising the amino acid sequence set forth in SEQ ID NO: 37 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 60; (e) a VH comprising the amino acid sequence set forth in SEQ ID NO: 38 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 61; (f) a VH comprising the amino acid sequence set forth in SEQ ID NO: 38 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 62; (g) a VH comprising the amino acid sequence set forth in SEQ ID NO: 38 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 63; (h) VH comprising the amino acid sequence set forth in SEQ ID NO: 39 and VL comprising the amino acid sequence set forth in SEQ ID NO: 60; (i) a VH comprising the amino acid sequence set forth in SEQ ID NO: 40 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 61; (j) VH and VL each comprising the amino acid sequence set forth in SEQ ID NO: 121 and SEQ ID NO: 63; (k) VH comprising the amino acid sequence set forth in SEQ ID NO: 120 and VL comprising the amino acid sequence set forth in SEQ ID NO: 61; (l) VH and VL each comprising the amino acid sequence set forth in SEQ ID NO: 112 and the amino acid sequence set forth in SEQ ID NO: 60; (m) a VH comprising the amino acid sequence set forth in SEQ ID NO: 113 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 60; (n) a VH comprising the amino acid sequence set forth in SEQ ID NO: 114 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 60; (o) a VH comprising the amino acid sequence set forth in SEQ ID NO: 115 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 60; (p) a VH comprising the amino acid sequence set forth in SEQ ID NO: 116 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 60; (q) a VH comprising the amino acid sequence set forth in SEQ ID NO: 117 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 60; (r) a VH comprising the amino acid sequence set forth in SEQ ID NO: 118 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 60; (s) a VH comprising the amino acid sequence set forth in SEQ ID NO: 119 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 60; and (t) VH comprising the amino acid sequence shown in SEQ ID NO: 364 and VL comprising the amino acid sequence shown in SEQ ID NO: 60 1. An isolated antibody or antigen-binding portion thereof comprising:

[0080] Embodiment 38. An antibody or antigen-binding portion thereof of embodiment 37, which binds to TIM3 at the same epitope as the subject antibody, for example, as determined by one or more methods provided herein.

[0081] Embodiment 39. (a) a VH comprising the amino acid sequence set forth in SEQ ID NO: 34 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 60; (b) a VH comprising the amino acid sequence set forth in SEQ ID NO: 35 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 61; (c) a VH comprising the amino acid sequence set forth in SEQ ID NO: 36 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 61; (d) a VH comprising the amino acid sequence set forth in SEQ ID NO: 37 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 60; (e) a VH comprising the amino acid sequence set forth in SEQ ID NO: 38 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 61; (f) a VH comprising the amino acid sequence set forth in SEQ ID NO: 38 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 62; (g) a VH comprising the amino acid sequence set forth in SEQ ID NO: 38 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 63; (h) VH comprising the amino acid sequence set forth in SEQ ID NO: 39 and VL comprising the amino acid sequence set forth in SEQ ID NO: 60; (i) a VH comprising the amino acid sequence set forth in SEQ ID NO: 40 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 61; (j) VH and VL each comprising the amino acid sequence set forth in SEQ ID NO: 121 and SEQ ID NO: 63; (k) VH and VL each comprising the amino acid sequence set forth in SEQ ID NO: 120 and the amino acid sequence set forth in SEQ ID NO: 61; (l) VH and VL each comprising the amino acid sequence set forth in SEQ ID NO: 112 and the amino acid sequence set forth in SEQ ID NO: 60; (m) a VH comprising the amino acid sequence set forth in SEQ ID NO: 113 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 60; (n) a VH comprising the amino acid sequence set forth in SEQ ID NO: 114 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 60; (o) a VH comprising the amino acid sequence set forth in SEQ ID NO: 115 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 60; (p) a VH comprising the amino acid sequence set forth in SEQ ID NO: 116 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 60; (q) a VH comprising the amino acid sequence set forth in SEQ ID NO: 117 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 60; (r) a VH comprising the amino acid sequence set forth in SEQ ID NO: 118 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 60; (s) a VH comprising the amino acid sequence set forth in SEQ ID NO: 119 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 60; and (t) VH comprising the amino acid sequence shown in SEQ ID NO: 364 and VL comprising the amino acid sequence shown in SEQ ID NO: 60 39. The antibody or antigen-binding portion thereof of embodiment 37 or 38, comprising a VH and a VL selected from the group consisting of:

[0082] Embodiment 40. The antibody or antigen-binding portion thereof of embodiment 37 or 38, comprising a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 and SEQ ID NO: 364, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 60.

[0083] Embodiment 41. The antibody or antigen-binding portion thereof of embodiment 37 or 38, comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 35 or SEQ ID NO: 120 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 61.

[0084] Embodiment 42. The antibody or antigen-binding portion thereof of embodiment 37 or 38, comprising a VH having the amino acid sequence set forth in SEQ ID NO: 36 and a VL having the amino acid sequence set forth in SEQ ID NO: 61.

[0085] Embodiment 43. The antibody or antigen-binding portion thereof of embodiment 37 or 38, comprising a VH having the amino acid sequence set forth in SEQ ID NO: 37 and a VL having the amino acid sequence set forth in SEQ ID NO: 60.

[0086] Embodiment 44. The antibody or antigen-binding portion thereof of embodiment 37 or 38, comprising a VH having the amino acid sequence set forth in SEQ ID NO: 38 or a VL having the amino acid sequence set forth in SEQ ID NO: 121 and SEQ ID NO: 61, SEQ ID NO: 63 or SEQ ID NO: 62.

[0087] Embodiment 45. The antibody or antigen-binding portion thereof of embodiment 37 or 38, comprising a VH having the amino acid sequence set forth in SEQ ID NO: 39 and a VL having the amino acid sequence set forth in SEQ ID NO: 60.

[0088] Embodiment 46. The antibody or antigen-binding portion thereof of embodiment 37 or 38, comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 40 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 61.

[0089] Embodiment 47. The antibody or antigen-binding portion thereof of any of the preceding embodiments, wherein the antibody is selected from the group consisting of IgG1, IgG2, IgG3, IgG4 or a variant thereof.

[0090] Embodiment 48. The antibody or antigen-binding portion thereof of embodiment 47, wherein the antibody is an IgG1 antibody.

[0091] Embodiment 49. The antibody or antigen-binding portion thereof of embodiment 47, wherein the antibody comprises an effectorless IgG1 Fc.

[0092] Embodiment 50. The antibody or antigen-binding portion thereof of embodiment 49, wherein the antibody or antigen-binding portion thereof comprises an effectorless IgG1 Fc comprising the following mutations: L234A, L235E, G237A and optionally A330S and P331S.

[0093] Embodiment 51. The antibody or antigen-binding portion thereof of any of the preceding embodiments, comprising a heavy chain constant region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 263 to 266.

[0094] Embodiment 52. The antibody or antigen-binding portion thereof of any of embodiments 1 to 51, wherein the antibody or antigen-binding portion thereof is a human or humanized antibody.

[0095] 53. An antibody (a1) heavy and light chain sequences comprising SEQ ID NOs: 301 (or 302) and 29, respectively; (a2) heavy and light chain sequences comprising SEQ ID NOs: 1 (or 8) and 29, respectively; (a3) heavy and light chain sequences comprising SEQ ID NOs: 15 (or 22) and 29, respectively; (a4) heavy and light chain sequences comprising SEQ ID NOs: 303 (or 304) and 29, respectively; (a5) heavy and light chain sequences comprising SEQ ID NOs: 72 (or 82) and 29, respectively; (a6) heavy and light chain sequences comprising SEQ ID NOs: 92 (or 102) and 29, respectively; (a7) heavy and light chain sequences comprising SEQ ID NOs: 305 (or 306) and 29, respectively; (a8) heavy and light chain sequences comprising SEQ ID NOs: 73 (or 83) and 29, respectively; (a9) heavy and light chain sequences comprising SEQ ID NOs: 93 (or 103) and 29, respectively; (a10) heavy and light chain sequences comprising SEQ ID NOs: 307 (or 308) and 29, respectively; (a11) heavy and light chain sequences comprising SEQ ID NOs: 74 (or 84) and 29, respectively; (a12) heavy and light chain sequences comprising SEQ ID NOs: 94 (or 104) and 29, respectively; (a13) heavy and light chain sequences comprising SEQ ID NOs: 309 (or 310) and 29, respectively; (a14) heavy and light chain sequences comprising SEQ ID NOs: 75 (or 85) and 29, respectively; (a15) heavy and light chain sequences comprising SEQ ID NOs: 95 (or 105) and 29, respectively; (a16) heavy and light chain sequences comprising SEQ ID NOs: 311 (or 312) and 29, respectively; (a17) heavy and light chain sequences comprising SEQ ID NOs: 76 (or 86) and 29, respectively; (a18) heavy and light chain sequences comprising SEQ ID NOs: 96 (or 106) and 29, respectively; (a19) heavy and light chain sequences comprising SEQ ID NOs: 313 (or 314) and 29, respectively; (a20) heavy and light chain sequences comprising SEQ ID NOs: 77 (or 87) and 29, respectively; (a21) heavy and light chain sequences comprising SEQ ID NOs: 97 (or 107) and 29, respectively; (a22) heavy and light chain sequences comprising SEQ ID NOs: 315 (or 316) and 29, respectively; (a23) heavy and light chain sequences comprising SEQ ID NOs: 78 (or 88) and 29, respectively; (a24) heavy and light chain sequences comprising SEQ ID NOs: 98 (or 108) and 29, respectively; (a25) heavy and light chain sequences comprising SEQ ID NOs: 317 (or 318) and 29, respectively; (a26) heavy and light chain sequences comprising SEQ ID NOs: 79 (or 89) and 29, respectively; (a27) heavy and light chain sequences comprising SEQ ID NOs: 99 (or 109) and 29, respectively; (a28) heavy and light chain sequences comprising SEQ ID NOs: 319 (or 320) and 29, respectively; (a29) heavy and light chain sequences comprising SEQ ID NOs: 349 (or 350) and 29, respectively; (a30) heavy and light chain sequences comprising SEQ ID NOs: 351 (or 352) and 29, respectively; (a31) heavy and light chain sequences comprising SEQ ID NOs: 353 (or 354) and 29, respectively; (b1) heavy and light chain sequences comprising SEQ ID NOs: 321 (or 322) and 30, respectively; (b2) heavy and light chain sequences comprising SEQ ID NOs: 2 (or 9) and 30, respectively; (b3) heavy and light chain sequences comprising SEQ ID NOs: 16 (or 23) and 30, respectively; (b4) heavy and light chain sequences comprising SEQ ID NOs: 323 (or 324) and 30, respectively; (b5) heavy and light chain sequences comprising SEQ ID NOs: 80 (or 90) and 30, respectively; (b6) heavy and light chain sequences comprising SEQ ID NOs: 100 (or 110) and 30, respectively; (b7) heavy and light chain sequences comprising SEQ ID NOs: 325 (or 326) and 30, respectively; (c1) heavy and light chain sequences comprising SEQ ID NOs: 327 (or 328) and 30, respectively; (c2) heavy and light chain sequences comprising SEQ ID NOs: 3 (or 10) and 30, respectively; (c3) heavy and light chain sequences comprising SEQ ID NOs: 17 (or 24) and 30, respectively; (c4) heavy and light chain sequences comprising SEQ ID NOs: 329 (or 330) and 30, respectively; (d1) heavy and light chain sequences comprising SEQ ID NOs: 331 (or 332) and 29, respectively; (d2) heavy and light chain sequences comprising SEQ ID NOs: 4 (or 11) and 29, respectively; (d3) heavy and light chain sequences comprising SEQ ID NOs: 18 (or 25) and 29, respectively; (d4) heavy and light chain sequences comprising SEQ ID NOs: 333 (or 334) and 29, respectively; (e1.1) heavy and light chain sequences comprising SEQ ID NOs: 335 (or 336) and 32, respectively; (e1.2) heavy and light chain sequences comprising SEQ ID NOs: 335 (or 336) and 33, respectively; (e1.3) heavy and light chain sequences comprising SEQ ID NOs: 335 (or 336) and 31, respectively; (e2) heavy and light chain sequences comprising SEQ ID NOs: 5 (or 12) and 33, respectively; (e3) heavy and light chain sequences comprising SEQ ID NOs: 19 (or 26) and 33, respectively; (e4) heavy and light chain sequences comprising SEQ ID NOs: 337 (or 338) and 33, respectively; (e5) heavy and light chain sequences comprising SEQ ID NOs: 81 (or 91) and 33, respectively; (e6) heavy and light chain sequences comprising SEQ ID NOs: 101 (or 111) and 33, respectively; (e7) heavy and light chain sequences comprising SEQ ID NOs: 339 (or 340) and 33, respectively; (f1) heavy and light chain sequences comprising SEQ ID NOs: 341 (or 342) and 29, respectively; (f2) heavy and light chain sequences comprising SEQ ID NOs: 6 (or 13) and 29, respectively; (f3) heavy and light chain sequences comprising SEQ ID NOs: 20 (or 27) and 29, respectively; (f4) heavy and light chain sequences comprising SEQ ID NOs: 343 (or 344) and 29, respectively; (g1) heavy and light chain sequences comprising SEQ ID NOs: 345 (or 346) and 29, respectively; (g2) heavy and light chain sequences comprising SEQ ID NOs: 7 (or 43) and 30, respectively; (g3) heavy and light chain sequences comprising SEQ ID NOs: 21 (or 28) and 30, respectively; or (g4) heavy and light chain sequences comprising SEQ ID NOs: 347 (or 348) and 30, respectively; 53. The antibody of any of embodiments 1 to 52, wherein the antibody specifically binds to human TIM3.

[0096] Embodiment 54. The antibody or antigen-binding portion thereof of any of embodiments 1 to 53, wherein the antibody or antigen-binding portion thereof has one or more of the following properties: (1) For example, as measured by Biacore, soluble human TIM3 has a K of, for example, 10 nM or less (e.g., 0.01 nM to 10 nM). D Combine with; (2) For example, when measured by Biacore, soluble cynomolgus monkey TIM3 has a K of, for example, 100 nM or less (e.g., 0.01 nM to 100 nM). D Combine with; (3) For example, an EC of 1 μg / mL or less (e.g., 0.01 μg / mL to 1 μg / mL) for membrane-bound human TIM3, as measured by flow cytometry. 50 Combine with; (4) For example, a K of 1 nM or less (e.g., 0.01 nM to 10 nM) for membrane-bound human TIM3, as measured by Scatchard analysis. D Combine with; (5) For example, an EC of 20 μg / mL or less (e.g., 0.01 μg / mL to 20 μg / mL) for membrane-bound cynomolgus monkey TIM3, as measured by flow cytometry. 50 Combine with; (6) For example, as measured by Scatchard analysis, membrane-bound cynomolgus monkey TIM3 has a K of, for example, 1 nM or less (e.g., 0.01 nM to 10 nM). D Combine with; (7) inducing or enhancing T cell activation (e.g., by blocking or reducing the inhibitory effect of TIM3), as evidenced by (i) increasing IFN-γ production by TIM3-expressing T cells (e.g., Th1 cells or TILs) and / or (ii) increasing the proliferation of TIM3-expressing T cells (e.g., Th1 cells or TILs); (8) stimulating T cell proliferation in mixed lymphocyte reaction (MLR) assays; (9) inhibiting phosphatidylserine binding to TIM3, e.g., as measured by a PS-hTIM3 “in tandem” blocking assay; (10) when bound to cellular TIM3, it does not internalize or downregulate cell surface TIM3; (11) binds to one of the following regions of the human TIM3 extracellular domain (SEQ ID NO: 290): (a) CPVFECG (SEQ ID NO: 296); (b) RIQIPGIMND (SEQ ID NO: 298); (c) CPVFECG and RIQIPGIMND (SEQ ID NOs: 296 and 298, respectively); and (d) WTSRYWLNGDFR (SEQ ID NO: 297); (12) reduced binding to human TIM3 in which one or more of the amino acids L48, C58, P59, V60, F61, E62, C63, G64, W78, S80, R81, W83, L84, G86, D87, R89, D104, R111, Q113, G116, M118, and D120 (numbered in SEQ ID NO: 286 ( FIG. 30 )) have been substituted with another amino acid, compared to binding to wild-type human TIM3; (13) competes for binding to human TIM3 in either or both orientations with an antibody comprising the VH and VL domains of 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, or any of TIM3.7, TIM3.8, TIM3.10, TIM3.11, TIM3.12, TIM3.13, TIM3.14, TIM3.15, TIM3.16, TIM3.17, and TIM3.18; (14) Human TIM3 region determined by HDX-MS 49 VPVCWGKGACPVFE 62 (SEQ ID NO: 367) and 111 RIQIPGIMNDEKFNLKL 127 binds to (SEQ ID NO: 368); (15) having a heavy chain and / or light chain variable region that interacts with at least 5, 10, 15, 20, or all of the following amino acids of human TIM3, P50, V51, C52, P59, V60, F61, E62, C63, G64, N65, V66, V67, L68, R69, D71, E72, D74, R111, Q113, G116, I117, M118, D120, and optionally T70 and / or I112, as determined by X-ray crystallography (e.g., as described in the Examples; numbering according to SEQ ID NO: 286 (Figure 30)); and / or (16) (a) exhibits reduced binding to human TIM3 in which one, two, three, four, five, six, seven, eight, or nine of the amino acids C58, P59, F61, E62, C63, R111, D120, and optionally D104 and Q113 (numbering according to SEQ ID NO: 286 (Figure 30)) have been substituted with another amino acid, compared to binding to wild-type human TIM3; (b) exhibits reduced binding to human TIM3 in which one, two, three, four, five, six, seven, eight, or nine of the amino acids C58, P59, F61, E62, C63, R111, D120, and optionally D104 and Q113 (numbering according to SEQ ID NO: 286 (Figure 30)) have been substituted with another amino acid, as determined by HDX-MS as described in the Examples. 49 VPVCWGKGACPVFE 62 (SEQ ID NO: 367), 111 RIQIPGIMNDEKFNLKL 127 (SEQ ID NO: 368) and 119 NDEKFNLKL 127 (c) binds to 13A3 or TIM3.18.IgG1.3 (SEQ ID NO: 373); and / or (d) competes with or cross-blocks the binding of 13A3 or TIM3.18.IgG1.3 to human TIM3.

[0097] Embodiment 55. A bispecific molecule comprising the antibody of any of the preceding embodiments, conjugated to a molecule having a second binding specificity.

[0098] Embodiment 56. A nucleic acid encoding the heavy and / or light chain variable region of the antibody or antigen-binding portion thereof of any of embodiments 1 to 54.

[0099] Embodiment 57. An expression vector comprising the nucleic acid molecule of embodiment 56.

[0100] Embodiment 58. A cell transformed with the expression vector of embodiment 57.

[0101] Embodiment 59. An immunoconjugate comprising the antibody of any of embodiments 1 to 54 conjugated to a drug.

[0102] Embodiment 60. A composition comprising the antibody or antigen-binding portion thereof, bispecific molecule or immunoconjugate of any of embodiments 1 to 55 and 59 and a carrier.

[0103] Embodiment 61. A kit comprising the antibody or antigen-binding portion thereof, or bispecific molecule, or immunoconjugate of any of embodiments 1 to 55 and 59 and instructions for use.

[0104] Embodiment 62. A method for producing an anti-TIM3 antibody or antigen-binding portion thereof, comprising expressing the antibody or antigen-binding portion thereof in a cell of embodiment 58 and isolating the antibody or antigen-binding portion thereof from the cell.

[0105] Embodiment 63. A method for stimulating an antigen-specific T cell response, comprising contacting a T cell with the antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate of any of embodiments 1 to 55 and 59, such that an antigen-specific T cell response is stimulated.

[0106] Embodiment 64. A method for activating or costimulating an effector T cell, comprising contacting the effector T cell with an anti-TIM3 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate of any of embodiments 1 to 55 and 59, and CD3, such that the effector T cell is activated or costimulated.

[0107] Embodiment 65. A method for increasing IFN-γ production in T cells, comprising contacting T cells with an effective amount of the antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate of any of embodiments 1 to 55 and 59.

[0108] Embodiment 66. A method for increasing T cell proliferation, comprising contacting a cell with an effective amount of the antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate of any of embodiments 1 to 55 and 59.

[0109] Embodiment 67. A method for increasing IFN-γ production by T cells in a subject, comprising administering to the subject an effective amount of the antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate of any of embodiments 1 to 55 and 59 so as to increase IFN-γ production from T cells.

[0110] Embodiment 68. A method for stimulating TIL activity in a subject, comprising administering to the subject a therapeutically effective amount of an anti-TIM3 antibody of any of embodiments 1 to 54.

[0111] Embodiment 69. A method for stimulating an immune response in a subject, comprising administering to the subject an antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate of any of embodiments 1 to 55 and 59, such that an immune response in the subject is stimulated.

[0112] Embodiment 70. The method of embodiment 69, wherein the subject has a tumor and an immune response against the tumor is stimulated.

[0113] Embodiment 71. A method of inhibiting tumor growth or reducing tumor size in a subject, comprising administering to the subject an antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate of any of embodiments 1 to 55 and 59, such that tumor growth in the subject is inhibited.

[0114] Embodiment 72. A method for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate of any of embodiments 1 to 55 and 59 to treat the cancer.

[0115] Embodiment 73. The method of embodiment 72, wherein the cancer is selected from the group consisting of bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, colon cancer, kidney cancer, head and neck cancer, lung cancer, gastric cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, lymphoma, leukemia, myeloma, sarcoma, and virus-associated cancer.

[0116] Embodiment 74. The method of embodiment 72 or 73, wherein the cancer is metastatic cancer, refractory cancer, or recurrent cancer.

[0117] Embodiment 75. The method of any of embodiments 67 to 74, further comprising administering one or more additional therapeutic agents.

[0118] Embodiment 76. The method of embodiment 75, wherein the additional therapeutic agent is an anti-PD-1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, an anti-GITR antibody, or an anti-PD-L1 antibody.

[0119] Embodiment 77. A method for detecting the presence of T-cell immunoglobulin and mucin domain-containing 3 (TIM3) in a sample, comprising contacting the sample with an antibody or antigen-binding portion thereof of any of embodiments 1 to 54 under conditions that allow the formation of a complex between the antibody or antigen-binding portion thereof and TIM3, and detecting the formation of the complex. [Brief explanation of the drawings]

[0120] [Figure 1A] The nucleotide sequence (SEQ ID NO: 167) and amino acid sequence (SEQ ID NO: 34) of the mature heavy chain variable (VH) region of anti-TIM3 monoclonal antibody 13A3 are shown. CDR1 (SEQ ID NO: 41), CDR2 (SEQ ID NO: 46), and CDR3 (SEQ ID NO: 53) are underlined, and the V, D, and J germline sequences are indicated.

[0121] [Figure 1B] The nucleotide sequence (SEQ ID NO: 193) and amino acid sequence (SEQ ID NO: 60) of the mature light chain variable (VL) region of anti-TIM3 monoclonal antibody 13A3 are shown. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66), and CDR3 (SEQ ID NO: 68) are underlined, and the V and J germline sequences are indicated.

[0122] [Figure 1C] The nucleotide sequence (sequence number 167) and amino acid sequence (sequence number 34) of the heavy chain VH region of anti-TIM3 monoclonal antibody 13A3 together with the signal sequence (sequence numbers 274 and 269, respectively) and the nucleotide sequence (sequence number 193) and amino acid sequence (sequence number 60) of the light chain VL region of anti-TIM3 monoclonal antibody 13A3 together with the signal sequence (sequence numbers 273 and 268, respectively) are shown.

[0123] [Figure 2A] The nucleotide sequence (SEQ ID NO: 168) and amino acid sequence (SEQ ID NO: 35) of the mature heavy chain variable (VH) region of anti-TIM3 monoclonal antibody 8B9 are shown. CDR1 (SEQ ID NO: 42), CDR2 (SEQ ID NO: 47), and CDR3 (SEQ ID NO: 54) are underlined, and the V, D, and J germline sequences are indicated.

[0124] [Figure 2B] The nucleotide sequence (SEQ ID NO: 194) and amino acid sequence (SEQ ID NO: 61) of the mature light chain variable (VL) region of anti-TIM3 monoclonal antibody 8B9 are shown. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66), and CDR3 (SEQ ID NO: 69) are underlined, and the V and J germline sequences are indicated.

[0125] [Figure 2C]The nucleotide sequence (sequence number 168) and amino acid sequence (sequence number 35) of the heavy chain VH region of anti-TIM3 monoclonal antibody 8B9 together with the signal sequence (sequence numbers 274 and 269, respectively) and the nucleotide sequence (sequence number 194) and amino acid sequence (sequence number 61) of the light chain VL region of anti-TIM3 monoclonal antibody 8B9 together with the signal sequence (sequence numbers 273 and 268, respectively) are shown.

[0126] [Figure 3A] The nucleotide sequence (SEQ ID NO: 169) and amino acid sequence (SEQ ID NO: 36) of the mature heavy chain variable (VH) region of anti-TIM3 monoclonal antibody 8C4 are shown. CDR1 (SEQ ID NO: 43), CDR2 (SEQ ID NO: 48), and CDR3 (SEQ ID NO: 55) are underlined, and the V, D, and J germline sequences are indicated.

[0127] [Figure 3B] The nucleotide sequence (SEQ ID NO: 194) and amino acid sequence (SEQ ID NO: 61) of the mature light chain variable (VL) region of anti-TIM3 monoclonal antibody 8C4 are shown. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66), and CDR3 (SEQ ID NO: 69) are underlined, and the V and J germline sequences are indicated.

[0128] [Figure 3C] The nucleotide sequence (sequence number 169) and amino acid sequence (sequence number 36) of the heavy chain VH region of anti-TIM3 monoclonal antibody 8C4 together with the signal sequence (sequence numbers 274 and 269, respectively) and the nucleotide sequence (sequence number 194) and amino acid sequence (sequence number 61) of the light chain VL region of anti-TIM3 monoclonal antibody 8C4 together with the signal sequence (sequence numbers 273 and 268, respectively) are shown.

[0129] [Figure 4A]The nucleotide sequence (SEQ ID NO: 170) and amino acid sequence (SEQ ID NO: 37) of the mature heavy chain variable (VH) region of anti-TIM3 monoclonal antibody 17C3 are shown. CDR1 (SEQ ID NO: 44), CDR2 (SEQ ID NO: 49), and CDR3 (SEQ ID NO: 56) are underlined, and the V, D, and J germline sequences are indicated.

[0130] [Figure 4B] The nucleotide sequence (SEQ ID NO: 193) and amino acid sequence (SEQ ID NO: 60) of the mature light chain variable (VL) region of anti-TIM3 monoclonal antibody 17C3 are shown. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66), and CDR3 (SEQ ID NO: 68) are underlined, and the V and J germline sequences are indicated.

[0131] [Figure 4C] The nucleotide sequence (sequence number 170) and amino acid sequence (sequence number 37) of the heavy chain VH region of anti-TIM3 monoclonal antibody 17C3 together with the signal sequence (sequence numbers 272 and 267, respectively) and the nucleotide sequence (sequence number 193) and amino acid sequence (sequence number 60) of the light chain VL region of anti-TIM3 monoclonal antibody 17C3 together with the signal sequence (sequence numbers 273 and 268, respectively) are shown.

[0132] [Figure 5A] The nucleotide sequence (SEQ ID NO: 171) and amino acid sequence (SEQ ID NO: 38) of the mature heavy chain variable (VH) region of anti-TIM3 monoclonal antibody 9F6 are shown. CDR1 (SEQ ID NO: 45), CDR2 (SEQ ID NO: 50), and CDR3 (SEQ ID NO: 57) are underlined, and the V, D, and J germline sequences are indicated.

[0133] [Figure 5B] The nucleotide sequence (SEQ ID NO: 195) and amino acid sequence (SEQ ID NO: 62) of the mature light chain variable (VL) region of VK1 of anti-TIM3 monoclonal antibody 9F6 are shown. CDR1 (SEQ ID NO: 65), CDR2 (SEQ ID NO: 67), and CDR3 (SEQ ID NO: 70) are underlined, and the V and J germline sequences are indicated.

[0134] [Figure 5C] The nucleotide sequence (SEQ ID NO: 196) and amino acid sequence (SEQ ID NO: 63) of the mature light chain variable (VL) region of VK2 of anti-TIM3 monoclonal antibody 9F6 are shown. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66), and CDR3 (SEQ ID NO: 71) are underlined, and the V and J germline sequences are indicated.

[0135] [Figure 5D] The nucleotide sequence (SEQ ID NO: 194) and amino acid sequence (SEQ ID NO: 61) of the mature light chain variable (VL) region of VK3 of anti-TIM3 monoclonal antibody 9F6 are shown. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66), and CDR3 (SEQ ID NO: 69) are underlined, and the V and J germline sequences are indicated.

[0136] [Figure 5E] The nucleotide sequence (sequence number 171) and amino acid sequence (sequence number 38) of the heavy chain VH region of VK1, VK2, and VK3 of anti-TIM3 monoclonal antibody 9F6, along with their signal sequences (sequence numbers 275 and 270, respectively), and the nucleotide sequence (sequence numbers 195, 196, and 194, respectively) and amino acid sequence (sequence numbers 62, 63, and 61, respectively) of the light chain VL region of anti-TIM3 monoclonal antibody 9F6, along with their signal sequences (sequence numbers 276 and 271, respectively) are shown.

[0137] [Figure 6A] The nucleotide sequence (SEQ ID NO: 172) and amino acid sequence (SEQ ID NO: 39) of the mature heavy chain variable (VH) region of anti-TIM3 monoclonal antibody 3G4 are shown. CDR1 (SEQ ID NO: 45), CDR2 (SEQ ID NO: 51), and CDR3 (SEQ ID NO: 58) are underlined, and the V, D, and J germline sequences are indicated.

[0138] [Figure 6B]The nucleotide sequence (SEQ ID NO: 193) and amino acid sequence (SEQ ID NO: 60) of the mature light chain variable (VL) region of anti-TIM3 monoclonal antibody 3G4 are shown. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66), and CDR3 (SEQ ID NO: 68) are underlined, and the V and J germline sequences are indicated.

[0139] [Figure 6C] The nucleotide sequence (sequence number 172) and amino acid sequence (sequence number 39) of the heavy chain VH region of anti-TIM3 monoclonal antibody 3G4 together with the signal sequence (sequence numbers 275 and 270, respectively) and the nucleotide sequence (sequence number 193) and amino acid sequence (sequence number 60) of the light chain VL region of anti-TIM3 monoclonal antibody 3G4 together with the signal sequence (sequence numbers 273 and 268, respectively) are shown.

[0140] [Figure 7A] The nucleotide sequence (SEQ ID NO: 173) and amino acid sequence (SEQ ID NO: 40) of the mature heavy chain variable (VH) region of anti-TIM3 monoclonal antibody 17C8 are shown. CDR1 (SEQ ID NO: 45), CDR2 (SEQ ID NO: 52), and CDR3 (SEQ ID NO: 59) are underlined, and the V, D, and J germline sequences are indicated.

[0141] [Figure 7B] The nucleotide sequence (SEQ ID NO: 194) and amino acid sequence (SEQ ID NO: 61) of the mature light chain variable (VL) region of anti-TIM3 monoclonal antibody 17C8 are shown. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66), and CDR3 (SEQ ID NO: 69) are underlined, and the V and J germline sequences are indicated.

[0142] [Figure 7C]The nucleotide sequence (sequence number 173) and amino acid sequence (sequence number 40) of the heavy chain VH region of anti-TIM3 monoclonal antibody 17C8 together with the signal sequence (sequence numbers 275 and 270, respectively) and the nucleotide sequence (sequence number 194) and amino acid sequence (sequence number 61) of the light chain VL region of anti-TIM3 monoclonal antibody 17C8 together with the signal sequence (sequence numbers 273 and 268, respectively) are shown.

[0143] [Figure 8A] The nucleotide sequence (SEQ ID NO:420) and amino acid sequence (SEQ ID NO:410) of the mature heavy chain variable (VH) region of anti-TIM3 monoclonal antibody 14H7 are shown. CDR1 (SEQ ID NO:45), CDR2 (SEQ ID NO:413), and CDR3 (SEQ ID NO:414) are underlined, and the V, D, and J germline sequences are indicated.

[0144] [Figure 8B] The nucleotide sequence (SEQ ID NO:427) and amino acid sequence (SEQ ID NO:417) of the mature light chain variable (VL) region of anti-TIM3 monoclonal antibody 14H7 are shown. CDR1 (SEQ ID NO:64), CDR2 (SEQ ID NO:66), and CDR3 (SEQ ID NO:69) are underlined, and the V and J germline sequences are indicated.

[0145] [Figure 8C] The nucleotide sequence (sequence number 420) and amino acid sequence (sequence number 410) of the heavy chain VH region of anti-TIM3 monoclonal antibody 14H7 together with the signal sequence (sequence numbers 362 and 361, respectively) and the nucleotide sequence (sequence number 427) and amino acid sequence (sequence number 417) of the light chain VL region of anti-TIM3 monoclonal antibody 14H7 together with the signal sequence (sequence numbers 363 and 361, respectively) are shown.

[0146] [Figure 9A]The nucleotide sequence (SEQ ID NO:421) and amino acid sequence (SEQ ID NO:411) of the mature heavy chain variable (VH) region of anti-TIM3 monoclonal antibody 23B3 are shown. CDR1 (SEQ ID NO:45), CDR2 (SEQ ID NO:415), and CDR3 (SEQ ID NO:416) are underlined, and the V, D, and J germline sequences are indicated.

[0147] [Figure 9B] The nucleotide sequence (SEQ ID NO: 193) and amino acid sequence (SEQ ID NO: 60) of the mature light chain variable (VL) region of VK1 of anti-TIM3 monoclonal antibody 23B3 are shown. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66), and CDR3 (SEQ ID NO: 68) are underlined, and the V and J germline sequences are indicated.

[0148] [Figure 9C] The nucleotide sequence (SEQ ID NO:428) and amino acid sequence (SEQ ID NO:418) of the mature light chain variable (VL) region of VK2 of the anti-TIM3 monoclonal antibody 23B3 are shown. CDR1 (SEQ ID NO:64), CDR2 (SEQ ID NO:66), and CDR3 (SEQ ID NO:419) are underlined, and the V and J germline sequences are indicated.

[0149] [Figure 9D] The nucleotide sequence (sequence number 421) and amino acid sequence (sequence number 411) of the heavy chain VH region of anti-TIM3 monoclonal antibody 23B3, along with the signal sequence (sequence numbers 362 and 361, respectively), and the nucleotide sequence (sequence numbers 193 and 428, respectively) and amino acid sequence (sequence numbers 60 and 418, respectively) of the light chain VL region of VK1 and VK2 of anti-TIM3 monoclonal antibody 23B3, along with the signal sequence (sequence numbers 363 and 361, respectively), are shown.

[0150] [Figure 10A] 1 shows a sequence alignment of the heavy chain variable (VH) regions of monoclonal antibodies 13A3, 8B9, 8C4, 17C3, 9F6, 3G4, 17C8, 23B3, and 14H7. Complementarity determining regions (CDRs) are boxed.

[0151] [Figure 10B] 1 is a list of the sequence numbers of the antibody VH region, each CDR, and variants thereof.

[0152] [Figure 11A] 1 shows a sequence alignment of the light chain variable (VL) regions of monoclonal antibodies 13A3, 8B9, 8C4, 17C3, 9F6_VK1, 9F6_VK2, 9F6_VK3, 3G4, 17C8, 23B3_VK1, 23B3_VK2, and 14H7. Complementarity determining regions (CDRs) are boxed.

[0153] [Figure 11B] 1 is a list of the VL regions of antibodies and the sequence numbers of each CDR.

[0154] [Figure 12-1] 1 shows a sequence alignment of the mature full-length heavy chains (HC) of monoclonal antibody TIM3.5 (13A3) and its exemplary variants: TIM3.13 (D101E), TIM3.14 (P102V), TIM3.15 (P102Y), TIM3.16 (P102L), TIM3.17 (N60Q / P102Y), TIM3.18 (N60Q / D101E), TIM3.10 (N60Q), TIM3.11 (N60S), and TIM3.12 (N60A). The VH region of each heavy chain is underlined. [Figure 12-2] Same as above.

[0155] [Figure 13] 1 shows a sequence alignment of the mature full-length HC of monoclonal antibody 9F6 and its exemplary variant TIM3.7(A108T). The VH region of each heavy chain is underlined.

[0156] [Figure 14] Figure 1 shows a sequence alignment of the mature full-length HC of monoclonal 8B9 and its exemplary variant TIM3.8(S61P). The VH region of each heavy chain is underlined.

[0157] [Figure 15] 1 shows a sequence alignment of the mature full-length HC of monoclonal antibody 23B3 and exemplary variant TIM3.25 (G6E / D79Y). The VH region of each heavy chain is underlined.

[0158] [Figure 16-1]

[0033] Figure 16 lists the SEQ ID NOs for the full-length heavy and light chains, variable regions, and CDRs of hybridoma-derived antibodies (13A3, 8B9, 8C4, 17C3, 9F6, 3G4, 17C8, 14H7, and 23B3) and recombinant (TIM3.2-TIM3.18, TIM3.24, and TIM3.25) anti-human TIM3 antibodies. The isotypes of the heavy and light chains are also shown. "Hn" is the hybridoma name. The heavy and light chains shown in Figure 16 can be derived from elements thereof, e.g., variable and constant regions, disclosed therein. When a SEQ ID NO is not in a column on page 2 or 3 of the table, it is provided in a column on the previous page or pages prior thereto. [Figure 16-2] Same as above. [Figure 16-3] Same as above. [Figure 16-4] Same as above.

[0159] [Figure 17A] Binding curves and EC50 values ​​of several anti-TIM3 antibodies to human TIM3-transfected CHO cells (Figure 17A) and activated human T cells (Figure 17B) are shown. In Figure 17A, the following anti-TIM3 antibodies are shown: 13A3, 17C3, 17C8, 3G4, 8B9, and 9F6. In Figure 17B, the following anti-TIM3 antibodies are shown: 13A3, 17C3, 17C8, 3G4, 8B9, 8C4, and 9F6. In both Figures 17A and 17B, isotype control antibodies human IgG1 (hG1), IgG2 (hG2), and IgG4 (hG4) were used as negative controls. [Figure 17B] Same as above.

[0160] [Figure 18]The binding curves and EC50 values ​​of the anti-TIM3 antibodies 14H7 and 23B3 for the anti-TIM3 antibody 13A3 binding to human TIM3-positive cells are compared. Isotype control antibodies IgG1, IgG1.1 (hG1.1), IgG2, and IgG4 were used as negative controls.

[0161] [Figure 19A] Binding analysis of anti-TIM3 antibodies to a cynomolgus monkey TIM3-transfected CHO cell line (Figure 19A) and activated cynomolgus monkey T cells (Figures 19B and 19C) is shown. In Figure 19A, binding curves and EC50 values ​​are provided for anti-TIM3 antibodies 13A3, 17C3, 17C8, 3G4, 8B9, and 9F6. In Figure 19B, binding curves and EC50 values ​​for anti-TIM3 antibody 13A3 are provided. In Figure 19C, 100 μg of relevant antibodies were tested for binding to activated cynomolgus monkey CD8+ T cells. Binding data are presented as mean fluorescence index (MFI). In Figures 19A-19C, relevant isotype control antibodies (hIgG1, hIgG2, and hIgG4) or no antibody treatment (No Ab) were used as negative controls. [Figure 19B] Same as above. [Figure 19C] Same as above.

[0162] [Figure 20] Flow cytometry histograms of the binding of anti-TIM3 antibodies 14H7 (left panel) and 23B3 (right panel) to CHO cells transfected with human TIM3 ("1") or cynomolgus monkey TIM3 ("2") are shown. CHO cells transfected with an isotype control antibody were used as a negative control ("3").

[0163] [Figure 21]Figure 1 shows anti-TIM3 activity (at various antibody concentrations, as indicated in the boxed legend) in promoting IFN-γ production from tumor-infiltrating leukocytes (TILs) in renal cell carcinoma (RCC). The eight bars for each antibody represent various concentrations of the indicated antibody. Data are shown for anti-TIM3 antibodies 13A3, 3G4, 17C3, 17C8, 8B9, and 9F6. An isotype control antibody was used as a control. The horizontal dotted line provides the background threshold level.

[0164] [Figure 22A] Anti-TIM3 activity (at various antibody concentrations, as indicated in the boxed legend) in promoting IFN-γ production from lung cancer TILs (Figure 22A, IFN-γ ELISA; Figure 22B, intracellular IFN-γ staining) is shown. In Figure 22A, individual bars for each antibody shown (13A3 and 3G4) represent various concentrations of the indicated antibody. In Figure 22B, the top panel shows CD4+ T cells, and the bottom panel shows CD8+ T cells. TIM3 levels were measured with 8B9 (x-axis). In Figure 22B, a non-TIM3-specific antibody (KLH-2F5-g4p) was used as a control. The x-axis shows TIM3 expression, and the y-axis shows IFN-γ expression. [Figure 22B] Same as above.

[0165] [Figure 23] Figure 1 shows the efficacy of anti-TIM3 antibodies (i.e., antibodies 13A3 and 3G4) in promoting IFN-γ secretion from TILs isolated from various patient tissues (i.e., kidney, pancreas, lung, and thyroid) and cultured in vitro with anti-TIM3 antibodies 13A3 or 3G4. IFN-γ secretion was measured in the presence of CHO-OKT3 cells. Data are presented as the fold increase over IFN-γ levels produced by TILs in the absence of anti-TIM3 antibodies.

[0166] [Figure 24]Figures 24A and 24B show cross-blocking data for anti-TIM3 antibodies on activated human T cells. In Figure 24A, anti-TIM3 antibodies 17C3 (dark gray), 8B9 (black), and 13A3 (light gray) are shown on activated human T cells preincubated with one of the following anti-TIM3 antibodies: 3G4, 8B9, 9F6, 17C3, 17C8, and 8C4. In Figure 24B, activated human T cells were preincubated with 14H7 or 23B3 antibody, and then binding of the 13A3 antibody was measured. In both Figures 24A and 24B, the geometric mean fluorescence intensity of binding of anti-TIM3 antibodies to activated human T cells is provided.

[0167] [Figure 25] The amino acid residues essential for binding of anti-TIM3 monoclonal antibodies 13A3, 3G4, 17C3, and 8B9 to human TIM3 are shown, with the signal sequence and transmembrane domains underlined.

[0168] [Figure 26-1] Figures 26A and B show that certain anti-TIM3 antibodies block the interaction of human TIM3 with PS-liposomes. Figure 26A shows a schematic diagram of the phosphatidylserine (PS)-hTIM3 "in tandem" blocking assay. Figure 26B shows the blockade of hTIM3-Fc binding to PS-liposomes by certain anti-TIM3 antibodies, as measured in the PS-hTIM3 "in tandem" blocking assay shown in Figure 26A. The top row shows data for anti-TIM3 antibodies 3G4, 13A3, 17C3, and 17C8. The bottom row shows data for anti-TIM3 antibodies 8B9, 8C4, and 9F6. The PS response time and whether hTIM3-Fc binds to PS-liposomes are provided in the table to the right of each graph. [Figure 26-2] Same as above. [Figure 26-3] Same as above.

[0169] [Figure 27]Figure 1 shows a summary of the functional activities of various anti-TIM3 antibodies (e.g., TIM3.5 (13A3), TIM3.4 (3G4), TIM3.2 (17C3), TIM3.9 (17C8), 9F6, TIM3.8 (8B9 with an S61P substitution in the VH), and TIM3.6 (8C4). Data from binding assays, T cell assays, TIL assays, and PS-TIM3 blocking assays are provided.

[0170] [Figure 28-1] A list of all SEQ ID NOs is provided, along with a description of the sequences represented by the SEQ ID NOs. [Figure 28-2] Same as above. [Figure 28-3] Same as above. [Figure 28-4] Same as above. [Figure 28-5] Same as above. [Figure 28-6] Same as above. [Figure 28-7] Same as above. [Figure 28-8] Same as above. [Figure 28-9] Same as above. [Figure 28-10] Same as above. [Figure 28-11] Same as above. [Figure 28-12] Same as above. [Figure 28-13] Same as above. [Figure 28-14] Same as above. [Figure 28-15] Same as above.

[0171] [Figure 29A]Figure 29A shows the antitumor activity of combined administration of anti-PD1 and anti-TIM3 antibodies in a CT26 colorectal tumor mouse model. Figure 29A shows tumor volumes at various time points after tumor implantation in mice (n=10 / group) treated with (i) control IgG (upper left panel), (ii) RMT3-23 anti-TIM3 antibody alone (upper right panel), (iii) RMP1-14 anti-PD1 antibody alone (lower left panel), and (iv) the combination of RMT3-23 anti-TIM3 and RMP1-14 anti-PD1 antibodies (lower right panel). Figure 29B shows the mean tumor volume as a function of time (days after tumor implantation) for mice treated with (i) RMT3-23 anti-TIM3 antibody alone, (ii) AbM anti-TIM3 antibody alone, (iii) RMP1-14 anti-PD1 antibody alone, (iv) the combination of RMT3-23 anti-TIM3 and RMP1-14 anti-PD1 antibodies, (v) the combination of AbM anti-TIM3 and RMP1-14 anti-PD1 antibodies, and (vi) an isotype control antibody. [Figure 29B] Same as above.

[0172] [Figure 30] Figure 1 shows a list of common peptides of hTIM3 used to map the epitopes of anti-TIM3 antibodies (13A3 and 3G4) using hydrogen / deuterium exchange mass spectrometry (HDX-MS). Each bar represents a digested peptide. Circled residues (i.e., N99, T145, and N172) indicate glycosylation sites.

[0173] [Figure 31] Figure 1 shows the human TIM3-binding regions of anti-TIM3 antibodies (13A3 and 3G4) identified using HDX-MX. The top panel shows the binding region of the 13A3 anti-TIM3 antibody. The bottom panel shows the binding region of the 3G4 anti-TIM3 antibody.

[0174] [Figure 32A]Figure 32 shows the results of a Scatchard analysis of TIM3.18.IgG1.3 binding to CHO cells ectopically expressing human or cynomolgus monkey TIM3. Figure 32A shows the I-TIM3 Ab standard curve. Figure 32B shows the amount of TIM3.18.IgG1.3 antibody binding to human (left panel) and cynomolgus monkey (right panel) TIM3-expressing CHO cells. Below each graph, estimated affinity values ​​and the number of TIM3 molecules expressed on the CHO cell surface are provided. These values ​​were calculated as described in Example 4. [Figure 32B] Same as above.

[0175] [Figure 33] Scatchard analysis of TIM3.18.IgG1.3 binding to activated Th1 cells from two separate donors (left and right panels) is shown. Estimated affinity values ​​and the number of TIM3 molecules expressed on the CHO cell surface are shown below each graph. These values ​​were calculated as described in Example 4.

[0176] [Figure 34A] Figure 34A shows that TIM3.18.IgG1.3 and TIM3.18.IgG1.3 Fab enhanced the proliferation of Th1 T cells in a polarized Th1 / irradiated CHO-OKT3 co-culture assay. Figure 34A shows the Th1 cell proliferation observed with various concentrations of TIM3.18.IgG1.3, 13A3 ("13A3-g4"), no antibody, or isotype control antibody (hIgG1.1 or hIgG4). Each bar for the antibody represents a different concentration. Figure 34B shows the Th1 cell proliferation observed with various concentrations of TIM3.18.IgG1.3 Fab, no antibody ("no ab"), or isotype control antibody IgG1.3 ("G1.3"). Each bar for the TIM3.18 antibody represents a different concentration. [Figure 34B] Same as above.

[0177] [Figure 35]Figure 1 shows TIM3.24- and TIM3.25-mediated enhancement of Th1 T cell proliferation in a polarized Th1 / irradiated CHO-OKT3 co-culture assay. The TIM3.18 antibody was included for comparison purposes. CHO cells not treated with any antibody ("No Ab") or treated with the isotype control IgG1.3 antibody ("IgG1.3") were used as negative controls. The horizontal dotted line indicates proliferation above the negative control.

[0178] [Figure 36] Figure 1 shows that the anti-TIM3 antibody TIM3.18.IgG1.3 (closed circles) in combination with nivolumab enhanced Th1 T cell proliferation in a polarized Th1 / irradiated CHO-OKT3-PD-L1 co-culture assay. CHO cells untreated with either antibody (open squares) or treated with the isotype control IgG1.3 antibody (grey squares) were used as negative controls.

[0179] [Figure 37] Figure 1 shows that the anti-TIM3 antibody TIM3.18.IgG1.3 enhanced interferon-γ secretion from renal cell carcinoma tumor-infiltrating lymphocytes (TILs) after stimulation with irradiated CHO-OKT3 cells. TILs stimulated with CHO in the presence of isotype control antibodies (hIgG4 or hIgG1.1f) or in the absence of complete antibody were used as negative controls. Each bar of the TIM3.18 antibody represents a different concentration of the antibody.

[0180] [Figure 38] Figure 1 shows that the anti-TIM3 antibody TIM3.18.IgG1.3 enhanced interferon-γ secretion in breast cancer TILs stimulated with irradiated CHO-OKT3 cells. Assays with no antibody or an isotype control antibody were used as controls. The horizontal dotted line indicates interferon-γ secretion above the negative control.

[0181] [Figure 39]Histograms of CD163, CD206, and TIM3 expression on M0 macrophages used in an AlloMLR (mixed lymphocyte reaction) assay are shown, the results of which are shown in Figure 40. Each histogram shows the fluorescence intensity of (1) CD163, CD206, or TIM3, (2) an isotype control antibody (i.e., negative control), and (3) background fluorescence ("unstained") (i.e., negative control).

[0182] [Figure 40] Cell proliferation in an AlloMLR assay performed in the presence of anti-TIM3 antibody TIM3.18.IgG1.3, isotype control, or in the absence of antibody is shown.

[0183] [Figure 41] A ribbon diagram of the structure of the TIM3:TIM3.18 Fab complex, as determined by crystallography, is shown, with the Fab fragment in light grey and TIM3 in dark grey.

[0184] [Figure 42] Figure 1 shows the structure of the TIM3:TIM3.18 Fab complex, as determined by crystallography. The Fab fragment is shown as a ribbon diagram. TIM3 is shown in white surface representation, and Fab contact residues are marked in dark grey.

[0185] [Figure 43]

[0023] Figure 1 is a diagram of the assay used to measure potential internalization by anti-TIM3 antibodies. The left panel shows a schematic overview of the assay. The right panel describes the calculation of internalization values.

[0186] [Figure 44] Anti-TIM3 antibody 13A3 (lower left panel) and certain of its variants (D101E - upper left panel; N60Q - upper right panel) do not induce receptor (ie, TIM3)-mediated internalization.

[0187] [Figure 45]Figures 45A and B show ribbon diagrams describing the epitopes of anti-TIM3 antibodies 13A3 (Figure 45A) and 3G4 (Figure 45B). The amino acid sequences of each epitope of the antibodies are provided below the ribbon diagrams. Different patterns identify specific regions of the anti-TIM3 antibodies that correspond to specific epitopes. DETAILED DESCRIPTION OF THE INVENTION

[0188] Detailed Description of the Invention In order that this specification may be more readily understood, certain terms are first defined. Further definitions are set forth throughout the detailed description.

[0189] It is understood that singular terms refer to one or more of that thing; for example, a "nucleotide sequence" is understood to refer to one or more nucleotide sequences. That is, the terms "a," "one or more," and "at least one" can be used interchangeably herein.

[0190] Furthermore, "and / or," as used herein, is to be construed as a specific disclosure of each of the two specified properties or components, with or without the other. Thus, the term "and / or" used herein in a phrase such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone) and "B" (alone). Similarly, the term "and / or" used in a phrase such as "A, B and / or C" is intended to include any of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0191] Wherever embodiments are described herein using the term "comprising," it is understood that similar embodiments other than those using the terms "consisting of" and / or "consisting essentially of" are also provided.

[0192] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used herein.

[0193] Units, prefixes, and symbols are written in the form accepted by the International System of Units (SI). Numerical ranges are inclusive of the numbers defining the range. Unless otherwise specified, nucleotide sequences are written left to right in 5' to 3' orientation. Amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of this disclosure, which may be obtained in light of the specification as a whole. Accordingly, the terms defined immediately below are more fully defined in light of the specification as a whole.

[0194] The term "about" is used herein to mean approximately, roughly, in the vicinity of, or within a range. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the numerical values ​​set forth. In general, the term "about" can modify a numerical value above or below the stated value by, for example, a 10 percent above or below (higher or lower) variance.

[0195] As used herein, the term "T cell immunoglobulin and mucin domain-containing 3" or "TIM3" refers to a receptor that is a member of the T cell immunoglobulin and mucin domain (TIM) family of proteins. The primary ligand for TIM3 contains phosphatidylserine (TIM3-L). TIM3 is also referred to as hepatitis A virus cellular receptor 2 (HAVCR2), T cell immunoglobulin mucin receptor 3, TIM-3, TIMD3, TIMD-3, kidney injury molecule-3, KIM-3, and CD366. The term "TIM3" includes any variant or isoform of TIM3 naturally expressed by cells. Thus, the antibodies described herein can cross-react with TIM3 from species other than humans (e.g., cynomolgus monkey TIM3). Alternatively, the antibodies are specific for human TIM3 and do not show any cross-reactivity with other species. TIM3 or any variants and isoforms thereof can be isolated from cells or tissues that naturally express it or can be produced recombinantly using techniques well known in the art and / or as described herein.

[0196] Two isoforms of human TIM3 have been identified. Isoform 1 (Accession No. NP_116171; SEQ ID NO: 286) consists of 301 amino acids and represents the reference sequence. Isoform 2 (Accession No. AAH20843; SEQ ID NO: 287) consists of 142 amino acids and is soluble. It lacks amino acid residues 143-301, which encode the transmembrane, cytoplasmic, and part of the extracellular domain of TIM3. Amino acid residues 132-142 also differ from the reference sequence.

[0197] Below are the amino acid sequences of the two known human TIM3 isoforms. (A) Human TIM3 isoform 1 (Accession No. NP_116171; SEQ ID NO: 286; encoded by the nucleotide sequence having Accession No. NM_032782.4; SEQ ID NO: 288; Figure 25): [ka] (B) Human TIM3 isoform 2 (Accession No. AAH20843; SEQ ID NO: 287; encoded by the nucleotide sequence having Accession No. BC020843.1; SEQ ID NO: 289): [ka]

[0198] The signal sequences of isoforms 1 and 2 correspond to amino acids 1 to 21 (underlined). Thus, mature isoforms 1 and 2 consist of amino acids 22 to 301 or 142, respectively. The extracellular domain of mature human TIM3 consists of amino acids 22 to 202 of SEQ ID NO: 286 and has the following amino acid sequence: [ka] (SEQ ID NO: 290).

[0199] The cynomolgus monkey TIM3 protein consists of the following amino acid sequence (including the signal sequence): [ka] (SEQ ID NO: 360)

[0200] The term "antibody," in some embodiments, refers to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). In some antibodies, such as naturally occurring IgG antibodies, the heavy chain constant region consists of a hinge and three domains, CH1, CH2, and CH3. In some antibodies, such as naturally occurring IgG antibodies, each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), separated by more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The heavy chain may or may not have a C-terminal lysine. Unless otherwise specified herein, amino acids in the variable regions are numbered using the Kabat numbering system, and those in the constant regions are numbered using the EU system.

[0201] As used herein, "IgG antibodies," e.g., human IgG1, IgG2, IgG3, and IgG4 antibodies, in some embodiments, have the structure of naturally occurring IgG antibodies, i.e., have the same number of heavy and light chains and disulfide bonds as naturally occurring IgG antibodies of the same subclass. For example, an anti-TIM3 IgG1, IgG2, IgG3, or IgG4 antibody consists of two heavy chains (HC) and two light chains (LC), in which the two HCs and LCs are linked by the same number and positions of disulfide bridges as present in naturally occurring IgG1, IgG2, IgG3, and IgG4 antibodies, respectively (unless the antibody has been mutated to remove disulfide bridges).

[0202] Antibodies are generally -5 ~10 -11 The dissociation constant (K D ) specifically binds to its cognate antigen with high affinity, reflected by approximately 10 -4 Any K greater than M D As used herein, an antibody that "specifically binds" to an antigen is generally considered to represent non-specific binding. -7 M or less, 10 -8 M or less, 5×10 -9 M or less or 10 -8 M~10 -10 K below M D "Substantially identical" refers to an antibody that binds to an antigen and a substantially identical antigen with high affinity, meaning that it does not bind to unrelated antigens with high affinity. An antigen is "substantially identical" to a given antigen if it shows a high degree of sequence identity with the antigen, for example, if it shows at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the sequence of the given antigen. For example, an antibody that specifically binds to human TIM3 may, in some embodiments, cross-react with TIM3 antigens from certain primate species (e.g., cynomolgus monkey TIM3), but not cross-react with TIM3 antigens from other species or antigens other than TIM3.

[0203] Immunoglobulins can be derived from any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. The IgG isotype is divided into subclasses in certain species: IgG1, IgG2, IgG3, and IgG4b in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. In some embodiments, the anti-TIM3 antibodies described herein are of the IgG1 subtype. Immunoglobulins, such as IgG1, exist in several allotypes, differing from each other by at most a few amino acids. "Antibody" includes, by way of example, both naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies; and completely synthetic antibodies.

[0204] As used herein, the term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human TIM3). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody, e.g., the anti-TIM3 antibodies described herein, include (i) a Fab fragment (a fragment resulting from papain cleavage) or a V L , V H (ii) a F(ab')2 fragment (a fragment resulting from pepsin cleavage) or a similar bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V H and an Fd fragment consisting of the CH1 domain; (iv) a V of a single arm of an antibody; L and V H Fv fragment consisting of domains, (v) V H (vi) isolated complementarity-determining regions (CDRs); and (vii) combinations of two or more isolated CDRs, optionally linked by synthetic linkers. In addition, Fv fragments, V L and V H The two domains may be encoded by separate genes, but may be linked using recombinant methods by a synthetic linker, such that they form a monovalent molecule. L and V HThese antibody fragments can be produced as a single protein chain in which the paired regions form a single Fv (scFv), known as a single-chain Fv (see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. These antibody fragments may be produced using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding portions may be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

[0205] A "bispecific" or "bifunctional antibody" is an artificial hybrid antibody having two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

[0206] As used herein, the term "monoclonal antibody" refers to an antibody from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are substantially similar and bind to the same epitope (e.g., the antibody exhibits a single binding specificity and affinity), except for possible variants that may arise during the production of the monoclonal antibody (such variants are generally present in minor amounts). The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population and is not to be construed as requiring production of the antibody by any particular method. The term "human monoclonal antibody" refers to an antibody from a substantially homogeneous antibody population that exhibits a single binding specificity and has variable and optionally constant regions derived from human germline immunoglobulin sequences. In certain embodiments, human monoclonal antibodies are produced by hybridomas, which include B cells obtained from a transgenic non-human animal, e.g., a transgenic mouse, whose genome comprises human heavy chain and light chain transgenes fused to an immortalized cell.

[0207] As used herein, the term "recombinant human antibody" includes fully human antibodies produced, expressed, generated, or isolated by recombinant means, e.g., (a) antibodies isolated from or hybridomas produced by animals (e.g., mice) that are transgenic or transchromosomal for human immunoglobulin genes, (b) antibodies isolated from host cells, e.g., transfectomas, transformed to express the antibody, (c) antibodies isolated from recombinant, combinatorial human antibody libraries, and (d) antibodies produced, expressed, generated, or isolated by any other means, including splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies contain variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but include subsequent rearrangements and mutations that occur, e.g., during antibody maturation. As is known from the literature (see, for example, Lonberg (2005) Nature Biotech. 23(9): 1117-1125), the variable region contains the antigen-binding domain, which is encoded by various genes that are rearranged to form an antibody specific to a certain foreign antigen. In addition to rearrangement, the variable region can be further modified by multiple single changes (called somatic mutation or hypermutation) to increase the affinity of the antibody to the foreign antigen. The constant region further changes the response to the antigen (i.e., isotype switching). Therefore, the rearranged and somatically mutated nucleic acid molecule encoding the light and heavy chain immunoglobulin polypeptides in response to the antigen may not have sequence identity with the original nucleic acid molecule, but instead is substantially identical or similar (i.e., has at least 80% identity).

[0208] A "human" antibody (HuMAb) refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The anti-TIM3 antibodies described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of other mammalian species, such as a mouse, have been grafted onto human framework sequences. The terms "human" antibody and "fully human" antibody are used interchangeably.

[0209] A "humanized" antibody refers to an antibody in which some, most, or all of the amino acids outside the CDR domains of a non-human antibody have been replaced with corresponding amino acids from a human immunoglobulin. In some embodiments of humanized forms of antibodies, some, most, or all of the amino acids outside the CDR domains are replaced with amino acids from a human immunoglobulin, while some, most, or all of the amino acids within one or more CDR regions remain unchanged. Small additions, deletions, insertions, substitutions, or modifications of amino acids are permissible as long as they do not eliminate the ability of the antibody to bind to a specific antigen. A "humanized" antibody retains antigen specificity similar to that of the original antibody.

[0210] A "chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, such as when the variable region is derived from a mouse antibody and the constant region is derived from a human antibody.

[0211] As used herein, "isotype" refers to the antibody class (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies) encoded by heavy chain constant region genes.

[0212] "Allotype" refers to naturally occurring variants within a particular isotype group, where the variants differ by several amino acids (see, e.g., Jefferis et al. (2009) mAbs 1:1). The anti-TIM3 antibodies described herein can be of any allotype. As used herein, antibodies designated as "IgG1f," "IgG1.1f," or "IgG1.3f" isotypes are IgG1, effectorless IgG1.1, and effectorless IgG1.3 antibodies, respectively, of allotype "f," i.e., having 214R, 356E, and 358M according to the EU index as in Kabat, as shown, for example, in SEQ ID NO: 3.

[0213] The terms "antibody that recognizes an antigen" and "antigen-specific antibody" are used interchangeably with the term "antibody that specifically binds to an antigen."

[0214] As used herein, an "isolated antibody" refers to an antibody that is substantially free of other proteins and cellular material.

[0215] As used herein, an antibody that "inhibits the binding of TIM3-L to TIM3" is intended to refer to an antibody that inhibits the binding of TIM3 to its ligand, e.g., phosphatidylserine, in a binding assay using, for example, CHO cells transfected with human TIM3 or TIM3-expressing activated T cells, and has a binding concentration of about 1 μg / mL or less, e.g., about 0.9 μg / mL or less, about 0.85 μg / mL or less, in an art-recognized method, e.g., a FACS-based binding assay described herein. EC50 of about 0.8 μg / mL or less, about 0.75 μg / mL or less, about 0.7 μg / mL or less, about 0.65 μg / mL or less, about 0.6 μg / mL or less, about 0.55 μg / mL or less, about 0.5 μg / mL or less, about 0.45 μg / mL or less, about 0.4 μg / mL or less, about 0.35 μg / mL or less, about 0.3 μg / mL or less, about 0.25 μg / mL or less, about 0.2 μg / mL or less, about 0.15 μg / mL or less, about 0.1 μg / mL or less, or about 0.05 μg / mL or less 50 It is an antibody that shows

[0216] A "effector function" refers to the interaction of an antibody Fc region with an Fc receptor or ligand, or the biochemical events resulting therefrom. Examples of "effector functions" include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, FcγR-mediated effector functions such as ADCC and antibody-dependent cellular phagocytosis (ADCP), and cell surface receptor (e.g., B cell receptor; BCR) downregulation. Such effector functions generally require the Fc region to associate with a binding domain (e.g., an antibody variable domain).

[0217] An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind IgG antibodies include receptors of the FcγR family, including allelic variants, or alternatively spliced ​​forms of these receptors. The FcγR family consists of three activating (FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory (FcγRIIB) receptor. The various properties of human FcγRs are known in the art. Most innate effector cell types co-express one or more activating FcγRs and the inhibitory FcγRIIB, whereas natural killer (NK) cells selectively express one activating Fc receptor (FcγRIII in mice and FcγRIIIA in humans) and do not express the inhibitory FcγRIIB in mice or humans. Human IgG1 binds to most human Fc receptors and is considered equivalent to mouse IgG2a in the types of activating Fc receptors it binds.

[0218] "Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates immunoglobulin binding to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or the first component (C1q) of the classical complement system. Thus, the Fc region includes the constant region of an antibody excluding the first constant region immunoglobulin domain (e.g., CH1 or CL). In IgG, IgA, and IgD antibody isotypes, the Fc region contains two identical protein fragments derived from the second (CH2) and third (CH3) constant domains of the heavy chain; IgM and IgE Fc regions contain three heavy chain constant domains (CH domains 2-4) on each polypeptide chain. For IgG, the Fc region includes immunoglobulin domains CH2 and CH3 and the hinge between the CH1 and CH2 domains. As defined herein, the boundaries of the Fc region of an immunoglobulin heavy chain vary, but the human IgG heavy chain Fc region stretches from amino acid residue D221 for IgG1, from V222 for IgG2, from L221 for IgG3, and from P224 for IgG4 to the carboxy terminus of the heavy chain, where numbering is according to the EU index as in Kabat. The CH2 domain of the human IgG Fc region stretches from amino acid 237 to amino acid 340, and the CH3 domain is located C-terminal to the CH2 domain in the Fc region, i.e., stretches from amino acid 341 to amino acid 447 or 446 (if the C-terminal lysine residue is absent) or 445 (if the C-terminal glycine and lysine residues are absent) of IgG. As used herein, an Fc region can be a native-sequence Fc, including any allotypic variant or variant Fc (e.g., a non-naturally occurring Fc). Fc also includes this region in the context of an Fc-containing protein polypeptide, such as an "Fc region-containing binding protein," either isolated or referred to as an "Fc fusion protein" (eg, an antibody or immunoadhesin).

[0219] A "native sequence Fc region" or "native sequence Fc" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions; native sequence human IgG2 Fc regions; native sequence human IgG3 Fc regions; and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof. Native sequence Fc includes the various allotypes of Fc (see, e.g., Jefferis et al. (2009) mAbs 1:1).

[0220] The term "epitope" or "antigenic determinant" refers to a site on an antigen (e.g., TIM3) to which an immunoglobulin or antibody specifically binds, as defined, for example, by the specific method used for identification. Epitopes can be formed both from contiguous amino acids (usually linear epitopes) or from noncontiguous amino acids juxtaposed by tertiary folding of a protein (usually conformational epitopes). Epitopes formed from contiguous amino acids are generally, but not always, retained by exposure to denaturing solvents, whereas epitopes formed by tertiary folding are generally lost by treatment with denaturing solvents. Epitopes generally include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods for determining which epitope is bound by an antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblot and immunoprecipitation assays, in which overlapping or adjacent peptides (e.g., from TIM3) are tested for reactivity with an antibody (e.g., an anti-TIM3 antibody). Methods for determining the spatial conformation of epitopes include techniques known in the art and described herein, for example, x-ray crystallography, antigenic variation analysis, two-dimensional nuclear magnetic resonance, and HDX-MS (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996)).

[0221] The term "epitope mapping" refers to the process of identifying molecular determinants for antibody-antigen recognition.

[0222] The term "binds to the same epitope" in reference to two or more antibodies means that these antibodies bind to the same segment of amino acid residues, as determined by some method. Techniques for determining whether an antibody binds to the "same epitope of TIM3" as an antibody described herein include epitope mapping methods, such as x-ray analysis of crystals of antigen:antibody complexes, which provide atomic resolution of the epitope, and hydrogen / deuterium exchange mass spectrometry (HDX-MS). Another method monitors the binding of antibodies to antigen fragments or mutational variations of the antigen, where loss of binding due to amino acid residue modification within the antigen sequence is often considered to be indicative of epitope components. Additionally, computational combinatorial methods of epitope mapping can also be used. These methods utilize the ability of the antibody of interest to affinity isolate specific short peptides from combinatorial phage-displayed peptide libraries. Antibodies with the same VH and VL or the same CDR1, 2, and 3 sequences are predicted to bind to the same epitope.

[0223] An antibody that "competes with another antibody for binding to a target" refers to an antibody that inhibits (partially or completely) the binding of the other antibody to the target. Whether two antibodies compete with each other for binding to a target, i.e., whether and to what extent one antibody inhibits the binding of the other antibody to the target, can be determined using known competition experiments, e.g., BIACORE®. (登録商標)This can be determined using surface plasmon resonance (SPR) analysis. In some embodiments, an antibody competes and inhibits the binding of another antibody to a target by at least 50%, 60%, 70%, 80%, 90%, or 100%. The level of inhibition or competition can vary depending on which antibody is the "blocking antibody" (i.e., the cold antibody that is first incubated with the target). Competition assays can be performed, for example, as described in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi: 10.1101 / pdb.prot4277 or in Chapter 11 of "Using Antibodies" by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999. Two antibodies "cross-compete" if they block each other in both directions, i.e., by at least 50%, regardless of whether one or the other antibody is first contacted with the antigen in a competition experiment.

[0224] Competitive binding assays to determine whether two antibodies compete or cross-compete for binding include competition for binding to TIM3-expressing T cells by, for example, flow cytometry, as described in the Examples. Other methods include SPR (e.g., BIACORE (登録商標)), solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli et al., Methods in Enzymology 9:242 (1983)); solid-phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid-phase direct label assay, solid-phase direct label sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid-phase direct label RIA using 1-125 label (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid-phase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and direct label RIA (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

[0225] As used herein, the terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to an antibody that binds to an epitope of a predetermined antigen. Generally, an antibody is (i) assayed using, for example, a BIACORE assay using a predetermined antigen, e.g., recombinant human TIM3, as the analyte and an antibody as the ligand. (登録商標) Approximately 10 as determined by surface plasmon resonance (SPR) technology on a 2000 instrument or Scatchard analysis of antibody binding to antigen-positive cells. -7 Less than M, e.g., about 10 -8 M, 10 -9 M or 10 -10 Equilibrium dissociation constant (K D ) and (ii) bind to the predetermined antigen with an affinity at least 2-fold greater than its affinity for binding to a non-specific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein). Thus, an antibody that "specifically binds to TIM3" binds to soluble or cell-bound human TIM3 with an affinity of at least 10 times greater than its affinity for binding to a non-specific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein).-7 M or less, for example, about 10 -8 M, 10 -9 M or 10 -10 K less than M or lower D An antibody that "cross-reacts with cynomolgus monkey TIM3" refers to an antibody that binds to cynomolgus monkey TIM3 at 10 -7 M or less, for example, about 10 -8 M, 10 -9 M or 10 -10 K less than M or lower D In certain embodiments, such antibodies that do not cross-react with TIM3 from non-human species exhibit essentially no detectable binding to these proteins in standard binding assays.

[0226] The term "k" used here assoc " or "k a " is intended to refer to the binding rate of a particular antibody-antigen interaction, while the term "k dis " or "k d " is intended to refer to the off-rate of a particular antibody-antigen interaction. As used herein, the term "K D " is intended to refer to the dissociation constant, which is k d vs. k a ratio (i.e., k d / k a ) and expressed as molar concentration (M). D The K value can be determined using methods well established in the art. D Available methods for the determination of (登録商標) Biosensor systems such as flow cytometry and Scatchard analysis.

[0227] As used herein, the term "high affinity" with respect to IgG antibodies refers to antibodies with a high affinity of 10 -8 M or less, 10 -9 M or less or 10 -10 K below M DHowever, "high affinity" binding may vary for other antibody isotypes. For example, "high affinity" binding for an IgM isotype is defined as 10 -10 M or less or 10 -8 K below M D An antibody having the following structure:

[0228] The term "EC" in the context of in vitro or in vivo assays using antibodies or antigen-binding fragments thereof 50 " refers to the concentration of an antibody or antigen-binding portion thereof that induces 50% of the maximal response, i.e., a response that is half the maximal response and half the baseline.

[0229] As used herein, the term "naturally occurring" in reference to an object means that the object is found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a natural source and has not been intentionally manipulated by humans in the laboratory is naturally occurring.

[0230] A "polypeptide" refers to a chain comprising at least two consecutively linked amino acid residues, with no upper limit on the chain length. One or more amino acid residues in a protein may contain modifications, such as, but not limited to, glycosylation, phosphorylation, or disulfide bond formation. A "protein" may include one or more polypeptides.

[0231] As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, and may be cDNA.

[0232] "Conservative amino acid substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, and include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, a predicted non-essential amino acid residue in an anti-TIM3 antibody is replaced with another amino acid residue from the same side chain family. Methods for identifying conservative nucleotide and amino acid substitutions that do not eliminate antigen binding are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0233] With respect to nucleic acids, the term "substantial homology" indicates that two nucleic acids or designated sequences thereof, with appropriate nucleotide insertions or deletions, when optimally aligned and compared, are identical in at least about 80% of the nucleotides, at least about 90%-95% of the nucleotides, or at least about 98%-99.5% of the nucleotides. Alternatively, substantial homology exists when the segments hybridize under selective hybridization conditions to a complementary strand.

[0234] With respect to polypeptides, the term "substantial homology" indicates that two polypeptides or designated sequences thereof, with appropriate amino acid insertions or deletions, when optimally aligned and compared, are identical in at least about 80% of the amino acids, at least about 90%-95% of the amino acids, or at least about 98%-99.5% of the amino acids.

[0235] The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions x 100), taking into account the number of gaps and the length of each gap that needed to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

[0236] The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at worldwideweb.gcg.com) using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity of two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4: 11-17 (1989)) incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm incorporated into the GAP program of the GCG software package (available at http: / / www.gcg.com) using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0237] The nucleic acid and protein sequences described herein can further be used as "query sequences" to search public databases, for example, to identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program at a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program at a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See worldwideweb.ncbi.nlm.nih.gov.

[0238] Nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. Nucleic acids are "isolated" or "made substantially pure" when they have been purified away from cellular components or other contaminants, such as other cellular nucleic acids (e.g., other chromosomal moieties) or proteins, by standard techniques, including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. See F. Ausubel, et al., ed., Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

[0239] Nucleic acids, e.g., cDNA, can be mutated by standard techniques to provide gene sequences. For coding sequences, these mutations can affect the amino acid sequence, as desired. In particular, DNA sequences that are substantially homologous to or derived from the naturally occurring V, D, J, constant, switch, and other such sequences described herein are contemplated (where "derived" indicates that the sequence is identical to or modified from another sequence).

[0240] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the host cell genome upon introduction into a host cell, and thereby be replicated co-introduced with the host genome. Moreover, some vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions are also included.

[0241] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell containing nucleic acid that is not naturally occurring in the cell, and may be a cell into which a recombinant expression vector has been introduced. It should be understood that such terms include not only the particular subject cell but also the progeny of such a cell. Some modifications may occur in progeny due to mutation or environmental influences, and in fact, such progeny may not be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0242] "Immune response" is understood in the art and generally to refer to a biological response in a vertebrate to foreign agents or abnormalities, e.g., cancer cells, which protects the organism from these agents and the diseases they cause. The immune response is mediated by the action of one or more cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by these cells or the liver, which results in the selective targeting, binding, damage, destruction, and / or elimination in the mammalian body of invading pathogens, pathogen-infected cells or tissues, cancer or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues. The immune response can be mediated by, for example, T cells, e.g., effector T cells, Th cells, CD4 + cells, CD8 + This includes the activation or inhibition of T cells or Treg cells or any other cells of the immune system, for example NK cells.

[0243] An "immunomodulator" or "immunoregulator" refers to an agent, e.g., an agent that targets a component of a signaling pathway that may be involved in regulating, controlling, or modifying an immune response. "Modulation," "control," or "modulation" of an immune response refers to any alteration of immune system cells or the activity of such cells (e.g., effector T cells, such as Th1 cells). Such modulation includes stimulation or suppression of the immune system, which may be achieved by increasing or decreasing the number of various cell types, increasing or decreasing the activity of these cells, or any other change that may occur within the immune system. Both inhibitory and stimulatory immunomodulators have been identified, some of which may have enhanced function in the tumor microenvironment. In certain embodiments, an immunomodulator targets a molecule on the surface of T cells. An "immunomodulatory target" or "immunoregulatory target" is a molecule, e.g., a cell surface molecule, that can be targeted for binding by a substance, agent, moiety, compound, or molecule, and whose activity is altered upon binding. Immunomodulatory targets include, for example, cell surface receptors ("immunomodulatory receptors") and receptor ligands ("immunomodulatory ligands").

[0244] "Immunotherapy" refers to the treatment of a subject having a disease or at risk of developing or recurring with methods involving eliciting, enhancing, suppressing or otherwise modifying the immune system or immune response.

[0245] "Immunostimulatory therapy" or "immunostimulatory therapy" refers to a therapy that results in an increase (induction or enhancement) of a subject's immune response, for example, to treat cancer.

[0246] "Enhancing an endogenous immune response" means increasing the efficacy or potency of the immune response present in a subject. This increased efficacy and potency can be achieved, for example, by overcoming mechanisms that suppress the endogenous host immune response or by stimulating mechanisms that enhance the endogenous host immune response.

[0247] "T Effector" ("T eff ") cells are T cells with cytolytic activity (e.g., CD4 + and CD8 +The term "anti-TIM3" refers to T cells, as well as T helper (Th) cells, e.g., Th1 cells, which secrete cytokines and activate and direct other immune cells, but does not include regulatory T cells (Treg cells). Certain anti-TIM3 antibodies described herein inhibit T eff Cells, e.g., CD4 + and CD8 + T eff Activates Th1 cells and Th2 cells.

[0248] The increased ability to stimulate an immune response or the immune system may be due to enhanced agonistic activity of T cell costimulatory receptors and / or enhanced antagonistic activity of inhibitory receptors. The increased ability to stimulate an immune response or the immune system may be due to an increased EC 50 Or the maximum level of activity, for example, may be reflected by a fold increase in assays measuring cytokine or chemokine release, cytolytic activity (determined directly on target cells or indirectly by CD107a or granzyme detection), and changes in proliferation. The ability to stimulate an immune response or immune system activity may be enhanced by at least 10%, 30%, 50%, 75%, 2-fold, 3-fold, 5-fold, or more.

[0249] As used herein, the term "linkage" refers to the attachment of two or more molecules. Linkage can be covalent or non-covalent. Linkage can also be genetic (i.e., recombinant fusion). Such linkage can be achieved using a wide range of art-recognized techniques, such as chemical conjugation and recombinant protein production.

[0250] As used herein, "administration" refers to the physical introduction of a composition containing a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Various routes of administration of the anti-TIM3 antibodies described herein include, for example, intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes of administration, such as by injection or infusion. The term "parenteral administration," as used herein, refers to modes of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. Alternatively, the antibodies described herein may be administered by topical, epithelial, or mucosal routes of administration, such as intranasal, oral, vaginal, rectal, sublingual, or topical administration, which are not parenteral. Administration can also be carried out, for example, once, multiple times and / or over one or more chronic periods.

[0251] As used herein, the term "T cell-mediated response" refers to the response of effector T cells (e.g., CD8 + cells) and helper T cells (e.g., CD4 + T cell-mediated responses refer to responses mediated by T cells, including T cells, including, for example, T cell cytotoxicity and proliferation.

[0252] As used herein, the term "cytotoxic T lymphocyte (CTL) response" refers to an immune response elicited by cytotoxic T cells. CTL responses are primarily CD8 + It is mediated by T cells.

[0253] As used herein, the terms "inhibition" or "blocking" (e.g., inhibiting / blocking the binding of TIM3-L to cellular TIM3) are used interchangeably and include both partial and complete inhibition / blocking. In certain embodiments, an anti-TIM3 antibody inhibits the binding of TIM3-L to TIM3 by at least about 50%, e.g., about 60%, 70%, 80%, 90%, 95%, 99%, or 100%, e.g., as determined further herein. In certain embodiments, an anti-TIM3 antibody inhibits the binding of TIM3-L to TIM3 by 50% or less, e.g., about 40%, 30%, 20%, 10%, 5%, or 1%, e.g., as determined further herein.

[0254] As used herein, the term "tumor growth inhibition" includes any measurable reduction in tumor growth, e.g., at least about 10%, e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 99% or 100% tumor growth inhibition.

[0255] As used herein, "cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division can lead to the formation of malignant tumors or cells that can invade adjacent tissues and metastasize to distant parts of the body via the lymphatic system or bloodstream.

[0256] As used herein, the terms "treat" and "treatment" refer to any type of intervention or process or administration of an active agent to a subject for the purpose of reversing, alleviating, ameliorating, arresting, or slowing the progression, development, severity, or recurrence of symptoms, complications, conditions, or biochemical manifestations associated with a disease, or preventing or prolonging overall survival. Treatment can be for subjects with a disease or for subjects without a disease (e.g., for prophylactic purposes).

[0257] "Hematopoietic malignancies" include lymphomas, leukemias, myelomas, or lymphoid malignancies, as well as cancers of the spleen and lymph nodes. Examples of lymphomas include both B-cell lymphomas (B-cell blood cancers) and T-cell lymphomas. B-cell lymphomas include both Hodgkin's lymphoma and most non-Hodgkin's lymphomas. Non-limiting examples of B-cell lymphomas include diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphocytic lymphoma (overlap with chronic lymphocytic leukemia), mantle cell lymphoma (MCL), Burkitt's lymphoma, mediastinal large B-cell lymphoma, Waldenstrom's macroglobulinemia, nodal marginal zone B-cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, and lymphomatoid granulomatosis. Non-limiting examples of T-cell lymphomas include extranodal T-cell lymphoma, cutaneous T-cell lymphoma, anaplastic large cell lymphoma, and angioimmunoblastic T-cell lymphoma. Hematological tumors also include leukemias, such as, but not limited to, secondary leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, and acute lymphoblastic leukemia. Hematological tumors further include myelomas, such as, but not limited to, multiple myeloma and smoldering multiple myeloma. Other hematological and / or B-cell or T-cell-related cancers are included in the term hematopoietic tumors.

[0258] The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve the desired effect. A "therapeutically effective amount" or "therapeutically effective dosage" of a drug or therapeutic agent is the amount of drug that, when used alone or in combination with other therapeutic agents, promotes disease regression as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or prevention of functional impairment or disability due to disease morbidity. A therapeutically effective amount or dosage of a drug includes a "prophylactically effective amount" or "prophylactically effective dosage," which is the amount of drug that, when administered alone or in combination with other therapeutic agents to a subject at risk of developing or recurring a disease, prevents the onset or recurrence of the disease. The ability of a therapeutic agent to promote disease regression or prevent the onset or recurrence of disease can be evaluated using a variety of methods known to skilled artisans, such as by assaying the activity of the drug in human subjects during therapeutic trials, in animal models predictive of efficacy in humans, or in in vitro assays.

[0259] For example, an anti-cancer agent is a drug that promotes cancer regression in a subject. In some embodiments, a therapeutically effective amount of a drug promotes cancer regression to the point of cancer elimination. "Promoting cancer regression" refers to the administration of an effective amount of a drug, alone or in combination with an anti-neoplastic agent, that results in a reduction in tumor growth or size, tumor necrosis, a reduction in the severity of at least one disease symptom, an increase in the frequency and duration of disease-free symptom intervals, prevention of disease-related functional or disability impairment, or other improvement of disease symptoms in a patient. Furthermore, the terms "effective" and "efficacy" in relation to treatment include both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of a drug to promote cancer regression in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (adverse effects) at the cellular, organ, and / or organismal level resulting from drug administration.

[0260] For example, in tumor treatment, a therapeutically effective amount or dosage of a drug inhibits cell proliferation or tumor growth by at least about 20%, at least about 40%, at least about 60%, or at least about 80% relative to an untreated subject. In some embodiments, a therapeutically effective amount or dosage of a drug completely inhibits cell proliferation or tumor growth, i.e., inhibits cell proliferation or tumor growth by 100%. The ability of a compound to inhibit tumor growth can be assessed using the assays described below. Alternatively, this property of a composition can be assessed by testing the ability of a compound to inhibit cell proliferation, and such inhibition can be measured in vitro by assays known to the skilled practitioner. In certain embodiments described herein, tumor regression can be observed and sustained for a period of at least about 20 days, at least about 40 days, or at least about 60 days.

[0261] The term "patient" includes human and other mammalian subjects receiving prophylactic or therapeutic treatment.

[0262] As used herein, the term "subject" includes any human or non-human animal. For example, the methods and compositions described herein can be used to treat a subject with cancer. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0263] As used herein, the term "weight-based" dosage or administration means that the dosage administered to a patient is calculated based on the patient's weight. For example, if a patient weighing 60 kg requires 3 mg / kg of anti-TIM3 antibody, the appropriate amount of anti-TIM3 antibody for administration (i.e., 180 mg) can be calculated and used.

[0264] The use of the term "fixed dose" in relation to the methods of the present invention means that two or more different antibodies (e.g., an anti-TIM3 antibody and a second antibody, e.g., a PD-1 or PD-L1 antibody) present in a single composition are present in the composition in a specific (fixed) ratio to each other. In certain embodiments, the fixed dose is based on antibody weight (e.g., mg). In certain embodiments, the fixed dose is based on antibody concentration (e.g., mg / ml). In certain embodiments, the ratio of two antibodies (e.g., anti-TIM3 and anti-PD1 or anti-PD-L1) is at least about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, about 1:100, about 1:120, about 1:140, about 1:160, about 1:180, about 1:200, about 1:210, about 1:220, about 1:230, about 1:240, about 1:250, about 1:260, about 1:270, about 1:280, about 1:290, about 1:300, about 1:310, about 1:320, about 1:330, about 1:340, about 1:350, about 1:360, about 1:370, about 1:380, about 1:390, about 1:400, about 1:410, about 1:420, about 1:430, about 1:440, about 1:450, about 1:460, about 1:470, about 1:480, about 1:490, about 1:500, about 1:510, about 1:520, about 1:530, about 1:540, about 1:550, about 1:560, about 1:570, about 1:580 1:180, about 1:200, about 200:1, about 180:1, about 160:1, about 140:1, about 120:1, about 100:1, about 90:1, about 80:1, about 70:1, about 60:1, about 50:1, about 40:1, about 30:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1 or about 2:1 mg of first antibody (e.g., anti-TIM3 antibody) to mg of second antibody. For example, a 2:1 ratio of anti-TIM3 antibody and PD-1 antibody, such as nivolumab, means that a vial or injection may contain about 480 mg of anti-TIM3 antibody and 240 mg of anti-PD-1 antibody, or about 2 mg / ml of anti-TIM3 antibody and 1 mg / ml of anti-PD-1 antibody.

[0265] The use of the term "fixed dose" in connection with the methods and dosages described herein refers to a dose administered to a patient regardless of the patient's weight or body surface area (BSA). A fixed dose is therefore provided as an absolute amount of agent (e.g., anti-TIM3 antibody) rather than a mg / kg dose. For example, a 60 kg person and a 100 kg person would receive the same dose of antibody (e.g., 480 mg of anti-TIM3 antibody).

[0266] As used herein, the terms "ug" and "uM" are used interchangeably with "μg" and "μM," respectively.

[0267] Various aspects described herein are described in further detail in the following subsections.

[0268] I. Anti-human TIM3 antibody Described herein are antibodies, e.g., fully human antibodies, characterized by specific functional properties or properties. For example, the antibody specifically binds to human TIM3, and more specifically, to a specific domain (e.g., a functional domain) within the extracellular domain of human TIM3. In some embodiments, the antibody specifically binds to the site of TIM3 to which TIM3-L binds. In some embodiments, the antibody is an antagonist antibody, i.e., inhibits or suppresses the T cell inhibitory activity of cells, e.g., T cells. In some embodiments, the anti-TIM3 antibody cross-reacts with TIM3 from one or more non-human primates, such as cynomolgus monkey TIM3. In some embodiments, the antibody specifically binds to the extracellular domain of human TIM3 and the extracellular domain of cynomolgus monkey TIM3. In some embodiments, the antibody binds to human TIM3 with high affinity.

[0269] The anti-TIM3 antibodies described herein exhibit one or more of the following functional properties: (a) binds to soluble and / or membrane-bound human TIM3; (b) binds to soluble and / or membrane-bound cynomolgus monkey TIM3; (c) induce or stimulate an immune response; (d) inducing or stimulating T cell activation, e.g., Th1 cell activation (e.g., evidenced by cytokine secretion and / or enhanced proliferation); (e) T cell proliferation (e.g., CD4 + , CD8 + induce or stimulate T cells, Th1 cells, or TILs); (f) induces or stimulates IFN-γ production by T cells, e.g., Th1 cells, or tumor-infiltrating lymphocytes (TILs), e.g., TILs from human kidney, lung, pancreatic, or breast cancer tumors, e.g., as determined by the assays described in the Examples; (g) blocks or inhibits the binding of human TIM3 to PtdSer, e.g., as determined by the assays described in the Examples; (h) when bound to cellular TIM3, it does not internalize or downregulate cell surface TIM3; (i) binds to the human TIM3 extracellular domain (i) CPVFECG (SEQ ID NO: 296); (ii) RIQIPGIMND (SEQ ID NO: 298); (iii) CPVFECG and RIQIPGIMND (SEQ ID NOs: 296 and 298, respectively); or (iv) WTSRYWLNGDFR (SEQ ID NO: 297); (j) competes with or cross-blocks binding to human TIM3 with an antibody to TIM3 described herein (e.g., 13A3, 8B9, 8C4, 3G4, 17C3, 17C8, 9F6, 14H7, 23B3, or TIM3.2 to any of TIM3.18, TIM3.24, and TIM3.25), e.g., as determined by an assay described in the Examples; (k) binds to human TIM3 but not to human TIM3 with one or more amino acid substitutions of the following amino acid residues: L48, C58, P59, V60, F61, E62, C63, G64, W78, S80, R81, W83, L84, G86, D87, R89, D104, R111, Q113, G116, M118, and D120 (numbering according to SEQ ID NO: 286 (Figure 25)); and (l) Human TIM3 region as determined by HDX-MS 49 VPVCWGKGACPVFE 62 (SEQ ID NO: 367) and 111 RIQIPGIMNDEKFNLKL 127 binds to (SEQ ID NO: 368); (m) having a heavy chain and / or light chain variable region that interacts with at least 5, 10, 15, 20, or all of the following amino acids of human TIM3, as determined by X-ray crystallography: P50, V51, C52, P59, V60, F61, E62, C63, G64, N65, V66, V67, L68, R69, D71, E72, D74, R111, Q113, G116, I117, M118, D120, and optionally T70 and / or I112; and / or (n) competes with or cross-blocks the binding of 13A3 or TIM3.18.IgG1.3 to human TIM3, for example, as described in the Examples.

[0270] In some embodiments, the anti-TIM3 antibodies described herein have high affinity for human TIM3, e.g., 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, 10 -12 M~10 -7 M, 10 -11 M~10 -7 M, 10 -10 M~10 -7 M or 10 -9 M~10 -7 K of M D In certain embodiments, the anti-TIM3 antibody binds to soluble human TIM3, e.g., by BIACORE TM (e.g., as described in the Examples) -7 M or less, 10 -8 M or less, 10 -9 M (1nM) or less, 10 -10 M or less, 10 -12 M~10 -7 M, 10 -11 M~10 -7 M, 10 -10 M~10 -7 M, 10 -9 M~10 -7 M or 10 -8 M~10 -7 K of M D In certain embodiments, the anti-TIM3 antibody binds to bound (e.g., cell membrane-bound) human TIM3, such as on activated human T cells, at a concentration of 10, as determined, for example, by flow cytometry and Scatchard plots. -7 M or less, 10 -8 M or less, 10 -9 M (1nM) or less, 5×10 -10 M or less, 10 -10 M or less, 10 -12 M~10 -7 M, 10 -11M~10 -8 M, 10 -10 M~10 -8 M, 10 -9 M~10 -8 M, 10 -11 M~10 -9 M or 10 -10 M~10 -9 K of M D In certain embodiments, the anti-TIM3 antibody binds to bound (e.g., cell membrane-bound) human TIM3, such as on activated human T cells, with an EC of 10 μg / mL or less, 5 μg / mL or less, 1 μg / mL or less, 0.9 μg / mL or less, 0.8 μg / mL or less, 0.7 μg / mL or less, 0.6 μg / mL or less, 0.5 μg / mL or less, 0.4 μg / mL or less, 0.3 μg / mL or less, 0.2 μg / mL or less, 0.1 μg / mL or less, 0.05 μg / mL or less, or 0.01 μg / mL or less, as determined, e.g., by flow cytometry. 50 In certain embodiments, the anti-TIM3 antibodies described herein bind to cynomolgus monkey TIM3 at, e.g., 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, 10 -12 M~10 -7 M, 10 -11 M~10 -7 M, 10 -10 M~10 -7 M or 10 -9 M~10 -7 K of M D In one embodiment, the anti-TIM3 antibody binds to soluble cynomolgus TIM3, e.g., by BIACORE TM (e.g., as described in the Examples) -7 M or less, 10 -8 M or less, 10 -9 M (1nM) or less, 10 -10 M or less, 10 -12 M~10 -7 M, 10 -11 M~10 -7 M, 10 -10 M~10 -7 M, 10-9 M~10 -7 M or 10 -8 M~10 -7 K of M D The anti-TIM3 antibody binds to membrane-bound cynomolgus monkey TIM3 with an EC2 of, for example, 100 nM or less, 10 nM or less, 100 nM to 0.01 nM, 100 nM to 0.1 nM, 100 nM to 1 nM, or 10 nM to 1 nM, as measured, for example, by flow cytometry (e.g., as described in the Examples). 50 In certain embodiments, the anti-TIM3 antibody can bind to bound (e.g., cell membrane-bound) cynomolgus monkey TIM3, such as on activated human T cells, at 10, as determined, for example, by flow cytometry and Scatchard plots. -7 M or less, 10 -8 M or less, 10 -9 M (1nM) or less, 5×10 -10 M or less, 10 -10 M or less, 10 -12 M~10 -7 M, 10 -11 M~10 -8 M, 10 -10 M~10 -8 M, 10 -9 M~10 -8 M, 10 -11 M~10 -9 M or 10 -10 M~10 -9 K of M D Combine with.

[0271] In certain embodiments, the anti-TIM3 antibodies described herein stimulate or enhance an immune response, e.g., by activating tumor T cells. For example, the anti-TIM3 antibodies may activate or costimulate cells, as evidenced by, e.g., enhanced cytokine (e.g., IFN-γ) secretion and / or enhanced proliferation, which may result from inhibition of TIM3-mediated T cell inhibitory activity. In certain embodiments, T cell activation or costimulation by the TIM3 antibodies occurs in the presence of CD3 stimulation. In certain embodiments, the anti-TIM3 antibodies increase IFN-γ secretion by 50%, 100% (i.e., 2-fold), 3-fold, 4-fold, 5-fold, or more, optionally up to 10-fold, 30-fold, or 100-fold, as measured, e.g., in primary human T cells and / or T cells expressing human TIM3, e.g., tumor-infiltrating lymphocytes (TILs).

[0272] In certain embodiments, the anti-TIM3 antibody inhibits binding of phosphatidylserine to human TIM3 in cells, e.g., CHO cells or T cells expressing activated human TIM3, with an EC of, e.g., 10 μg / ml or less, 1 μg / ml or less, 0.01 μg / ml to 10 μg / ml, 0.1 μg / ml to 10 μg / ml, or 0.1 μg / ml to 1 μg / ml. 50 and inhibits.

[0273] In some embodiments, the anti-TIM3 antibodies described herein bind to an epitope, e.g., a conformational epitope, in the extracellular portion of human TIM3, e.g., the Ig-like domain of the extracellular region, i.e., amino acids 22-202 of SEQ ID NO: 286 (Figure 25). In some embodiments, the anti-TIM3 antibodies bind to an epitope located at amino acids 22-120 of the human TIM3 extracellular domain (SEQ ID NO: 286) or 1-99 of mature human TIM3 (SEQ ID NO: 290) (see Examples). In some embodiments, the anti-TIM3 antibodies bind to an epitope at or within a region consisting of amino acids 58-64 of human TIM3 having SEQ ID NO: 286 (CPVFECG, SEQ ID NO: 296; see Figure 25), which corresponds to SEQ ID NO: 286 of mature human TIM3. In certain embodiments, the anti-TIM3 antibody binds to or an epitope within a region consisting of amino acids 111-120 of human TIM3 having SEQ ID NO:286 (RIQIPGIMND, SEQ ID NO:298; see Figure 25), which corresponds to SEQ ID NO:286 of mature human TIM3. In certain embodiments, the anti-TIM3 antibody binds to or an epitope within a region consisting of amino acids 58-64 of human TIM3 having SEQ ID NO:286 (CPVFECG, SEQ ID NO:296) or a region consisting of amino acids 111-120 of human TIM3 having SEQ ID NO:286 (RIQIPGIMND, SEQ ID NO:298; see Figure 25). In certain embodiments, the anti-TIM3 antibody binds to or an epitope within a region consisting of amino acids 78-89 of human TIM3 having SEQ ID NO:286 (WTSRYWLNGDFR, SEQ ID NO:297; see Figure 25), which corresponds to SEQ ID NO:286 of mature human TIM3.

[0274] In certain embodiments, the anti-TIM3 antibody binds to substantially the same epitope as 13A3, i.e., an epitope (or region of human TIM3) comprising one or more of amino acid residues C58, P59, F61, E62, C63, R111, and D120 of SEQ ID NO: 286 (Figure 25). In certain embodiments, the anti-TIM3 antibody binds to an epitope (or region of human TIM3) comprising one or more of amino acid residues C58, P59, F61, E62, C63, D104, R111, Q113, and D120 of SEQ ID NO: 286 (Figure 25). In certain embodiments, the anti-TIM3 antibody does not significantly bind, or binds with significantly reduced binding affinity, to a human TIM3 protein in which one or more of amino acid residues C58, P59, F61, E62, C63, R111, and D120 of SEQ ID NO: 286 have been changed to another amino acid, e.g., a non-conservative amino acid substitution. In certain embodiments, the anti-TIM3 antibody does not significantly bind, or binds with significantly reduced binding affinity, to a human TIM3 protein in which one or more of amino acid residues C58, P59, F61, E62, C63, D104, R111, Q113, and D120 of SEQ ID NO: 286 have been changed to another amino acid, e.g., a non-conservative amino acid substitution.

[0275] In certain embodiments, the anti-TIM3 antibody binds to substantially the same epitope as 3G4, i.e., an epitope (or region of human TIM3) comprising one or more of amino acid residues C58, P59, V60, F61, E62, C63, G116, and M118 of SEQ ID NO: 286 (Figure 25). In certain embodiments, the anti-TIM3 antibody binds to an epitope (or region of human TIM3) comprising one or more of amino acid residues C58, P59, V60, F61, E62, C63, D104, G116, and M118 of SEQ ID NO: 286 (Figure 25). In certain embodiments, the anti-TIM3 antibody does not significantly bind, or binds with significantly reduced binding affinity, to a human TIM3 protein in which one or more of amino acid residues C58, P59, V60, F61, E62, C63, G116, and M118 of SEQ ID NO: 286 have been changed to another amino acid, e.g., a non-conservative amino acid substitution. In certain embodiments, the anti-TIM3 antibody does not significantly bind, or binds with significantly reduced binding affinity, to a human TIM3 protein in which one or more of amino acid residues C58, P59, V60, F61, E62, C63, D104, G116, and M118 of SEQ ID NO: 286 have been changed to another amino acid, e.g., a non-conservative amino acid substitution.

[0276] In certain embodiments, the anti-TIM3 antibody binds to substantially the same epitope as 17C3, i.e., an epitope (or region of human TIM3) comprising one or more of amino acid residues C58, P59, V60, F61, E62, C63, G64, and G116 of SEQ ID NO: 286 (Figure 25). In certain embodiments, the anti-TIM3 antibody binds to an epitope (or region of human TIM3) comprising one or more of amino acid residues C58, P59, V60, F61, E62, C63, G64, D104, and G116 of SEQ ID NO: 286 (Figure 25). In certain embodiments, the anti-TIM3 antibody does not significantly bind, or binds with significantly reduced binding affinity, to a human TIM3 protein in which one or more of amino acid residues C58, P59, V60, F61, E62, C63, G64, and G116 of SEQ ID NO: 286 have been changed to another amino acid, e.g., a non-conservative amino acid substitution. In certain embodiments, the anti-TIM3 antibody does not significantly bind, or binds with significantly reduced binding affinity, to a human TIM3 protein in which one or more of amino acid residues C58, P59, V60, F61, E62, C63, G64, D104, and G116 of SEQ ID NO: 286 have been changed to another amino acid, e.g., a non-conservative amino acid substitution.

[0277] In certain embodiments, the anti-TIM3 antibody binds to substantially the same epitope as 8B9, i.e., an epitope (or region of human TIM3) comprising one or more of amino acid residues L48, W78, S80, R81, W83, G86, D87, and R89 of SEQ ID NO: 286 (Figure 25). In certain embodiments, the anti-TIM3 antibody binds to an epitope (or region of human TIM3) comprising one or more of amino acid residues L48, W78, S80, R81, W83, L84, G86, D87, and R89 of SEQ ID NO: 286 (Figure 25). In certain embodiments, the anti-TIM3 antibody binds to substantially the same epitope as 8B9, i.e., an epitope (or region of human TIM3) including one or more of amino acid residues L48, W78, S80, R81, W83, G86, D87, R89, and D104 of SEQ ID NO: 286 (Figure 25). In certain embodiments, the anti-TIM3 antibody does not bind significantly, or binds with significantly reduced binding affinity, to a human TIM3 protein in which one or more of amino acid residues L48, W78, S80, R81, W83, G86, D87, and R89 of SEQ ID NO: 286 have been changed to another amino acid, e.g., a non-conservative amino acid substitution (Figure 25). In certain embodiments, the anti-TIM3 antibody does not significantly bind, or binds with significantly reduced binding affinity, to a human TIM3 protein in which one or more of amino acid residues L48, W78, S80, R81, W83, L84, G86, D87, and R89 of SEQ ID NO: 286 have been changed to another amino acid, e.g., a non-conservative amino acid substitution (Figure 25). In certain embodiments, the anti-TIM3 antibody does not significantly bind, or binds with significantly reduced binding affinity, to a human TIM3 protein in which one or more of amino acid residues L48, W78, S80, R81, W83, G86, D87, R89, and D104 of SEQ ID NO: 286 have been changed to another amino acid, e.g., a non-conservative amino acid substitution (Figure 25).

[0278] In one embodiment, anti-TIM3 antibody c competes with (or inhibits) binding to any of the anti-TIM3 antibodies having the CDRs or variable regions of human TIM3 described herein, e.g., antibodies 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3, and TIM3.2 to TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24, and TIM3.25. In certain embodiments, the anti-TIM3 antibody inhibits binding of antibodies 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3 or any of TIM3.2 to TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24 and TIM3.25 to human TIM3 by at least 50%, 60%, 70%, 80%, 90% or 100%. In certain embodiments, 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3, or any of TIM3.2 to TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24, and TIM3.25 inhibits binding of an anti-TIM3 antibody to human TIM3 by at least 50%, 60%, 70%, 80%, 90% or 100%. In certain embodiments, the anti-TIM3 antibody inhibits binding of 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3, or any of TIM3.2 to TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24, and TIM3.25 to human TIM3 by at least 50%, 60%, 70%, 80%, 90%, or 100%; and 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3, or any of TIM3.2 to TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24, and TIM3.25 inhibits binding of an anti-TIM3 antibody to human TIM3 by at least 50%, 60%, 70%, 80%, 90%, or 100% (e.g., competes in both directions).

[0279] In certain embodiments, the anti-TIM3 antibody has one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or all of the following properties: (1) For example, when measured using the Biacore described in the Examples, soluble human TIM3 has a K of, for example, 10 nM or less (e.g., 0.01 nM to 10 nM). D Combine with; (2) For example, when measured using a Biacore as described in the Examples, soluble cynomolgus monkey TIM3 has a K of, for example, 100 nM or less (for example, 0.01 nM to 100 nM). D Combine with; (3) For example, when measured by flow cytometry as described in the Examples, membrane-bound human TIM3 has an EC of, for example, 1 μg / mL or less (for example, 0.01 μg / mL to 1 μg / mL). 50 Combine with; (4) For example, as measured by Scatchard analysis described in the Examples, the K D Combine with; (5) For example, when measured by flow cytometry as described in the Examples, membrane-bound cynomolgus monkey TIM3 has an EC of, for example, 20 μg / mL or less (e.g., 0.01 μg / mL to 20 μg / mL). 50 Combine with; (6) For example, as measured by Scatchard analysis described in the Examples, the K D Combine with; (7) (i) increasing IFN-γ production by TIM3-expressing T cells (e.g., Th1 cells or TILs) and / or (ii) inducing or enhancing T cell activation (e.g., by blocking or reducing the inhibitory effect of TIM3), as evidenced by enhanced T cell (e.g., Th1 cells or TILs) proliferation, e.g., as described in the Examples; (8) stimulating T cell proliferation in a mixed lymphocyte reaction (MLR) assay, e.g., as described in the Examples; (9) inhibiting the binding of phosphatidylserine to TIM3, e.g., as measured by a PS-hTIM3 “in tandem” blocking assay, e.g., as described in the Examples; (10) when bound to cellular TIM3, it does not internalize or downregulate cell surface TIM3; (11) For example, as described in the Examples, it binds to one of the following regions of the human TIM3 extracellular domain (SEQ ID NO: 290): (a) CPVFECG (SEQ ID NO: 296); (b) RIQIPGIMND (SEQ ID NO: 298); (c) CPVFECG and RIQIPGIMND (SEQ ID NOs: 296 and 298, respectively); and (d) WTSRYWLNGDFR (SEQ ID NO: 297); (12) Decreased binding to wild-type human TIM3, e.g., human TIM3 in which one or more of the amino acids L48, C58, P59, V60, F61, E62, C63, G64, W78, S80, R81, W83, L84, G86, D87, R89, D104, R111, Q113, G116, M118, and D120 (numbered in SEQ ID NO: 286 ( FIG. 25 )) have been substituted with another amino acid, compared to the binding described in the Examples; (13) competes in either or both orientations for binding to human TIM3 with 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3, TIM3.7, TIM3.8, TIM3.10, TIM3.11, TIM3.12, TIM3.13, TIM3.14, TIM3.15, TIM3.16, TIM3.17, TIM3.18, and TIM3.25, e.g., an antibody comprising any of the VH and VL domains described in the Examples; (14) For example, as described in the Examples, the human TIM3 region was determined by HDX-MS. 49 VPVCWGKGACPVFE 62 (SEQ ID NO: 367) and 111 RIQIPGIMNDEKFNLKL 127 binds to (SEQ ID NO: 368); (15) having a heavy chain and / or light chain variable region that interacts with at least 5, 10, 15, 20, or all of the following amino acids of human TIM3, P50, V51, C52, P59, V60, F61, E62, C63, G64, N65, V66, V67, L68, R69, D71, E72, D74, R111, Q113, G116, I117, M118, D120, and optionally T70 and / or I112, as determined by X-ray crystallography (e.g., as described in the Examples; numbering according to SEQ ID NO: 286 (Figure 25)); and / or (16) (a) exhibits reduced binding to human TIM3 in which one, two, three, four, five, six, seven, eight, or nine of the amino acids C58, P59, F61, E62, C63, R111, D120, and optionally D104 and Q113 (numbering according to SEQ ID NO: 286 (FIG. 25)) have been substituted with another amino acid, compared to binding to wild-type human TIM3; (b) exhibits reduced binding to human TIM3 in which one, two, three, four, five, six, seven, eight, or nine of the amino acids C58, P59, F61, E62, C63, R111, D120, and optionally D104 and Q113 (numbering according to SEQ ID NO: 286 (FIG. 25)) have been substituted with another amino acid, as determined by HDX-MS as described in the Examples. 49 VPVCWGKGACPVFE 62 (SEQ ID NO: 367), 111 RIQIPGIMNDEKFNLKL 127 (SEQ ID NO: 368) and 119 NDEKFNLKL 127 (SEQ ID NO: 373); and / or (c) competes with or cross-blocks the binding of 13A3 or TIM3.18.IgG1.3 to human TIM3, e.g., as described in the Examples; (17) hTIM3-IgV was measured using the method described in Example 22 and found to have a 5 × 10 -8 M, 2 x 10 -8 M, 10 -8 M or 5x10 -9 K below M D Combine with; (18) hTIM3-IgV was measured by the method described in Example 22. -7 M, 2 x 10 -8 M or 10 -8 K below M D Combine with; (19) 5 × 10 TIM3-ECD cells were measured using the method described in Example 22. -7 M, 2 x 10-8 M or 10 -8 K below M D and / or (20) hTIM3-ECD was measured using the method described in Example 22, and 8 × 10 -8 M, 5 x 10 -8 M or 10 -8 K below M D Combine with.

[0280] Thus, an antibody that exhibits one or more of these functional properties (e.g., biochemical, immunochemical, cellular, physiological or other biological activity, etc.) as determined by methods known in the art and described herein is understood to exhibit a statistically significant difference in the particular activity compared to that observed in the absence of the antibody (e.g., or in the presence of a control antibody of an irrelevant species). In certain embodiments, the anti-TIM3 antibody-induced increase in a measured parameter in an assay (e.g., T cell proliferation, cytokine production) results in a statistically significant increase of the measured parameter by at least 10%, e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% (i.e., 2-fold), 3-fold, 5-fold, or 10-fold; in certain embodiments, the antibodies described herein may increase the measured parameter by more than 92%, 94%, 95%, 97%, 98%, 99%, 100% (i.e., 2-fold), 3-fold, 5-fold, or 10-fold, relative to the same assay performed in the absence of the antibody. Conversely, anti-TIM3 antibody-induced reduction in a measured parameter in an assay (e.g., tumor volume, TIM3-L binding to human TIM3) results in a statistically significant reduction of the measured parameter by at least 10%, e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, and in certain embodiments, the antibodies described herein may reduce the measured parameter by more than 92%, 94%, 95%, 97%, 98%, or 99%, for example, compared to the same assay performed in the absence of the antibody.

[0281] Standard assays for assessing the binding ability of antibodies to TIM3 of various species are known in the art and include, for example, ELISA, Western blot, and RIA. Suitable assays are described in detail in the Examples. Antibody binding kinetics (e.g., binding affinity) can also be assessed by standard assays known in the art, such as Biacore analysis. Assays for assessing the effect of antibodies on the functional properties of TIM3 (e.g., ligand binding, T cell proliferation, cytokine production) are described below and in further detail in the Examples.

[0282] In certain embodiments, the anti-TIM3 antibody is not a native or naturally occurring antibody, e.g., the anti-TIM3 antibody has post-translational modifications that differ from those of naturally occurring antibodies, e.g., by having more, fewer, or different types of post-translational modifications.

[0283] In certain embodiments, the anti-TIM3 antibody lacks agonist activity, as determined, for example, by cross-linking the anti-TIM3 antibody in a CHO-OKT3-CD32:T cell coculture experiment, where such an antibody does not enhance activity over anti-TIM3 alone. In certain embodiments, the anti-TIM3 antibody blocks the interaction of TIM3 with its ligand without promoting agonist activity.

[0284] In certain embodiments, the anti-TIM3 antibody enhances IL-12 production from LPS-treated monocytes or dendritic cells.

[0285] In one embodiment, the anti-TIM3 antibody inhibits tumor-infiltrating CD8+ cells that co-express PD-1 and TIM3 upon combination treatment. + Restore T cells and thus CD8 + Avoid T cell depletion.

[0286] II. Examples of anti-TIM3 antibodies Certain anti-TIM3 antibodies described herein are antibodies, e.g., monoclonal, recombinant, and / or human antibodies, having the CDR and / or variable region sequences of antibodies 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3, or any of TIM3.2 through TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24, and TIM3.25, isolated and structurally characterized as described herein, and antibodies having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or at least 99% identity) to the variable region or CDR sequences thereof. See PCT / US2017 / 041946, incorporated herein by reference in its entirety. The VH amino acid sequences of 13A3, 8B9, 8C4, 17C3, 9F6, 3G4, 17C8, 14H7, and 23B3 are set forth in SEQ ID NOs: 34-40, 410, and 411, respectively. The VH amino acid sequences of mutant forms of 13A3, 8B9, 9F6, and 23B3 are set forth in SEQ ID NOs: 112-121, 364, and 412. The VH amino acid sequences of VTIM3.20 and TIM3.21 are set forth in SEQ ID NO: 449. The VH amino acid sequences of VTIM3.22 and TIM3.23 are set forth in SEQ ID NO: 456. The VL amino acid sequences of V13A3, 17C3, and 3G4 are set forth in SEQ ID NO: 60. The VL amino acid sequences of V8B9, 8C4, and 17C8 are set forth in SEQ ID NO: 61. The VL amino acid sequence of 9F6 is set forth in SEQ ID NOs: 61, 62, and 63. The VL V14H7 amino acid sequence is set forth in SEQ ID NO: 417. The VL V23B3 amino acid sequence is set forth in SEQ ID NOs: 60 and 418. The VL amino acid sequences of the mutant forms of 13A3, 8B9, 9F6, and 23B3 correspond to those of the corresponding unmutated antibodies. The VL VTIM3.20 and TIM3.22 amino acid sequences are set forth in SEQ ID NO: 450. The VL VTIM3.21 and TIM3.23 amino acid sequences are set forth in SEQ ID NO: 63. A summary of SEQ ID NO assignments is provided in Figure 16.

[0287] Accordingly, provided herein is an isolated antibody or antigen-binding portion thereof comprising a heavy chain and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 34-40, 112-121, 364, 410-412, 449 and 456.

[0288] Also provided is an isolated antibody or antigen-binding portion thereof comprising a heavy chain and a light chain variable region, wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 60-63, 417, 418, and 450.

[0289] What is offered here is (a) heavy and light chain variable region sequences comprising SEQ ID NOs: 34 and 60, respectively; (b) heavy and light chain variable region sequences comprising SEQ ID NOs: 35 and 61, respectively; (c) heavy and light chain variable region sequences comprising SEQ ID NOs: 36 and 61, respectively; (d) heavy and light chain variable region sequences comprising SEQ ID NOs: 37 and 60, respectively; (e) heavy and light chain variable region sequences comprising SEQ ID NOs: 38 and 61, respectively; (f) heavy and light chain variable region sequences comprising SEQ ID NOs: 38 and 62, respectively; (g) heavy and light chain variable region sequences comprising SEQ ID NOs: 38 and 63, respectively; (h) heavy and light chain variable region sequences comprising SEQ ID NOs: 39 and 60, respectively; (i) heavy and light chain variable region sequences comprising SEQ ID NOs: 40 and 61, respectively; (j) heavy and light chain variable region sequences comprising SEQ ID NOs: 121 and 63, respectively; (k) heavy and light chain variable region sequences comprising SEQ ID NOs: 120 and 61, respectively; (l) heavy and light chain variable region sequences comprising SEQ ID NOs: 112 and 60, respectively; (m) heavy and light chain variable region sequences comprising SEQ ID NOs: 113 and 60, respectively; (n) heavy and light chain variable region sequences comprising SEQ ID NOs: 114 and 60, respectively; (o) heavy and light chain variable region sequences comprising SEQ ID NOs: 115 and 60, respectively; (p) heavy and light chain variable region sequences comprising SEQ ID NOs: 116 and 60, respectively; (q) heavy and light chain variable region sequences comprising SEQ ID NOs: 117 and 60, respectively; (r) heavy and light chain variable region sequences comprising SEQ ID NOs: 118 and 60, respectively; (s) heavy and light chain variable region sequences comprising SEQ ID NOs: 119 and 60, respectively; (t) heavy and light chain variable region sequences comprising SEQ ID NOs: 364 and 60, respectively; (u) heavy and light chain variable region sequences comprising SEQ ID NOs: 410 and 417, respectively; (v) heavy and light chain variable region sequences comprising SEQ ID NOs: 411 and 60, respectively; (w) heavy and light chain variable region sequences comprising SEQ ID NOs: 411 and 418, respectively; (x) heavy and light chain variable region sequences comprising SEQ ID NOs: 412 and 60, respectively; (y) heavy and light chain variable region sequences comprising SEQ ID NOs: 449 and 450, respectively; (z) heavy and light chain variable region sequences comprising SEQ ID NOs: 449 and 63, respectively; (aa) heavy and light chain variable region sequences comprising SEQ ID NOs: 456 and 450, respectively; or (bb) heavy and light chain variable region sequences comprising SEQ ID NOs: 456 and 63, respectively An isolated anti-human TIM3 antibody or an antigen-binding portion thereof, comprising:

[0290] Anti-TIM3 antibodies may comprise heavy and light chain CDR1, CDR2, and CDR3 of any one of 13A3, 8B9, 8C4, 17C3, 9F6, 3G4, 17C8, 14H7, 23B3, TIM3.2 to TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24, and TIM3.25, or any combination thereof. The amino acid sequences of VH CDR1 of 13A3, 8B9, 8C4, and 17C3 are set forth in SEQ ID NOs: 41 to 44, respectively. The amino acid sequences of VH CDR1 of 9F6, 3G4, 17C8, 14H7, and 23B3 are set forth in SEQ ID NO: 45. The amino acid sequence of VH CDR1 of the mutated 13A3 antibody (i.e., TIM3.10-TIM3.18) is the same as that of the non-mutated 13A3 antibody, i.e., SEQ ID NO: 41. The amino acid sequence of VH CDR1 of the mutated 8B9 antibody (i.e., TIM3.8) is the same as that of the non-mutated 8B9 antibody, i.e., SEQ ID NO: 42. The amino acid sequence of VH CDR1 of the mutated 9F6 antibody (i.e., TIM3.7) is the same as that of the non-mutated 9F6 antibody, i.e., SEQ ID NO: 45. The amino acid sequence of VH CDR1 of the mutated 23B3 antibody (i.e., TIM3.25) is the same as that of the non-mutated 23B3 antibody, i.e., SEQ ID NO: 45.

[0291] The amino acid sequences of VH CDR2 of 13A3, 8B9, 8C4, 17C3, 9F6, 3G4, 17C8, 14H7, and 23B3 are set forth in SEQ ID NOs: 46-52, 413, and 415, respectively. The amino acid sequences of VH CDR2 of mutated 13A3 antibodies TIM3.10, TIM3.17, and TIM3.18 are set forth in SEQ ID NO: 122. The amino acid sequences of VH CDR2 of mutated 13A3 antibodies TIM3.11 and TIM3.12 are set forth in SEQ ID NOs: 123 and 124, respectively. The amino acid sequences of VH CDR2 of mutated 13A3 antibodies TIM3.13 and TIM3.16 are those of the non-mutated 13A3 antibody, i.e., SEQ ID NO: 46. The amino acid sequence of VH CDR2 of the mutated 8B9 antibody (i.e., TIM3.8) is set forth in SEQ ID NO: 125. The amino acid sequence of VH CDR2 of the mutated 9F6 antibody (i.e., TIM3.7) is the same as that of the non-mutated 9F6 antibody, i.e., SEQ ID NO: 50. The amino acid sequence of VH CDR2 of the mutated 23B3 antibody (i.e., TIM3.25) is the same as that of the non-mutated 23B3 antibody, i.e., SEQ ID NO: 415.

[0292] The amino acid sequences of the VH CDR3 of 13A3, 8B9, 8C4, 17C3, 9F6, 3G4, 17C8, 14H7, and 23B3 are set forth in SEQ ID NOs: 53 to 59, 414, and 416, respectively. The amino acid sequences of the VH CDR3 of the mutated 13A3 antibodies TIM3.10 to TIM3.12 are those of the non-mutated 13A3 antibody, i.e., SEQ ID NO: 53. The amino acid sequences of the VH CDR3 of the mutated 13A3 antibodies TIM3.13 and TIM3.18 are set forth in SEQ ID NO: 126. The amino acid sequences of the VH CDR3 of the mutated 13A3 antibodies TIM3.15 and TIM3.17 are set forth in SEQ ID NO: 128. The amino acid sequences of the VH CDR3 of the mutated 13A3 antibodies TIM3.14 and TIM3.16 are set forth in SEQ ID NOs: 127 and 129, respectively. The amino acid sequence of the VH CDR3 of the mutated 8B9 antibody (i.e., TIM3.8) is that of the non-mutated 8B9 antibody, i.e., SEQ ID NO: 54. The amino acid sequence of the VH CDR3 of the mutated 9F6 antibody (i.e., TIM3.7) is the same as that of the non-mutated 9F6 antibody, i.e., SEQ ID NO: 57. The amino acid sequence of the VH CDR3 of the mutated 23B3 antibody (i.e., TIM3.25) is the same as that of the non-mutated 23B3 antibody, i.e., SEQ ID NO: 416.

[0293] The amino acid sequences of VL CDR1 of 13A3, 8B9, 8C4, 17C3, 3G4, 17C8, 14H7, and 23B3 are set forth in SEQ ID NO: 64. The amino acid sequences of VL CDR1 of 9F6 are set forth in SEQ ID NOs: 64 and 65. The amino acid sequences of VL CDR2 of 13A3, 8B9, 8C4, 17C3, 3G4, 17C8, 14H7, and 23B3 are set forth in SEQ ID NO: 66. The amino acid sequences of VL CDR2 of 9F6 are set forth in SEQ ID NOs: 66 and 67. The amino acid sequences of VL CDR3 of 13A3, 17C3, and 3G4 are set forth in SEQ ID NO: 68. The amino acid sequence of VL CDR3 of 8B9, 8C4, 17C8, and 14H7 is set forth in SEQ ID NO: 69. The amino acid sequences of VL CDR3 of 9F6 are set forth in SEQ ID NOs: 69, 70, and 71. The amino acid sequence of the VL CDR3 of 23B3 is set forth in SEQ ID NOs: 68 and 419. The amino acid sequences of the VL CDRs of mutated antibodies 13A3, 8B9, 9F6, and 23B3 are those of the corresponding unmutated antibodies. Figure 16 provides a list of SEQ ID NOs for the CDRs of the anti-TIM3 antibodies described herein.

[0294] CDR regions are delineated using the Kabat system (Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). Kabat is the most common numbering system, a scheme referred to as the EU index or EU numbering system, which is based on the sequence numbering of the first human IgG1 to be sequenced (EU antibody; Edelman et al. 1969). Based on the Kabat numbering scheme disclosed herein, antibody numbering can be converted to other systems known in the art, such as the Chotia, IMGT, Martin (extended Chotia), or AHo numbering schemes.

[0295] Given that each of these antibodies binds to human TIM3 and that antigen-binding specificity is primarily provided by the CDR1, 2, and 3 regions, VH CDR1, 2, and 3 sequences and VL CDR1, 2, and 3 sequences, such as those in Figure 16, can be "mixed and matched" (i.e., CDRs from different antibodies can be mixed and matched, but each antibody must contain VH CDR1, 2, and 3 and VL CDR1, 2, and 3) to create other anti-TIM3 binding molecules described herein. TIM3 binding of such "mixed and matched" antibodies can be tested using the binding assays described above and in the Examples (e.g., ELISAs). In certain embodiments, when VH CDR sequences are mixed and matched, the CDR1, CDR2, and / or CDR3 sequence from a particular VH sequence is replaced with a structurally similar CDR sequence(s). Similarly, when VL CDR sequences are mixed and matched, the CDR1, CDR2, and / or CDR3 sequence from a particular VL sequence is replaced with a structurally similar CDR sequence(s). It will be readily apparent to one of skill in the art that novel VH and VL sequences can be generated by replacing one or more VH and / or VL CDR region sequences with structurally similar sequences from the CDR sequences disclosed herein for any of monoclonal antibodies 13A3, 8B9, 8C4, 17C3, 9F6, 3G4, 17C8, 14H7, 23B3, and TIM3.2 to TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24, and TIM3.25.

[0296] What is offered here is (a) a heavy chain variable region CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 41 to 45 and 469; (b) a heavy chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 46 to 52, 122 to 125, 413, 415, and 470; (c) a heavy chain variable region CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 59, 126 to 129, 414, 416, and 471; (d) a light chain variable region CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 64-65 and 472; (e) a light chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66 to 67 and 473; and (f) a light chain variable region CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 68 to 71, 419, and 474; or an antigen-binding portion thereof, wherein the antibody specifically binds to human TIM3.

[0297] In one embodiment, the anti-human TIM3 antibody comprises heavy and light chain variable regions, wherein the heavy chain variable region CDR1, CDR2, and CDR3 regions are (a) SEQ ID NOs: 41, 46, 53; (b) SEQ ID NOs: 42, 47, 54; (c) SEQ ID NOs: 43, 48, 55; (d) SEQ ID NOs: 44, 49, 56; (e) SEQ ID NOs: 45, 50, 57; (f) SEQ ID NOs: 45, 51, 58; (g) SEQ ID NOs: 45, 52, 59; (h) SEQ ID NOs: 41, 122, 53; (i) SEQ ID NOs: 41, 123, 53; (j) SEQ ID NOs: 41, 124, 53; (k) SEQ ID NOs: 41, 46, 126; (l) SEQ ID NOs: 41, 46, 127; (m) SEQ ID NOs: 41, 46, 128; (n) SEQ ID NOs: 41, 46, 129; (o) SEQ ID NOs: 41, 122, 128; (p) SEQ ID NOs: 41, 122, 126; (q) SEQ ID NOs: 45, 413, 414; (r) SEQ ID NOs: 45, 415, 416; or (s) SEQ ID NOs: 469, 470 and 471; wherein the antibody specifically binds to human TIM3.

[0298] In one embodiment, the anti-human TIM3 antibody comprises heavy and light chain variable regions, wherein the light chain variable region CDR1, CDR2, and CDR3 regions are (a) SEQ ID NOs: 64, 66, 68; (b) SEQ ID NOs: 64, 66, 69; (c) SEQ ID NOs: 65, 67, 70; (d) SEQ ID NOs: 64, 66, 71; (e) SEQ ID NOs: 64, 66, 419; or (f) SEQ ID NOs: 472, 473, 474, wherein the antibody specifically binds to human TIM3.

[0299] In certain embodiments, the anti-TIM3 antibody comprises a heavy chain and a light chain variable region, wherein: (a1) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 41, 46, and 53, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 68, respectively; (a2) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 41, 122, and 53, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 68, respectively; (a3) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 41, 123, and 53, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 68, respectively; (a4) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 41, 124, and 53, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 68, respectively; (a5) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 41, 46, and 126, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 68, respectively; (a6) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 41, 46, and 127, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 68, respectively; (a7) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 41, 46, and 128, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 68, respectively; (a8) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 41, 46, and 129, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 68, respectively; (a9) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 41, 122, and 128, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 68, respectively; (a10) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 41, 122, and 126, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 68, respectively; (b1) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 42, 47, and 54, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 69, respectively; (b2) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 42, 125, and 54, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 69, respectively; (c) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 43, 48, and 55, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 69, respectively; (d) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 44, 49, and 56, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 68, respectively; (e1) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 45, 50, and 57, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 69, respectively; (e2) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 45, 50, and 57, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 71, respectively; (e3) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 45, 50, and 57, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 65, 67, and 70, respectively; (f) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 45, 51, and 58, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 68, respectively; (g) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 45, 52, and 59, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 69, respectively; (h) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 45, 413, and 414, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 69, respectively; (i1) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 45, 415, and 416, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 68, respectively; (i2) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 45, 415, and 416, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 419, respectively; (j1) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 469, 470, and 471, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 472, 473, and 474, respectively; or (j2) the heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 469, 470, and 471, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 71, respectively; Here, the antibody specifically binds to human TIM3.

[0300] One or more of the VH domains or CDRs thereof described herein can be combined with constant domains to form a heavy chain, e.g., a full-length heavy chain. Similarly, one or more of the VL domains or CDRs thereof described herein can be combined with constant domains to form a light chain, e.g., a full-length light chain. A full-length heavy chain (other than the optional C-terminal lysine (K) or other than the C-terminal glycine and lysine (GK)) and a full-length light chain combine to form a full-length antibody.

[0301] The VH domains described herein may be fused to the constant domain of a naturally occurring or modified human IgG, e.g., IgG1, IgG2, IgG3, or IgG4, e.g., as further described herein. For example, the VH domain may have the following wild-type human IgG1 constant domain amino acid sequence: [ka] (SEQ ID NO: 291) or an allotypic variant of SEQ ID NO: 291, having the following amino acid sequence: [ka] (SEQ ID NO: 277; "IgG1f" allotype-specific amino acid residues are bold and underlined) The VH domain may comprise the amino acid sequence of any of the VH domains described herein fused to a human IgG, e.g., IgG1, constant region, such as one having the following sequence:

[0302] The VH domain of the anti-TIM3 antibody may comprise an effector-less constant region, for example, the following effector-less human IgG1 constant domain amino acid sequence: [ka] (SEQ ID NO: 294; "IgG1.1f" with underlined substitutions L234A, L235E, G237A, A330S and P331S) or [ka] (SEQ ID NO: 295; "IgG1.3f" with underlined substitutions L234A, L235E and G237A) The VH domain may comprise the amino acid sequence of any of the VH domains described herein fused to the VH domain.

[0303] For example, an allotypic variant of IgG1 comprises K97R, D239E, and / or L241M (underlined and bolded above), numbered according to SEQ ID NOs: 277, 294, and 295. Within a full-length heavy region, e.g., 8C4 (SEQ ID NO: 3), and according to EU numbering, these amino acid substitutions are numbered K214R, D356E, and L358M. In certain embodiments, the constant region of an anti-TIM3 antibody may further comprise one or more mutations or substitutions at amino acids L117, A118, G120, A213, and P214 (underlined above) numbered according to SEQ ID NOs: 277, 294, and 295, or L234, A235, G237, A330, and P331 according to EU numbering. In certain embodiments, the constant region of the anti-TIM3 antibody comprises one or more mutations or substitutions at amino acids L117A, A118E, G120A, A213S, and P214S, or L234A, L235E, G237A, A330S, and P331S according to EU numbering, of SEQ ID NO: 291. The constant region of the anti-TIM3 antibody can also comprise one or more mutations or substitutions at L117A, A118E, and G120A, or L234A, L235E, and G237A according to EU numbering, of SEQ ID NO: 291.

[0304] Alternatively, the VH domain of the anti-TIM3 antibody may comprise a human IgG4 constant region, for example, the following human IgG4 amino acid sequence or a variant thereof: [ka] (SEQ ID NO: 292, including S228P) The VH domain may comprise the amino acid sequence of any of the VH domains described herein fused to

[0305] The VL domains described herein can be fused to the constant domain of a human kappa or lambda light chain. For example, the VL domain of an anti-TIM3 antibody can be fused to the following human IgG1 kappa light chain amino acid sequence: [ka] (SEQ ID NO: 278) The VL domain may comprise the amino acid sequence of any of the VL domains described herein fused to

[0306] In some embodiments, the heavy chain constant region includes a lysine or other amino acid at the C-terminus, e.g., the next-to-last amino acid in the heavy chain, LSPGK (SEQ ID NO: 279). In some embodiments, the heavy chain constant region lacks one or more amino acids at the C-terminus, e.g., has the C-terminal sequence LSPG (SEQ ID NO: 280) or LSP (SEQ ID NO: 281).

[0307] Examples of heavy and light chain amino acid sequences correspond to SEQ ID NOs: 1-28, 72-111, 301-354 for the heavy chain and SEQ ID NOs: 29-30 and 32-33 for the light chain.

[0308] What is offered here is (a1) heavy and light chain sequences comprising SEQ ID NOs: 301 (or 302) and 29, respectively; (a2) heavy and light chain sequences comprising SEQ ID NOs: 1 (or 8) and 29, respectively; (a3) heavy and light chain sequences comprising SEQ ID NOs: 15 (or 22) and 29, respectively; (a4) heavy and light chain sequences comprising SEQ ID NOs: 303 (or 304) and 29, respectively; (a5) heavy and light chain sequences comprising SEQ ID NOs: 72 (or 82) and 29, respectively; (a6) heavy and light chain sequences comprising SEQ ID NOs: 92 (or 102) and 29, respectively; (a7) heavy and light chain sequences comprising SEQ ID NOs: 305 (or 306) and 29, respectively; (a8) heavy and light chain sequences comprising SEQ ID NOs: 73 (or 83) and 29, respectively; (a9) heavy and light chain sequences comprising SEQ ID NOs: 93 (or 103) and 29, respectively; (a10) heavy and light chain sequences comprising SEQ ID NOs: 307 (or 308) and 29, respectively; (a11) heavy and light chain sequences comprising SEQ ID NOs: 74 (or 84) and 29, respectively; (a12) heavy and light chain sequences comprising SEQ ID NOs: 94 (or 104) and 29, respectively; (a13) heavy and light chain sequences comprising SEQ ID NOs: 309 (or 310) and 29, respectively; (a14) heavy and light chain sequences comprising SEQ ID NOs: 75 (or 85) and 29, respectively; (a15) heavy and light chain sequences comprising SEQ ID NOs: 95 (or 105) and 29, respectively; (a16) heavy and light chain sequences comprising SEQ ID NOs: 311 (or 312) and 29, respectively; (a17) heavy and light chain sequences comprising SEQ ID NOs: 76 (or 86) and 29, respectively; (a18) heavy and light chain sequences comprising SEQ ID NOs: 96 (or 106) and 29, respectively; (a19) heavy and light chain sequences comprising SEQ ID NOs: 313 (or 314) and 29, respectively; (a20) heavy and light chain sequences comprising SEQ ID NOs: 77 (or 87) and 29, respectively; (a21) heavy and light chain sequences comprising SEQ ID NOs: 97 (or 107) and 29, respectively; (a22) heavy and light chain sequences comprising SEQ ID NOs: 315 (or 316) and 29, respectively; (a23) heavy and light chain sequences comprising SEQ ID NOs: 78 (or 88) and 29, respectively; (a24) heavy and light chain sequences comprising SEQ ID NOs: 98 (or 108) and 29, respectively; (a25) heavy and light chain sequences comprising SEQ ID NOs: 317 (or 318) and 29, respectively; (a26) heavy and light chain sequences comprising SEQ ID NOs: 79 (or 89) and 29, respectively; (a27) heavy and light chain sequences comprising SEQ ID NOs: 99 (or 109) and 29, respectively; (a28) heavy and light chain sequences comprising SEQ ID NOs: 319 (or 320) and 29, respectively; (a29) heavy and light chain sequences comprising SEQ ID NOs: 349 (or 350) and 29, respectively; (a30) heavy and light chain sequences comprising SEQ ID NOs: 351 (or 352) and 29, respectively; (a31) heavy and light chain sequences comprising SEQ ID NOs: 353 (or 354) and 29, respectively; (b1) heavy and light chain sequences comprising SEQ ID NOs: 321 (or 322) and 30, respectively; (b2) heavy and light chain sequences comprising SEQ ID NOs: 2 (or 9) and 30, respectively; (b3) heavy and light chain sequences comprising SEQ ID NOs: 16 (or 23) and 30, respectively; (b4) heavy and light chain sequences comprising SEQ ID NOs: 323 (or 324) and 30, respectively; (b5) heavy and light chain sequences comprising SEQ ID NOs: 80 (or 90) and 30, respectively; (b6) heavy and light chain sequences comprising SEQ ID NOs: 100 (or 110) and 30, respectively; (b7) heavy and light chain sequences comprising SEQ ID NOs: 325 (or 326) and 30, respectively; (c1) heavy and light chain sequences comprising SEQ ID NOs: 327 (or 328) and 30, respectively; (c2) heavy and light chain sequences comprising SEQ ID NOs: 3 (or 10) and 30, respectively; (c3) heavy and light chain sequences comprising SEQ ID NOs: 17 (or 24) and 30, respectively; (c4) heavy and light chain sequences comprising SEQ ID NOs: 329 (or 330) and 30, respectively; (d1) heavy and light chain sequences comprising SEQ ID NOs: 331 (or 332) and 29, respectively; (d2) heavy and light chain sequences comprising SEQ ID NOs: 4 (or 11) and 29, respectively; (d3) heavy and light chain sequences comprising SEQ ID NOs: 18 (or 25) and 29, respectively; (d4) heavy and light chain sequences comprising SEQ ID NOs: 333 (or 334) and 29, respectively; (e1.1) heavy and light chain sequences comprising SEQ ID NOs: 335 (or 336) and 32, respectively; (e1.2) heavy and light chain sequences comprising SEQ ID NOs: 335 (or 336) and 33, respectively; (e1.3) heavy and light chain sequences comprising SEQ ID NOs: 335 (or 336) and 31, respectively; (e2) heavy and light chain sequences comprising SEQ ID NOs: 5 (or 12) and 33, respectively; (e3) heavy and light chain sequences comprising SEQ ID NOs: 19 (or 26) and 33, respectively; (e4) heavy and light chain sequences comprising SEQ ID NOs: 337 (or 338) and 33, respectively; (e5) heavy and light chain sequences comprising SEQ ID NOs: 81 (or 91) and 33, respectively; (e6) heavy and light chain sequences comprising SEQ ID NOs: 101 (or 111) and 33, respectively; (e7) heavy and light chain sequences comprising SEQ ID NOs: 339 (or 340) and 33, respectively; (f1) heavy and light chain sequences comprising SEQ ID NOs: 341 (or 342) and 29, respectively; (f2) heavy and light chain sequences comprising SEQ ID NOs: 6 (or 13) and 29, respectively; (f3) heavy and light chain sequences comprising SEQ ID NOs: 20 (or 27) and 29, respectively; (f4) heavy and light chain sequences comprising SEQ ID NOs: 343 (or 344) and 29, respectively; (g1) heavy and light chain sequences comprising SEQ ID NOs: 345 (or 346) and 30, respectively; (g2) heavy and light chain sequences comprising SEQ ID NOs: 7 (or 14) and 30, respectively; (g3) heavy and light chain sequences comprising SEQ ID NOs: 21 (or 28) and 30, respectively; (g4) heavy and light chain sequences comprising SEQ ID NOs: 347 (or 348) and 30, respectively; (h1) heavy and light chain sequences comprising SEQ ID NOs: 386 (or 387) and 408, respectively; (h2) heavy and light chain sequences comprising SEQ ID NOs: 388 (or 389) and 408, respectively; (h3) heavy and light chain sequences comprising SEQ ID NOs: 390 (or 391) and 408, respectively; (h4) heavy and light chain sequences comprising SEQ ID NOs: 392 (or 393) and 408, respectively; (i1.1) heavy and light chain sequences comprising SEQ ID NOs: 394 (or 395) and 29, respectively; (i1.2) heavy and light chain sequences comprising SEQ ID NOs: 394 (or 395) and 409, respectively; (i2) heavy and light chain sequences comprising SEQ ID NOs: 396 (or 397) and 29, respectively; (i3) heavy and light chain sequences comprising SEQ ID NOs: 398 (or 399) and 29, respectively; (i4) heavy and light chain sequences comprising SEQ ID NOs: 400 (or 401) and 29, respectively; (i5) heavy and light chain sequences comprising SEQ ID NOs: 402 (or 403) and 29, respectively; (i6) heavy and light chain sequences comprising SEQ ID NOs: 404 (or 405) and 29, respectively; (i7) heavy and light chain sequences comprising SEQ ID NOs: 406 (or 407) and 29, respectively; (j1) heavy and light chain sequences comprising SEQ ID NOs: 451 (or 461) and 453, respectively; (j2) heavy and light chain sequences comprising SEQ ID NOs: 452 (or 462) and 453, respectively; (k1) heavy chain and light chain sequences comprising SEQ ID NOs: 454 (or 463) and 33, respectively; (k2) heavy and light chain sequences comprising SEQ ID NOs: 455 (or 464) and 33, respectively; (l1) heavy and light chain sequences comprising SEQ ID NOs: 457 (or 465) and 453, respectively; (l2) heavy and light chain sequences comprising SEQ ID NOs: 458 (or 466) and 453, respectively; (m1) heavy and light chain sequences comprising SEQ ID NOs: 459 (or 467) and 33, respectively; or (m2) heavy and light chain sequences comprising SEQ ID NOs: 460 (or 468) and 33, respectively an isolated anti-human TIM3 antibody or antigen-binding portion thereof, comprising:

[0309] In certain embodiments, the anti-TIM3 antibody comprises a combination of heavy and light chain sequences as set forth herein, e.g., in the paragraph above, where the antibody comprises two heavy chains and two light chains and may further comprise at least one disulfide bond linking the two heavy chains together. The antibody may also comprise a disulfide bond linking each of the light chains to each of the heavy chains.

[0310] Heavy and light chains comprising amino acid sequences at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, or 70% identical to any of the heavy and light chains (or variable regions thereof) set forth herein, e.g., SEQ ID NOS: 1-33, 72-111, 301-354, 386-409, 451, 452, 453, 454, 455, 457, 458, 459, 460, and 461-468, can be used to generate anti-human TIM3 antibodies with desired characteristics, e.g., those further described herein. Exemplary variants include, for example, allotypic variants in the constant domain and / or those containing mutations in the variable or constant regions, e.g., mutations disclosed herein. Heavy and light chains containing amino acid sequences that differ by up to 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid (by substitution, addition, or deletion) from any of the heavy or light chains (or variable regions thereof) set forth herein can be used to generate anti-human TIM3 antibodies with desired characteristics, such as those further described herein.

[0311] In certain embodiments, the antibody exhibits one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, or all of the following functional properties: (1) For example, when measured using the Biacore described in the Examples, soluble human TIM3 has a K of, for example, 10 nM or less (e.g., 0.01 nM to 10 nM). D Combine with; (2) For example, when measured using a Biacore as described in the Examples, soluble cynomolgus monkey TIM3 has a K of, for example, 100 nM or less (for example, 0.01 nM to 100 nM). D Combine with; (3) For example, when measured by flow cytometry as described in the Examples, membrane-bound human TIM3 has an EC of, for example, 1 μg / mL or less (for example, 0.01 μg / mL to 1 μg / mL). 50 Combine with; (4) For example, as measured by Scatchard analysis described in the Examples, the K D Combine with; (5) For example, when measured by flow cytometry as described in the Examples, membrane-bound cynomolgus monkey TIM3 has an EC of, for example, 20 μg / mL or less (e.g., 0.01 μg / mL to 20 μg / mL). 50 Combine with; (6) For example, as measured by Scatchard analysis described in the Examples, the K D Combine with; (7) inducing or enhancing T cell activation (e.g., by blocking or reducing the inhibitory effect of TIM3), as evidenced by, for example, (i) increasing IFN-γ production by TIM3-expressing T cells (e.g., Th1 cells or TILs) and / or (ii) increasing proliferation of TIM3-expressing T cells (e.g., Th1 cells or TILs), as described in the Examples; (8) stimulating T cell proliferation in a mixed lymphocyte reaction (MLR) assay, e.g., as described in the Examples; (9) inhibiting the binding of phosphatidylserine to TIM3, e.g., as measured by a PS-hTIM3 “in tandem” blocking assay as described in the Examples; (10) when bound to cellular TIM3, it does not internalize or downregulate cell surface TIM3; (11) For example, as described in the Examples, it binds to one of the following regions of the human TIM3 extracellular domain (SEQ ID NO: 290): (a) CPVFECG (SEQ ID NO: 296); (b) RIQIPGIMND (SEQ ID NO: 298); (c) CPVFECG and RIQIPGIMND (SEQ ID NOs: 296 and 298, respectively); and (d) WTSRYWLNGDFR (SEQ ID NO: 297); (12) As described in the Examples, the binding to human TIM3 in which one or more of the amino acids L48, C58, P59, V60, F61, E62, C63, G64, W78, S80, R81, W83, L84, G86, D87, R89, D104, R111, Q113, G116, M118, and D120 (numbered in SEQ ID NO: 286 ( FIG. 25 )) have been substituted with another amino acid is reduced compared to the binding to wild-type human TIM3; (13) competes for binding to human TIM3 in either or both orientations with an antibody comprising the VH and VL domains of any of 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3, TIM3.7, TIM3.8, TIM3.10, TIM3.11, TIM3.12, TIM3.13, TIM3.14, TIM3.15, TIM3.16, TIM3.17, TIM3.18, and TIM3.25 described in the Examples; (14) For example, as described in the Examples, the human TIM3 region was determined by HDX-MS. 49 VPVCWGKGACPVFE 62 (SEQ ID NO: 367) and 111 RIQIPGIMNDEKFNLKL 127 binds to (SEQ ID NO: 368); (15) Having a heavy chain and / or light chain variable region that interacts with at least 5, 10, 15, 20, or all of the following amino acids of human TIM3, P50, V51, C52, P59, V60, F61, E62, C63, G64, N65, V66, V67, L68, R69, D71, E72, D74, R111, Q113, G116, I117, M118, D120, and optionally T70 and / or I112, as determined by X-ray crystallography (e.g., as described in the Examples; numbering according to SEQ ID NO: 286 (Figure 25)); (16) (a) exhibits reduced binding to human TIM3 in which one, two, three, four, five, six, seven, eight, or nine of the amino acids C58, P59, F61, E62, C63, R111, D120, and optionally D104 and Q113 (numbering according to SEQ ID NO: 286 (FIG. 25)) have been substituted with another amino acid, compared to binding to wild-type human TIM3; (b) exhibits reduced binding to human TIM3 in which one, two, three, four, five, six, seven, eight, or nine of the amino acids C58, P59, F61, E62, C63, R111, D120, and optionally D104 and Q113 (numbering according to SEQ ID NO: 286 (FIG. 25)) have been substituted with another amino acid, as determined by HDX-MS as described in the Examples. 49 VPVCWGKGACPVFE 62 (SEQ ID NO: 367), 111 RIQIPGIMNDEKFNLKL 127 (SEQ ID NO: 368) and 119 NDEKFNLKL 127 (SEQ ID NO: 373); and / or (c) competes with or cross-blocks the binding of 13A3 or TIM3.18.IgG1.3 to human TIM3, e.g., as described in the Examples; (17) hTIM3-IgV was measured using the method described in Example 22 and found to have a 5 × 10 -8 M, 2 x 10 -8 M, 10 -8 M or 5x10 -9 K below M D Combine with; (18) hTIM3-IgV was measured by the method described in Example 22. -7 M, 2 x 10 -8 M or 10 -8 K below M D Combine with; (19) 5 × 10 TIM3-ECD cells were measured using the method described in Example 22. -7 M, 2 x 10 -8M or 10 -8 K below M D and / or (20) hTIM3-ECD was measured using the method described in Example 22, and 8 × 10 -8 M, 5 x 10 -8 M or 10 -8 K below M D Combine with.

[0312] Such antibodies include, for example, human antibodies, humanized antibodies, or chimeric antibodies.

[0313] In certain embodiments, the anti-TIM3 antibodies described herein bind to a conformational epitope.

[0314] In certain embodiments, the anti-TIM3 antibodies described herein comprise the following region of the mature human TIM3 extracellular domain (SEQ ID NO: 290), which corresponds to amino acid residues 1-99 of the mature human TIM3 extracellular domain (SEQ ID NO: 290) or amino acids 22-120 of human TIM3 having SEQ ID NO: 286: [ka] (SEQ ID NO: 299).

[0315] In certain embodiments, the anti-TIM3 antibodies described herein bind to amino acid residues in the next region of the mature human TIM3 extracellular domain (SEQ ID NO: 290), CPVFECG (SEQ ID NO: 296), which corresponds to amino acid residues 37-43 of the mature human TIM3 extracellular domain (SEQ ID NO: 290).

[0316] In certain embodiments, the anti-TIM3 antibodies described herein bind to amino acid residues in the next region of the mature human TIM3 extracellular domain (SEQ ID NO: 290), WTSRYWLNGDFR (SEQ ID NO: 297), which corresponds to amino acid residues 57-83 of the mature human TIM3 extracellular domain (SEQ ID NO: 290).

[0317] In certain embodiments, the anti-TIM3 antibodies described herein bind to amino acid residues in the next region of the mature human TIM3 extracellular domain (SEQ ID NO: 290), RIQIPGIMND (SEQ ID NO: 298), which corresponds to amino acid residues 90-99 of the mature human TIM3 extracellular domain (SEQ ID NO: 290).

[0318] In certain embodiments, the anti-TIM3 antibody has the same pattern of binding to wild-type and mutant human TIM3 as one or more of antibodies 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3, and TIM3.2-TIM3.18, TIM3.24, and TIM3.25. In certain embodiments, the anti-TIM3 antibody binds to amino acid residues within the following regions of the mature human TIM3 extracellular domain (SEQ ID NO: 290): CPVFECG (SEQ ID NO: 296), WTSRYWLNGDFRKGDVSLTIENVTLAD (SEQ ID NO: 297), and / or RIQIPGIMND (SEQ ID NO: 298).

[0319] In certain embodiments, the anti-TIM3 antibody is: (1) IgG1-associated IgG2-associated IgG3-associated IgG4-associated IgG5-associated IgG6-associated IgG7-associated IgG8-associated IgG1-associated IgG2-associated IgG4-associated IgG5-associated IgG6-associated IgG1-associated IgG2-associated IgG4-associ 49 VPVCWGKGACPVFE 62 (SEQ ID NO: 367) and 111 RIQIPGIMNDEKFNLKL 127 (SEQ ID NO: 368) or (2) 40 YTPAAPGNLVPVCWGKGACPVFE 62 (SEQ ID NO: 369), 66 VVLRTDERDVNY 77 (SEQ ID NO: 370), 78 WTSRYWLNGDFRKGDVSL 95 (SEQ ID NO: 371), 110 CRIQIPGIMNDEKFNLKL 127 (SEQ ID NO: 372) and 119 NDEKFNLKL 127(SEQ ID NO: 373). In certain embodiments, the anti-TIM3 antibody interacts with the regions of amino acid residues 40-62 and 111-127 of hTIM3, but does not significantly interact with other regions, e.g., the region N-terminal to amino acid residue Y40, the region located at amino acid residues E62-R111, and the region C-terminal to amino acid residue L127, as determined by HDX-MS, e.g., as described in the Examples.

[0320] In certain embodiments, the anti-TIM3 antibody exhibits reduced binding to human TIM3 in which one or more of amino acids L48, C58, P59, V60, F61, E62, C63, G64, W78, S80, R81, W83, L84, G86, D87, R89, D104, R111, Q113, G116, M118, and D120 (numbered in SEQ ID NO: 286 ( FIG. 25 )) have been substituted with another amino acid compared to binding to wild-type human TIM3, e.g., as determined by HDX-MS as described in the Examples, and the antibody is 49 VPVCWGKGACPVFE 62 (SEQ ID NO: 367) and 111 RIQIPGIMNDEKFNLKL 127 (SEQ ID NO: 368) or (2) 40 YTPAAPGNLVPVCWGKGACPVFE 62 (SEQ ID NO: 369), 66 VVLRTDERDVNY 77 (SEQ ID NO: 370), 78 WTSRYWLNGDFRKGDVSL 95 (SEQ ID NO: 371), 110 CRIQIPGIMNDEKFNLKL 127 (SEQ ID NO: 372) and 119 NDEKFNLKL 127 (SEQ ID NO: 373).

[0321] In certain embodiments, the anti-TIM3 antibody has a similar pattern of binding to wild-type and mutant human TIM3 as TIM3.18.IgG1.3 or 13A3, i.e., the antibody: (i) As determined by HDX-MS, (1)49 VPVCWGKGACPVFE 62 (SEQ ID NO: 367), 111 RIQIPGIMNDEKFNLKL 127 (SEQ ID NO: 368) and 119 NDEKFNLKL 127 (SEQ ID NO: 373), for example, (a) a peptide having a sequence located at the N-terminus of amino acid residue 49; (b) a peptide having a sequence located at amino acid residues 62 to 111 (e.g., 78 WTSRYWLNGDFRKGDVSL 95 (SEQ ID NO: 371); and (c) does not significantly bind to a peptide having a sequence located C-terminal to amino acid residue 127 (e.g., as described in the Examples); (ii) does not bind to human TIM3 with one or more of the following amino acid mutations or has significantly reduced binding to human TIM3 with one or more of the following amino acid mutations (e.g., as described in the Examples): C58, P59, F61, E62, C63, R111, D120, and optionally D104 and Q113 (numbering according to SEQ ID NO: 286 (Figure 25)); and / or (iii) have heavy and / or light chain variable regions that interact with at least 5, 10, 15, 20 or all of the following amino acids of human TIM3 as determined by X-ray crystallography: P50, V51, C52, P59, V60, F61, E62, C63, G64, N65, V66, V67, L68, R69, D71, E72, D74, R111, Q113, G116, I117, M118, D120 and optionally T70 and / or I112 (e.g., as described in the Examples; numbering according to SEQ ID NO: 286 (Figure 25)).

[0322] In certain embodiments, the anti-TIM3 antibody comprises a heavy chain and a light chain, wherein the heavy chain is selected from the group consisting of SEQ ID NOs: 72-111, 305-320, 325-326, 339-340, 349-354, 402-407, 451, 452, 454, 455, 457, 458, 459, 460, and 461-468, and / or the light chain is selected from the group consisting of SEQ ID NOs: 29-33 and 453.

[0323] As further described herein, the heavy chain constant region of the anti-TIM3 antibodies described herein can be of any isotype, e.g., IgG1, IgG2, IgG3, and IgG4, or combinations and / or modifications thereof. The anti-TIM3 antibodies can have effector function, or can have reduced or no effector function. In certain embodiments, the anti-TIM3 antibodies comprise a modified heavy chain constant region that enhances the properties of the antibody.

[0324] In certain embodiments, the anti-TIM3 antibody comprises a heavy chain and a light chain, wherein the heavy chain is selected from the group consisting of SEQ ID NOs: 72-111, 349-352, 402-405, 451, 452, 454, 455, 457, 458, 459, 460, and 461-468, and / or the light chain is selected from the group consisting of SEQ ID NOs: 29-33 and 453.

[0325] III. Antibodies with specific germline sequences In certain embodiments, the anti-TIM3 antibody comprises a heavy chain variable region from a particular germline heavy chain immunoglobulin gene and / or a light chain variable region from a particular germline light chain immunoglobulin gene.

[0326] As provided herein, human antibodies specific for TIM3 have been produced that contain heavy chain variable regions that are the product of or derived from human germline VH 4-39 gene, VH 4-59 gene, VH 1-46 gene, VH 3-11, VH 4-17 gene, VH 3-10 gene, VH 6-19 gene, VH 6-13 gene, VH 4-23, VH JH4b, VH JH5b gene and / or VH JH6b gene. Accordingly, provided herein is an isolated monoclonal antibody, or antigen-binding portion thereof, comprising a heavy chain variable region that is the product of or derived from a human VH germline gene selected from the group consisting of VH 4-39, VH 4-59, VH 1-46, VH 3-11, VH 4-17, VH 3-10, VH6-19, VH 6-13, VH 4-23, VH JH4b, VH JH5b, VH JH6b, and any combination thereof.

[0327] Human antibodies specific for TIM3 have been produced that contain a light chain variable region that is the product of or derived from a human germline VK A27 gene, VK JK5 gene, VK JK4 gene, VK JK3, VK L18 gene, and / or VK JK1 gene. Accordingly, provided herein is an isolated monoclonal antibody, or antigen-binding portion thereof, that contains a light chain variable region that is the product of or derived from a human VK germline gene selected from the group consisting of VK A27, VK JK5, VK JK4, VK JK3, VK L18, VK JK1, and any combination thereof.

[0328] The anti-TIM3 antibodies described herein include those that contain a heavy chain variable region that is the product of, or derived from, one of the above human germline VH genes, and also contain a light chain variable region that is the product of, or derived from, one of the above human germline VK genes, as shown in the figures.

[0329] As used herein, a human antibody includes heavy and light chain variable regions that are "the product" or "derived" from a particular germline sequence if the antibody variable regions are obtained from a system that uses human germline immunoglobulin genes. Such systems include immunizing transgenic mice carrying human immunoglobulin genes with the antigen of interest or screening a phage-displayed human immunoglobulin gene library with the antigen of interest. A human antibody that is "the product" or "derived" from a human germline immunoglobulin sequence can itself be identified by comparing the amino acid sequence of the human antibody with that of a human germline immunoglobulin and selecting the human germline immunoglobulin sequence that is closest in sequence to the human antibody sequence (i.e., most closely in percent identity). A human antibody that is "the product" or "derived" from a particular human germline immunoglobulin sequence can contain amino acid differences compared to the germline sequence, for example, due to naturally occurring somatic mutations or deliberate introduction of site-specific mutations. However, a selected human antibody will generally be at least 90% identical in amino acid sequence to the amino acid sequence encoded by a human germline immunoglobulin gene and will contain amino acid residues that identify it as human when compared to germline immunoglobulin amino acid sequences of other species (e.g., murine germline sequences). In some cases, a human antibody may be at least 95%, or at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Generally, a human antibody derived from a particular human germline sequence will not display more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In some cases, a human antibody will not display more than 5, or even more than 4, 3, 2, or 1 amino acid differences from the amino acid sequence encoded by the germline immunoglobulin gene.

[0330] IV. Homologous antibodies Included herein are antibodies comprising heavy and light chain variable regions having amino acid sequences that are homologous to the amino acid sequences of the anti-TIM3 antibodies described herein, wherein the antibodies retain the desired functional properties of the anti-TIM3 antibodies described herein.

[0331] For example, an isolated anti-TIM3 antibody, or antigen-binding portion thereof, can comprise a heavy chain variable region and a light chain variable region, (a) the heavy chain variable region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 34-40, 112-121, 364, 410-412, 449, and 456, or contains 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-50 amino acid alterations (i.e., amino acid substitutions, additions, or deletions) relative to an amino acid sequence selected from the group consisting of SEQ ID NOs: 34-40, 112-121, 364, 410-412, 449, and 456, wherein optionally the heavy chain variable region comprises the CDR sequence of one of the anti-TIM3 antibodies described herein; (b) the light chain variable region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 60-63, 417, and 418, or contains 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-50 amino acid alterations (i.e., amino acid substitutions, additions, or deletions) relative to an amino acid sequence selected from the group consisting of SEQ ID NOs: 60-63, 417, 418, and 450, wherein optionally the light chain variable region comprises the CDR sequence of one of the anti-TIM3 antibodies described herein; (c) the antibody specifically binds to human TIM3; and (d) the antibody exhibits one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, seventeen, eighteen, nineteen, or all of the following functional properties: (1) For example, when measured using the Biacore described in the Examples, soluble human TIM3 has a K of, for example, 10 nM or less (e.g., 0.01 nM to 10 nM). D Combine with; (2) For example, when measured using a Biacore as described in the Examples, soluble cynomolgus monkey TIM3 has a K of, for example, 100 nM or less (for example, 0.01 nM to 100 nM). DCombine with; (3) For example, when measured by flow cytometry as described in the Examples, membrane-bound human TIM3 has an EC of, for example, 1 μg / mL or less (for example, 0.01 μg / mL to 1 μg / mL). 50 Combine with; (4) For example, as measured by Scatchard analysis described in the Examples, the K D Combine with; (5) For example, when measured by flow cytometry as described in the Examples, membrane-bound cynomolgus monkey TIM3 has an EC of, for example, 20 μg / mL or less (e.g., 0.01 μg / mL to 20 μg / mL). 50 Combine with; (6) For example, as measured by Scatchard analysis described in the Examples, the K D Combine with; (7) inducing or enhancing T cell activation (e.g., by blocking or reducing the inhibitory effect of TIM3), as evidenced by, for example, (i) increasing IFN-γ production by TIM3-expressing T cells (e.g., Th1 cells or TILs) and / or (ii) increasing proliferation of TIM3-expressing T cells (e.g., Th1 cells or TILs), as described in the Examples; (8) stimulating T cell proliferation in a mixed lymphocyte reaction (MLR) assay, e.g., as described in the Examples; (9) inhibiting the binding of phosphatidylserine to TIM3, e.g., as measured by a PS-hTIM3 “in tandem” blocking assay, e.g., as described in the Examples; (10) when bound to cellular TIM3, it does not internalize or downregulate cell surface TIM3; (11) For example, as described in the Examples, it binds to one of the following regions of the human TIM3 extracellular domain (SEQ ID NO: 290): (a) CPVFECG (SEQ ID NO: 296); (b) RIQIPGIMND (SEQ ID NO: 298); (c) CPVFECG and RIQIPGIMND (SEQ ID NOs: 296 and 298, respectively); and (d) WTSRYWLNGDFR (SEQ ID NO: 297); (12) For example, as described in the Examples, the binding to human TIM3 in which one or more of the amino acids L48, C58, P59, V60, F61, E62, C63, G64, W78, S80, R81, W83, L84, G86, D87, R89, D104, R111, Q113, G116, M118, and D120 (numbered in SEQ ID NO: 286 ( FIG. 25 )) have been substituted with other amino acids is reduced compared to the binding to wild-type human TIM3; (13) Competes in either or both directions for binding to human TIM3 with an antibody comprising the VH and VL domains of any of 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3, or TIM3.7, TIM3.8, TIM3.10, TIM3.11, TIM3.12, TIM3.13, TIM3.14, TIM3.15, TIM3.16, TIM3.17, TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, and TIM3.25 described in the Examples; (14) For example, as described in the Examples, the human TIM3 region was determined by HDX-MS. 49 VPVCWGKGACPVFE 62 (SEQ ID NO: 367) and 111 RIQIPGIMNDEKFNLKL 127 binds to (SEQ ID NO: 368); (15) Having a heavy chain and / or light chain variable region that interacts with at least 5, 10, 15, 20, or all of the following amino acids of human TIM3, P50, V51, C52, P59, V60, F61, E62, C63, G64, N65, V66, V67, L68, R69, D71, E72, D74, R111, Q113, G116, I117, M118, D120, and optionally T70 and / or I112, as determined by X-ray crystallography (e.g., as described in the Examples; numbering according to SEQ ID NO: 286 (Figure 25)); (16) (a) exhibits reduced binding to human TIM3 in which one, two, three, four, five, six, seven, eight, or nine of the amino acids C58, P59, F61, E62, C63, R111, D120, and optionally D104 and Q113 (numbering according to SEQ ID NO: 286 (FIG. 25)) have been substituted with another amino acid, compared to binding to wild-type human TIM3; (b) exhibits reduced binding to human TIM3 in which one, two, three, four, five, six, seven, eight, or nine of the amino acids C58, P59, F61, E62, C63, R111, D120, and optionally D104 and Q113 (numbering according to SEQ ID NO: 286 (FIG. 25)) have been substituted with another amino acid, as determined by HDX-MS as described in the Examples. 49 VPVCWGKGACPVFE 62 (SEQ ID NO: 367), 111 RIQIPGIMNDEKFNLKL 127 (SEQ ID NO: 368) and 119 NDEKFNLKL 127 (SEQ ID NO: 373); and / or (c) competes with or cross-blocks the binding of 13A3 or TIM3.18.IgG1.3 to human TIM3, e.g., as described in the Examples; (17) hTIM3-IgV was measured using the method described in Example 22 and found to have a 5 × 10 -8 M, 2 x 10 -8 M, 10 -8 M or 5x10 -9 K below M D Combine with; (18) hTIM3-IgV was measured by the method described in Example 22. -7 M, 2 x 10 -8 M or 10 -8 K below M D Combine with; (19) 5 × 10 TIM3-ECD cells were measured using the method described in Example 22. -7 M, 2 x 10 -8 M or 10 -8 K below M D and / or (20) hTIM3-ECD was measured using the method described in Example 22, and 8 × 10 -8 M, 5 x 10 -8 M or 10 -8 K below M D Combine with.

[0332] In certain embodiments, the antibody may exhibit one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or all of the functional properties listed above in (1) to (20). The antibody may be, for example, a human antibody, a humanized antibody, or a chimeric antibody.

[0333] The isolated anti-TIM3 antibody, or antigen-binding portion thereof, can comprise a heavy chain and a light chain, wherein: (a) Heavy chain SEQ ID NOS: 1 to 28, 72 to 111, and 349 to 352, 388 to 391 (14H7 / TIM3.24 HC IgG1.1f and IgG1.3f), 396 to 399 (23B3 HC IgG1.1f and IgG1.3f), 402 to 405 (TIM3.25 HC IgG1f and IgG1.3f), 451, 452, 461, 462 (TIM3.20 HC IgG1f and IgG1.3f), 454, 455, 463, 464 (TIM3.21 HC IgG1f and IgG1.3f), 457, 458, 465, 466 (TIM3.22 HC or SEQ ID NOs: 1-28, 72-111, 349-352, 388-391 (14H7 / TIM3.24 HC IgG1.1f and IgG1.3f), 396-399 (23B3 HC IgG1.1f and IgG1.3f), 402-405 (TIM3.25 HC IgG1.1f and IgG1.3f), 451, 452, 461, 462 (TIM3.20 HC IgG1.1f and IgG1.3f), and 459, 460, 467, 468 (TIM3.23 HC IgG1f and IgG1.3f), or SEQ ID NOs: 1-28, 72-111, 349-352, 388-391 (14H7 / TIM3.24 HC IgG1.1f and IgG1.3f), 396-399 (23B3 HC IgG1.1f and IgG1.3f), 402-405 (TIM3.25 HC IgG1.1f and IgG1.3f), 451, 452, 461, 462 (TIM3.20 HC IgG.1f and IgG1.3f), 454, 455, 463, 464 (TIM3.21 HC IgG1f and IgG1.3f), 457, 458, 465, 466 (TIM3.22 HC IgG1f and IgG1.3f), and 459, 460, 467, 468 (TIM3.23 HC and IgG1.3f), with the proviso that, in certain embodiments, if the sequence is an effectorless heavy chain, the mutations that render the heavy chain effectorless for the IgG1.1 constant region are not modified (e.g., R214, A234, E235, A237, S330, and S331 are not modified), and IgG1.R214, A234, and E235 of the three constant regions are unmodified, and optionally the heavy chain variable region comprises the CDR sequences of one of the anti-TIM3 antibodies described herein; (b) the light chain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-33, 408, 409 and 453, or contains 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25 or 1-50 amino acid alterations (i.e., amino acid substitutions, additions, or deletions) relative to an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-33, 408, 409 and 453, wherein optionally the light chain variable region comprises the CDR sequence of one of the anti-TIM3 antibodies described herein; (c) the antibody specifically binds to human TIM3; and (d) the antibody exhibits one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or all of the following functional properties: (1) For example, when measured using the Biacore described in the Examples, soluble human TIM3 has a K of, for example, 10 nM or less (e.g., 0.01 nM to 10 nM). D Combine with; (2) For example, when measured using a Biacore as described in the Examples, soluble cynomolgus monkey TIM3 has a K of, for example, 100 nM or less (for example, 0.01 nM to 100 nM). D Combine with; (3) For example, when measured by flow cytometry as described in the Examples, membrane-bound human TIM3 has an EC of, for example, 1 μg / mL or less (for example, 0.01 μg / mL to 1 μg / mL). 50 Combine with; (4) For example, as measured by Scatchard analysis described in the Examples, the K D Combine with; (5) For example, when measured by flow cytometry as described in the Examples, membrane-bound cynomolgus monkey TIM3 has an EC of, for example, 20 μg / mL or less (e.g., 0.01 μg / mL to 20 μg / mL).50 Combine with; (6) For example, as measured by Scatchard analysis described in the Examples, the K D Combine with; (7) inducing or enhancing T cell activation (e.g., by blocking or reducing the inhibitory effect of TIM3), as evidenced by, for example, (i) increasing IFN-γ production by TIM3-expressing T cells (e.g., Th1 cells or TILs) and / or (ii) increasing proliferation of TIM3-expressing T cells (e.g., Th1 cells or TILs), as described in the Examples; (8) stimulating T cell proliferation in a mixed lymphocyte reaction (MLR) assay, e.g., as described in the Examples; (9) inhibiting the binding of phosphatidylserine to TIM3, e.g., as measured by a PS-hTIM3 “in tandem” blocking assay, e.g., as described in the Examples; (10) when bound to cellular TIM3, it does not internalize or downregulate cell surface TIM3; (11) For example, as described in the Examples, it binds to one of the following regions of the human TIM3 extracellular domain (SEQ ID NO: 290): (a) CPVFECG (SEQ ID NO: 296); (b) RIQIPGIMND (SEQ ID NO: 298); (c) CPVFECG and RIQIPGIMND (SEQ ID NOs: 296 and 298, respectively); and (d) WTSRYWLNGDFR (SEQ ID NO: 297); (12) Reduced binding to human TIM3 in which one or more of the amino acids L48, C58, P59, V60, F61, E62, C63, G64, W78, S80, R81, W83, L84, G86, D87, R89, D104, R111, Q113, G116, M118, and D120 (numbered in SEQ ID NO: 286 ( FIG. 25 )) have been substituted with another amino acid, compared to binding to wild-type human TIM3, e.g., as described in the Examples; (13) Competes in either or both directions for binding to human TIM3 with an antibody comprising the VH and VL domains of any of 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3, TIM3.7, TIM3.8, TIM3.10, TIM3.11, TIM3.12, TIM3.13, TIM3.14, TIM3.15, TIM3.16, TIM3.17, TIM3.18, and TIM3.25 described in the Examples; (14) For example, as described in the Examples, the human TIM3 region was determined by HDX-MS. 49 VPVCWGKGACPVFE 62 (SEQ ID NO: 367) and 111 RIQIPGIMNDEKFNLKL 127 binds to (SEQ ID NO: 368); (15) Having a heavy chain and / or light chain variable region that interacts with at least 5, 10, 15, 20, or all of the following amino acids of human TIM3, P50, V51, C52, P59, V60, F61, E62, C63, G64, N65, V66, V67, L68, R69, D71, E72, D74, R111, Q113, G116, I117, M118, D120, and optionally T70 and / or I112, as determined by X-ray crystallography (e.g., as described in the Examples; numbering according to SEQ ID NO: 286 (Figure 25)); (16) For example, as described in the Examples, (a) exhibits reduced binding to human TIM3 in which one, two, three, four, five, six, seven, eight, or nine of the amino acids C58, P59, F61, E62, C63, R111, D120, and optionally D104 and Q113 (numbering according to SEQ ID NO: 286 ( FIG. 25 )) have been substituted with another amino acid, compared to binding to wild-type human TIM3; (b) exhibits reduced binding to human TIM3 in which one, two, three, four, five, six, seven, eight, or nine of the amino acids C58, P59, F61, E62, C63, R111, D120, and optionally D104 and Q113 (numbering according to SEQ ID NO: 286 ( FIG. 25 )) have been substituted with another amino acid, as determined by HDX-MS, as described in the Examples. 49 VPVCWGKGACPVFE 62 (SEQ ID NO: 367), 111 RIQIPGIMNDEKFNLKL 127 (SEQ ID NO: 368) and 119 NDEKFNLKL 127(SEQ ID NO: 373); and / or (c) competes with or cross-blocks the binding of 13A3 or TIM3.18.IgG1.3 to human TIM3; (17) hTIM3-IgV was measured using the method described in Example 22 and found to have a 5 × 10 -8 M, 2 x 10 -8 M, 10 -8 M or 5x10 -9 K below M D Combine with; (18) hTIM3-IgV was measured by the method described in Example 22. -7 M, 2 x 10 -8 M or 10 -8 K below M D Combine with; (19) 5 × 10 TIM3-ECD cells were measured using the method described in Example 22. -7 M, 2 x 10 -8 M or 10 -8 K below M D and / or (20) hTIM3-ECD was measured using the method described in Example 22, and 8 × 10 -8 M, 5 x 10 -8 M or 10 -8 K below M D Combine with.

[0334] Also provided are anti-TIM3 antibodies comprising a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1, a VL CDR2 and / or a VL CDR3 that differ from the corresponding CDRs of any of 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3, or TIM3.2 to TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24 and TIM3.25 by 1, 2, 3, 4, 5, 1 to 2, 1 to 3, 1 to 4, or 1 to 5 amino acid changes (i.e., amino acid substitutions, additions, or deletions). In certain embodiments, the anti-TIM3 antibody contains 1 to 5 amino acid changes in each of 1, 2, 3, 4, 5, or 6 of the CDRs relative to the corresponding sequence of 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3, or any of TIM3.2 to TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24, and TIM3.25. In certain embodiments, the anti-TIM3 antibody contains a total of 1 to 5 amino acid changes across all CDRs compared to the CDRs of 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3, or any of TIM3.2 to TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24, and TIM3.25.

[0335] In certain embodiments, the anti-TIM3 antibody comprises VH and VL CDRs consisting of those of 13A3, wherein one or more of the amino acids in one or more CDRs are those of one of the other anti-TIM3 antibodies disclosed herein.

[0336] For example, in one embodiment, an anti-TIM3 antibody comprises a VH CDR1 that contains one or more amino acid modifications relative to SRSYYWG (SEQ ID NO: 41), and may comprise, for example, the following degenerate sequence: X1X2X3X4YX5X6 (SEQ ID NO: 282) (wherein X1 is any amino acid, e.g., S or none; X2 is any amino acid, e.g., R or none; X3 is any amino acid, e.g., S, R, or D; X4 is any amino acid, e.g., Y or H; X5 is any amino acid, e.g., W or M; and X6 is any amino acid, e.g., G, N, S, or H).

[0337] In some embodiments, the anti-TIM3 antibody comprises a VH CDR2 that contains one or more amino acid modifications relative to SIYYSGFTYYNPSLKS (SEQ ID NO: 46), e.g., the following degenerate sequence: X1IX2X3X4GX5X6X7X8YX9X 10 X 11 X 12 X 13 X 14 (SEQ ID NO: 283) (wherein X1 is any amino acid, e.g., S, Y, I, or F; X2 is any amino acid, e.g., Y, H, N, or S; X3 is any amino acid, e.g., Y, P, G, T, S, or N; X4 is any amino acid, e.g., S, T, R, or G; X5 is any amino acid, e.g., F, S, or D; X6 is any amino acid, e.g., S, T, or I; X7 is any amino acid, e.g., I, or none; X8 is any amino acid, e.g., Y, N, or I; X9 is any amino acid, e.g., N, Q, S, or A; X 10 is any amino acid, e.g., P, S, Q, or D; X 11 is any amino acid, e.g., S or K; X 12 is any amino acid, e.g., L, F, or V; X 13 is any amino acid, e.g., K or Q; and X 14 may contain any amino acid, e.g., S or G).

[0338] In some embodiments, the anti-TIM3 antibody comprises a VH CDR3 that contains one or more amino acid modifications relative to GGPYGDYAHWFDP (SEQ ID NO: 53), e.g., the following degenerate sequence: X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 (SEQ ID NO: 284) (wherein X1 is any amino acid, e.g., D, E, G, or not; X2 is any amino acid, e.g., F, G, R, or not; X3 is any amino acid, e.g., Y, M, I, or not; X4 is any amino acid, e.g., G, S, V, or not; X5 is any amino acid, e.g., G, T, R, S, or not; X6 is any amino acid, e.g., G, S, or not; X7 is any amino acid, e.g., M, N, W, or not; X8 is any amino acid, e.g., Y, S, E, N, or not; X9 is any amino acid, e.g., Y, or not; X 10 is any amino acid, e.g., F, P, or Y; X 11 is any amino acid, e.g., Y or F; X 12 is any amino acid, e.g., G; X 13 is any amino acid, e.g., D; X 14 is any amino acid, e.g., Y; X 15 is any amino acid, e.g., A or not; X 16 is any amino acid, e.g., H; X 17 is any amino acid, e.g., W; X 18 is any amino acid, e.g., F, M, or none; X 19 is any amino acid, e.g., D or E; and X 20 may contain any amino acid, e.g., P, I, V, Y, or L).

[0339] In certain embodiments, the anti-TIM3 antibody comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO:64 or SEQ ID NO:65.

[0340] In certain embodiments, the anti-TIM3 antibody comprises a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO:66 or SEQ ID NO:67.

[0341] In some embodiments, the anti-TIM3 antibody comprises a VL CDR3 containing one or more amino acid modifications relative to QQYGSSPIT (SEQ ID NO: 68), and may include, for example, the following degenerate sequence: QQX1X2SX3X4X5X6 (SEQ ID NO: 285) (wherein X1 is any amino acid, e.g., F or Y; X2 is any amino acid, e.g., N or G; X3 is any amino acid, e.g., Y or S; X4 is any amino acid, e.g., P or none; X5 is any amino acid, e.g., I, R, L or none; and X6 is any amino acid, e.g., T or none).

[0342] 13A3, 8B9, 8C4, 17C3, 9F6, 3G4, 8C4, 14H7, 23B3, or any of TIM3.2 to TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24, and TIM3.25, for example, the VH and VL regions of SEQ ID NOs: 34 to 40, 112 to 121, 364, and 410 to 412, and SEQ ID NOs: 60 to 63, 417, and 418, respectively, or the VH and VL regions of SEQ ID NOs: 1 to 28, 72 to 111, 349 to 352, 388 to 391, 396 to 399, or 402 to 405, and SEQ ID NOs: Antibodies having sequences homologous to the heavy and light chains or CDRs of SEQ ID NOs: 29-33, 408, or 409 can be obtained by mutagenizing (e.g., site-directed or PCR-mediated mutagenesis) a nucleic acid molecule encoding SEQ ID NOs: 167-173 and / or 193-196, or SEQ ID NOs: 134-161, 430-437, and / or 162-166 and 442-444, and then testing for retention of function of the encoded modified antibody (i.e., the functions set forth in (1) to (16) above) using the functional assays described herein.

[0343] V. Antibodies with Conservative Modifications An anti-TIM3 antibody can comprise a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences, and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein one or more of these CDR sequences comprise an amino acid sequence identified based on an anti-TIM3 antibody described herein (e.g., 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3, or any of TIM3.2 to TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24, and TIM3.25), or a conservative modification thereof, wherein the antibody retains the desired functional properties of the anti-TIM3 antibodies described herein. Thus, an isolated anti-TIM3 antibody, or antigen-binding portion thereof, can comprise a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein: (a) the heavy chain variable region CDR3 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-59, 126-129, 414, 416, and 471, and conservative modifications thereof, e.g., amino acid sequences with 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, or 1-5 conservative amino acid substitutions, wherein optionally the heavy chain variable region comprises the CDR sequence of one of the anti-TIM3 antibodies described herein; (b) the light chain variable region CDR3 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 68-71, 419, and 474, and conservative modifications thereof, e.g., amino acid sequences with 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, or 1-5 conservative amino acid substitutions, wherein optionally the light chain variable region comprises the CDR sequence of one of the anti-TIM3 antibodies described herein; (c) the antibody specifically binds to human TIM3; and (d) the antibody exhibits one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or all of the following properties: (1) For example, when measured using the Biacore described in the Examples, soluble human TIM3 has a K of, for example, 10 nM or less (e.g., 0.01 nM to 10 nM).D Combine with; (2) For example, when measured using a Biacore as described in the Examples, soluble cynomolgus monkey TIM3 has a K of, for example, 100 nM or less (for example, 0.01 nM to 100 nM). D Combine with; (3) For example, when measured by flow cytometry as described in the Examples, membrane-bound human TIM3 has an EC of, for example, 1 μg / mL or less (for example, 0.01 μg / mL to 1 μg / mL). 50 Combine with; (4) For example, as measured by Scatchard analysis described in the Examples, the K D Combine with; (5) For example, when measured by flow cytometry as described in the Examples, membrane-bound cynomolgus monkey TIM3 has an EC of, for example, 20 μg / mL or less (e.g., 0.01 μg / mL to 20 μg / mL). 50 Combine with; (6) For example, as measured by Scatchard analysis described in the Examples, the K D Combine with; (7) inducing or enhancing T cell activation (e.g., by blocking or reducing the inhibitory effect of TIM3), as evidenced by, for example, (i) increasing IFN-γ production by TIM3-expressing T cells (e.g., Th1 cells or TILs) and / or (ii) increasing proliferation of TIM3-expressing T cells (e.g., Th1 cells or TILs), as described in the Examples; (8) stimulating T cell proliferation in a mixed lymphocyte reaction (MLR) assay, e.g., as described in the Examples; (9) inhibiting the binding of phosphatidylserine to TIM3, e.g., as measured by a PS-hTIM3 “in tandem” blocking assay, e.g., as described in the Examples; (10) when bound to cellular TIM3, it does not internalize or downregulate cell surface TIM3; (11) For example, as described in the Examples, it binds to one of the following regions of the human TIM3 extracellular domain (SEQ ID NO: 290): (a) CPVFECG (SEQ ID NO: 296); (b) RIQIPGIMND (SEQ ID NO: 298); (c) CPVFECG and RIQIPGIMND (SEQ ID NOs: 296 and 298, respectively); and (d) WTSRYWLNGDFR (SEQ ID NO: 297); (12) Reduced binding to human TIM3 in which one or more of the amino acids L48, C58, P59, V60, F61, E62, C63, G64, W78, S80, R81, W83, L84, G86, D87, R89, D104, R111, Q113, G116, M118, and D120 (numbered in SEQ ID NO: 286 ( FIG. 25 )) have been substituted with another amino acid, compared to binding to wild-type human TIM3, e.g., as described in the Examples; (13) Competes in either or both directions for binding to human TIM3 with an antibody comprising the VH and VL domains of any of 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3, or TIM3.7, TIM3.8, TIM3.10, TIM3.11, TIM3.12, TIM3.13, TIM3.14, TIM3.15, TIM3.16, TIM3.17, TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, and TIM3.25 described in the Examples; (14) For example, as described in the Examples, the human TIM3 region was determined by HDX-MS. 49 VPVCWGKGACPVFE 62 (SEQ ID NO: 367) and 111 RIQIPGIMNDEKFNLKL 127 binds to (SEQ ID NO: 368); (15) Having a heavy chain and / or light chain variable region that interacts with at least 5, 10, 15, 20, or all of the following amino acids of human TIM3, P50, V51, C52, P59, V60, F61, E62, C63, G64, N65, V66, V67, L68, R69, D71, E72, D74, R111, Q113, G116, I117, M118, D120, and optionally T70 and / or I112, as determined by X-ray crystallography (e.g., as described in the Examples; numbering according to SEQ ID NO: 286 (Figure 25)); (16) (a) exhibits reduced binding to human TIM3 in which one, two, three, four, five, six, seven, eight, or nine of the amino acids C58, P59, F61, E62, C63, R111, D120, and optionally D104 and Q113 (numbering according to SEQ ID NO: 286 (FIG. 25)) have been substituted with another amino acid, compared to binding to wild-type human TIM3; (b) exhibits reduced binding to human TIM3 in which one, two, three, four, five, six, seven, eight, or nine of the amino acids C58, P59, F61, E62, C63, R111, D120, and optionally D104 and Q113 (numbering according to SEQ ID NO: 286 (FIG. 25)) have been substituted with another amino acid, as determined by HDX-MS as described in the Examples. 49 VPVCWGKGACPVFE 62 (SEQ ID NO: 367), 111 RIQIPGIMNDEKFNLKL 127 (SEQ ID NO: 368) and 119 NDEKFNLKL 127 (SEQ ID NO: 373); and / or (c) competes with or cross-blocks the binding of 13A3 or TIM3.18.IgG1.3 to human TIM3, e.g., as described in the Examples; (17) hTIM3-IgV was measured using the method described in Example 22 and found to have a 5 × 10 -8 M, 2 x 10 -8 M, 10 -8 M or 5x10 -9 K below M D Combine with; (18) hTIM3-IgV was measured by the method described in Example 22. -7 M, 2 x 10 -8 M or 10 -8 K below M D Combine with; (19) 5 × 10 TIM3-ECD cells were measured using the method described in Example 22. -7 M, 2 x 10 -8M or 10 -8 K below M D and / or (20) hTIM3-ECD was measured using the method described in Example 22, and 8 × 10 -8 M, 5 x 10 -8 M or 10 -8 K below M D Combine with.

[0344] In one embodiment, the heavy chain variable region CDR2 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 46 to 52, 122 to 125, 413, 415, and 470, and conservative modifications thereof, for example, amino acid sequences with 1, 2, 3, 4, 5, 1 to 2, 1 to 3, 1 to 4, or 1 to 5 conservative amino acid substitutions; and the light chain variable region CDR2 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 66 to 67 and 473, and conservative modifications thereof, for example, amino acid sequences with 1, 2, 3, 4, 5, 1 to 2, 1 to 3, 1 to 4, or 1 to 5 conservative amino acid substitutions.

[0345] In one embodiment, the heavy chain variable region CDR1 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41 to 45 and 469 and conservative modifications thereof, for example, amino acid sequences with 1, 2, 3, 4, 5, 1 to 2, 1 to 3, 1 to 4, or 1 to 5 conservative amino acid substitutions; and the light chain variable region CDR1 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 64 to 65 and 472 and conservative modifications thereof, for example, amino acid sequences with 1, 2, 3, 4, 5, 1 to 2, 1 to 3, 1 to 4, or 1 to 5 conservative amino acid substitutions.

[0346] In some embodiments, the antibody may exhibit one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or all of the functional properties listed above in (1) to (20). Such an antibody may be, for example, a human antibody, a humanized antibody, or a chimeric antibody.

[0347] Conservative amino acid substitutions can be made in portions of an antibody other than or in addition to CDRs. For example, conservative amino acid modifications can be made in the framework regions or Fc region. The variable region or heavy or light chain can contain 1, 2, 3, 4, 5, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 25, or 1 to 50 conservative amino acid substitutions compared to the anti-TIM3 antibody sequences provided herein. In some embodiments, the anti-TIM3 antibody contains a combination of conservative and non-conservative amino acid modifications.

[0348] VI. Antibodies that bind to or compete for binding to the same epitope Also provided are antibodies that compete for binding to human TIM3 with one or more of the specific anti-TIM3 antibodies described herein (e.g., antibodies 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3 and / or TIM3.2-TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24 and TIM3.25). Such competing antibodies can be identified based on their ability to competitively inhibit the binding of monoclonal antibodies 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3, and / or TIM3.2-TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24, and TIM3.25 to one or more of human TIM3 in a standard TIM3 binding assay. For example, a standard ELISA assay or a competitive ELISA assay can be used, in which recombinant human TIM3 protein is immobilized on a plate, various concentrations of an unlabeled first antibody are added, the plate is washed, a labeled second antibody is added, and the amount of label is measured. If increasing concentrations of the unlabeled (first) antibody (also referred to as a "blocking antibody") inhibit the binding of the labeled (second) antibody, the first antibody is said to inhibit or compete with the binding of the second antibody to the target on the plate. Additionally or alternatively, Biacore analysis can be used to assess the ability of an antibody to compete: The ability of a test antibody to inhibit the binding of an anti-TIM3 antibody described herein to TIM3 indicates that the test antibody can compete with that antibody for binding to human TIM3.

[0349] Accordingly, provided herein are anti-TIM3 antibodies that inhibit binding of the anti-TIM3 antibodies described herein to TIM3 on cells, e.g., activated T cells, by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% and / or to TIM3 on human cells, e.g., activated T cells, by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, as measured by ELISA or FACS, e.g., using the assays described in the next paragraph.

[0350] For example, an exemplary competition experiment to determine whether a first antibody blocks (i.e., "competes") the binding of a second antibody can be performed as described in the Examples or as follows: Activated human T cells are prepared as follows: Peripheral blood mononuclear cells (PBMCs) are isolated from human whole blood using a Ficoll gradient and activated with 10 μg / mL phytohemagglutinin (PHA-L) (USBiol #P3370-30) and 200 IU / mL recombinant IL-2 (Peprotech #200-02) for 3 days. The activated T cells are resuspended in FACS buffer (PBS containing 5% fetal bovine serum) and plated at 10 5Cells are seeded per well. The plate is placed on ice, followed by the addition of unconjugated primary antibodies at concentrations ranging from 0 to 50 μg / mL (three-fold titration starting from a maximum concentration of 50 μg / mL). An irrelevant IgG can be used as an isotype control for the primary antibody and added at the same concentration (three-fold titration starting from a maximum concentration of 50 μg / mL). A sample preincubated with 50 μg / mL unlabeled secondary antibody can be used as a positive control for complete blocking (100% inhibition), while a sample without antibody in the primary incubation can be used as a negative control (no competition; 0% inhibition). After a 30-minute incubation, labeled, e.g., biotinylated, secondary antibody is added at a concentration of 2 μg / mL per well without washing. The samples are incubated on ice for an additional 30 minutes. Unbound antibody is removed by washing the cells with FACS buffer. The cell-bound labeled secondary antibody is detected with a reagent that detects the label, such as PE-conjugated streptavidin (Invitrogen, catalog #S21388) for biotin detection. Samples are acquired on a FACS Calibur Flow Cytometer (BD, San Jose) and analyzed by FLOWJO. (登録商標) The results are analyzed using software (Tree Star, Inc., Ashland, OR). Results can be expressed as % inhibition (i.e., 100% minus the amount of label at each concentration divided by the amount of label obtained in the absence of blocking antibody). Typically, the same experiment is then performed in reverse, i.e., first antibody vs. second antibody and second antibody vs. first antibody.

[0351] In certain embodiments, an antibody at least partially (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) or completely (100%) blocks the binding of another antibody, e.g., human TIM3 or a portion thereof, to its target, regardless of whether the inhibition occurs when one or the other antibody is the first antibody. First and second antibodies "cross-block" each other's binding to their targets when the antibodies compete with each other in both directions, i.e., in a competition experiment where the first antibody is added first and in a competition experiment where the second antibody is added first.

[0352] In some embodiments, the anti-TIM3 antibody binds to the same epitope as an anti-TIM3 antibody described herein (e.g., 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3, and / or TIM3.2-TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24, and TIM3.25), as determined, for example, by an epitope mapping technique. Techniques for determining antibodies that bind to the "same epitope on TIM3" as an anti-TIM3 antibody described herein include, for example, epitope mapping methods, such as x-ray analysis of a crystal of the antigen:antibody complex, which provides atomic resolution of the epitope. Another method is to monitor the binding of the antibody to antigen fragments or mutational variations of the antigen, where loss of binding due to amino acid residue modification within the antigen sequence is often considered an indication of epitope identity (see Figure 25). Furthermore, computerized combinatorial methods of epitope mapping can also be used. The method can also utilize the ability of a target antibody to affinity isolate specific short peptides (in their native three-dimensional form or in denatured form) from a combinatorial phage-displayed peptide library. The peptides are then considered as leads to define the epitopes corresponding to the antibodies used to screen the peptide library. For epitope mapping, computerized algorithms have been developed and have been shown to map conformationally discontinuous epitopes.

[0353] Antibodies that compete for binding with or bind to the same epitope as the anti-TIM3 antibodies described herein can be identified using methods known in the art. For example, mice can be immunized with the human TIM3 described herein to produce hybridomas, and the resulting monoclonal antibodies can be screened for their ability to compete with the binding of the antibodies described herein to human TIM3. Mice can also be immunized with a small fragment of TIM3 containing the epitope to which the antibody binds. The epitope or a region containing the epitope can be localized, for example, by screening for binding to a series of overlapping peptides spanning TIM3. Alternatively, the method of Jespers et al., Biotechnology 12:899, 1994, can be used to guide the selection of antibodies that have the same epitope as the anti-TIM3 antibodies described herein and therefore have similar properties. Using phage display, the heavy chain of a first anti-TIM3 antibody is paired with a repertoire of (human) light chains to select for TIM3-binding antibodies, and the new light chain is then paired with a repertoire of (human) heavy chains to select for (human) TIM3-binding antibodies that have the same epitope or epitope region as the anti-TIM3 antibodies described herein. Alternatively, variants of the antibodies described herein can be obtained by mutagenesis of the cDNA encoding the heavy and light chains of the antibodies.

[0354] Alanine scanning mutagenesis or some other form of point mutagenesis of amino acid residues as described by Cunningham and Wells (1989) Science 244: 1081-1085 of TIM3 can also be used to obtain TIM3 antibody binding characteristics.

[0355] The binding characteristics of a specific antibody can also be determined by assessing the binding of the antibody to peptides containing fragments of TIM3, such as native or denatured fragments. A series of overlapping peptides encompassing the sequence of TIM3 (e.g., human TIM3) can be synthesized and screened for binding, for example, by direct ELISA, competitive ELISA (assessing the ability of peptides to inhibit antibody binding to TIM3 bound to wells of a microtiter plate), or chip.

[0356] The binding characteristics of anti-TIM3 antibodies can also be obtained by MS-based protein footprinting methods, such as hydrogen / deuterium exchange mass spectrometry (HDX-MS) and Fast Photochemical Oxidation of Proteins (FPOP). HDX-MS can be performed, for example, as described in WO2015 / 18735 and Wei et al. (2014) Drug Discovery Today 19:95, which are expressly incorporated herein by reference. FPOP can be performed, for example, as described in Hambley and Gross (2005) J. American Soc. Mass Spectrometry 16:2057, which are expressly incorporated herein by reference.

[0357] The binding characteristics of anti-TIM3 antibodies can also be obtained by structural methods, such as X-ray crystal structure determination (e.g., WO2005 / 044853), molecular modeling, and nuclear magnetic resonance (NMR) spectroscopy (including NMR determination of the HD exchange rate of the labile amide hydrogen of TIM3 when free and when bound in complex with the antibody of interest) (Zinn-Justin et al. (1992) Biochemistry 31, 11335-11347; Zinn-Justin et al. (1993) Biochemistry 32, 6884-6891).

[0358] For X-ray crystallography, crystallization can be achieved using any of the methods known in the art, including microbatch (e.g., Chayen (1997) Structure 5: 1269-1274), hanging drop-vapor diffusion (e.g., McPherson (1976) J. Biol. Chem. 251:6300-6303), seeding, and dialysis (e.g., Giege et al. (1994) Acta Crystallogr. D50:339-350; McPherson (1990) Eur. J. Biochem. 189: 1-23). ​​Protein preparations at a concentration of at least about 1 mg / mL or about 10 mg / mL to about 20 mg / mL are desirable. Crystallization is best achieved with a precipitant solution containing polyethylene glycol 1000-20,000 (PEG; average molecular weight of about 1000 to about 20,000 Da), about 5000 to about 7000 Da, or about 6000 Da, in a concentration range of about 10% to about 30% (w / v). It may also be desirable to include a protein stabilizer, such as glycerol, in a concentration range of about 0.5% to about 20%. A suitable salt, such as sodium chloride, lithium chloride, or sodium citrate, may also be desirable in the precipitant solution, in a concentration range of about 1 mM to about 1000 mM. The precipitant is buffered to a pH of about 3.0 to about 5.0. Specific buffers useful in precipitant solutions vary and are well known in the art (Scopes, Protein Purification: Principles and Practice, Third ed., (1994) Springer-Verlag, New York). Examples of useful buffers include, but are not limited to, HEPES, Tris, MES, and acetic acid. Crystals can be grown over a wide range of temperatures, including 2°C, 4°C, 8°C, and 26°C.

[0359] Antibody:antigen crystals can be examined using well-known X-ray diffraction techniques and refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Blundell & Johnson (1985) Meth. Enzymol. 114 & 115, HW Wyckoff et al., eds., Academic Press; U.S. Patent Application Publication 2004 / 0014194) and BUSTER (Bricogne (1993) Acta Cryst. D49:37-60; Bricogne (1997) Meth. Enzymol. 276A:361-423, Carter & Sweet, eds.; Roversi et al. (2000) Acta Cryst. D56: 1313-1323), the disclosures of which are incorporated herein by reference in their entireties.

[0360] The anti-TIM3 antibody can bind to the same epitope as any of the anti-TIM3 antibodies having the amino acid sequences described herein, as determined by epitope mapping techniques, such as those described herein.

[0361] Antibodies that bind to human TIM3, and optionally cynomolgus TIM3, with binding characteristics similar to the anti-TIM3 antibodies described herein, as determined by one of the methods used in the Examples, are encompassed herein.

[0362] In certain embodiments, the anti-TIM3 antibodies described herein bind to an epitope in the extracellular portion of human TIM3, e.g., a conformational epitope in the Ig-like or IgV domain of the extracellular region, i.e., amino acids 22-130 of SEQ ID NO: 286 (Figure 25). In certain embodiments, the anti-TIM3 antibodies bind to an epitope located at amino acids 22-120 of the human TIM3 extracellular domain (SEQ ID NO: 286) or 1-99 of mature human TIM3 (SEQ ID NO: 290) (see Examples). In certain embodiments, the anti-TIM3 antibodies bind to an epitope at or within a region consisting of amino acids 58-64 of human TIM3 having SEQ ID NO: 286 (CPVFECG, SEQ ID NO: 296; see Figure 25), which corresponds to SEQ ID NO: 286 of mature human TIM3. In certain embodiments, the anti-TIM3 antibody binds to an epitope within or in a region consisting of amino acids 111-120 of human TIM3 having SEQ ID NO:286 (RIQIPGIMND, SEQ ID NO:298; see Figure 25), which corresponds to SEQ ID NO:286 of mature human TIM3. In certain embodiments, the anti-TIM3 antibody binds to an epitope within or in a region consisting of amino acids 58-64 of human TIM3 having SEQ ID NO:286 (CPVFECG, SEQ ID NO:296) and a region consisting of amino acids 111-120 of human TIM3 having SEQ ID NO:286 (RIQIPGIMND, SEQ ID NO:298; see Figure 25). In certain embodiments, the anti-TIM3 antibody binds to an epitope within or in a region consisting of amino acids 78-89 of human TIM3 having SEQ ID NO:286 (WTSRYWLNGDFR, SEQ ID NO:297; see Figure 25), which corresponds to SEQ ID NO:286 of mature human TIM3.

[0363] In certain embodiments, the anti-TIM3 antibody binds to substantially the same epitope as 13A3. In certain embodiments, the anti-TIM3 antibody binds to an epitope (or a region of human TIM3) comprising one or more of amino acid residues C58, P59, F61, E62, C63, R111, and D120 of SEQ ID NO: 286 (Figure 25). In certain embodiments, the anti-TIM3 antibody binds to an epitope (or a region of human TIM3) comprising one or more of amino acid residues C58, P59, F61, E62, C63, D104, R111, Q113, and D120 of SEQ ID NO: 286 (Figure 25). In certain embodiments, the anti-TIM3 antibody does not significantly bind, or binds with significantly reduced binding affinity, to a human TIM3 protein in which one or more of amino acid residues C58, P59, F61, E62, C63, R111, and D120 of SEQ ID NO: 286 have been changed to another amino acid, e.g., a non-conservative amino acid substitution. In certain embodiments, the anti-TIM3 antibody does not significantly bind, or binds with significantly reduced binding affinity, to a human TIM3 protein in which one or more of amino acid residues C58, P59, F61, E62, C63, D104, R111, Q113, and D120 of SEQ ID NO: 286 have been changed to another amino acid, e.g., a non-conservative amino acid substitution.

[0364] In certain embodiments, the anti-TIM3 antibody binds to substantially the same epitope as 3G4. In certain embodiments, the anti-TIM3 antibody binds to an epitope (or a region of human TIM3) comprising one or more of amino acid residues C58, P59, V60, F61, E62, C63, G116, and M118 of SEQ ID NO: 286 (Figure 25). In certain embodiments, the anti-TIM3 antibody binds to an epitope (or a region of human TIM3) comprising one or more of amino acid residues C58, P59, V60, F61, E62, C63, D104, G116, and M118 of SEQ ID NO: 286 (Figure 25). In certain embodiments, the anti-TIM3 antibody does not significantly bind, or binds with significantly reduced binding affinity, to a human TIM3 protein in which one or more of amino acid residues C58, P59, V60, F61, E62, C63, G116, and M118 of SEQ ID NO: 286 have been changed to another amino acid, e.g., a non-conservative amino acid substitution. In certain embodiments, the anti-TIM3 antibody does not significantly bind, or binds with significantly reduced binding affinity, to a human TIM3 protein in which one or more of amino acid residues C58, P59, V60, F61, E62, C63, D104, G116, and M118 of SEQ ID NO: 286 have been changed to another amino acid, e.g., a non-conservative amino acid substitution.

[0365] In certain embodiments, the anti-TIM3 antibody binds to substantially the same epitope as 17C3. In certain embodiments, the anti-TIM3 antibody binds to an epitope (or a region of human TIM3) comprising one or more of amino acid residues C58, P59, V60, F61, E62, C63, G64, and G116 of SEQ ID NO: 286 (Figure 25). In certain embodiments, the anti-TIM3 antibody binds to an epitope (or a region of human TIM3) comprising one or more of amino acid residues C58, P59, V60, F61, E62, C63, G64, D104, and G116 of SEQ ID NO: 286 (Figure 25). In certain embodiments, the anti-TIM3 antibody does not significantly bind, or binds with significantly reduced binding affinity, to a human TIM3 protein in which one or more of amino acid residues C58, P59, V60, F61, E62, C63, G64, and G116 of SEQ ID NO: 286 have been changed to another amino acid, e.g., a non-conservative amino acid substitution. In certain embodiments, the anti-TIM3 antibody does not significantly bind, or binds with significantly reduced binding affinity, to a human TIM3 protein in which one or more of amino acid residues C58, P59, V60, F61, E62, C63, G64, D104, and G116 of SEQ ID NO: 286 have been changed to another amino acid, e.g., a non-conservative amino acid substitution.

[0366] In certain embodiments, the anti-TIM3 antibody binds to substantially the same epitope as 8B9. In certain embodiments, the anti-TIM3 antibody binds to an epitope (or a region of human TIM3) comprising one or more of amino acid residues L48, W78, S80, R81, W83, G86, D87, and R89 of SEQ ID NO: 286 (Figure 25). In certain embodiments, the anti-TIM3 antibody binds to an epitope (or a region of human TIM3) comprising one or more of amino acid residues L48, W78, S80, R81, W83, L84, G86, D87, and R89 of SEQ ID NO: 286 (Figure 25). In certain embodiments, the anti-TIM3 antibody binds to substantially the same epitope as 8B9. In certain embodiments, the anti-TIM3 antibody binds to an epitope (or region of human TIM3) that includes one or more of amino acid residues L48, W78, S80, R81, W83, G86, D87, R89, and D104 (Figure 25) of SEQ ID NO: 286. In certain embodiments, the anti-TIM3 antibody does not bind significantly, or binds only with significantly reduced binding affinity, to a human TIM3 protein in which one or more of amino acid residues L48, W78, S80, R81, W83, G86, D87, and R89 of SEQ ID NO: 286 have been changed to another amino acid, e.g., a non-conservative amino acid substitution. In certain embodiments, the anti-TIM3 antibody does not significantly bind, or binds with significantly reduced binding affinity, to a human TIM3 protein in which one or more of amino acid residues L48, W78, S80, R81, W83, L84, G86, D87, and R89 of SEQ ID NO: 286 have been changed to another amino acid, e.g., a non-conservative amino acid substitution. In certain embodiments, the anti-TIM3 antibody does not significantly bind, or binds with significantly reduced binding affinity, to a human TIM3 protein in which one or more of amino acid residues L48, W78, S80, R81, W83, G86, D87, R89, and D104 of SEQ ID NO: 286 have been changed to another amino acid, e.g., a non-conservative amino acid substitution (Figure 25).

[0367] In certain embodiments, the anti-TIM3 antibody competes with (or inhibits) the binding of human TIM3 to an anti-TIM3 antibody having a CDR or variable region described herein, e.g., antibodies 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3, and any of TIM3.2 to TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24, and TIM3.25. In certain embodiments, the anti-TIM3 antibody inhibits binding of antibodies 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3 or any of TIM3.2 to TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24 and TIM3.25 to human TIM3 by at least 50%, 60%, 70%, 80%, 90% or 100%. In certain embodiments, 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3, or any of TIM3.2 to TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24, and TIM3.25 inhibits binding of an anti-TIM3 antibody to human TIM3 by at least 50%, 60%, 70%, 80%, 90% or 100%. In certain embodiments, the anti-TIM3 antibody inhibits binding of 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3, or any of TIM3.2 to TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24, and TIM3.25 to human TIM3 by at least 50%, 60%, 70%, 80%, 90%, or 100%, and 3A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3, or any of TIM3.2 to TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24, and TIM3.25 inhibits binding of an anti-TIM3 antibody to human TIM3 by at least 50%, 60%, 70%, 80%, 90%, or 100% (e.g., competes in both directions).

[0368] In certain embodiments, the anti-TIM3 antibody competes for binding to human TIM3 with an anti-TIM3 antibody comprising the CDRs or variable regions of anti-TIM3 antibodies 13A3, 3G4, 17C3, 17C8, 9F6, 14H7, and 23B3 (and variants thereof). In certain embodiments, the anti-TIM3 antibody does not compete for binding to human TIM3 with an anti-TIM3 antibody comprising the CDRs or variable regions of anti-TIM3 antibodies 8B9 and 8C4 (and variants thereof).

[0369] VII. Engineered and Modified Antibodies Also provided are engineered, modified antibodies that can be produced using as starting materials antibodies having one or more of the VH and / or VL sequences disclosed herein to design modified antibodies, which may have altered properties from the starting antibody. Antibodies can be engineered by modifying one or more residues within one or both variable regions (i.e., VH and / or VL), for example, within one or more CDR regions and / or within one or more framework regions. Additionally or alternatively, antibodies can be engineered by modifying residues within the constant regions, for example, to alter the effector functions of the antibody.

[0370] One type of variable region engineering that can be performed is CDR grafting. Antibodies interact with target antigens primarily through amino acid residues located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within the CDRs are more diverse between individual antibodies than the sequences outside the CDRs. Because CDR sequences are largely responsible for antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a particular naturally occurring antibody by constructing an expression vector containing CDR sequences from that particular naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al. (1986) Nature 321:522-525; Queen, C. et al. (1989) Proc. Natl. Acad. Sci. USA 86: 10029-10033; Winter, U.S. Patent No. 5,225,539 and Queen et al., U.S. Patents Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0371] Accordingly, certain embodiments described herein relate to an isolated monoclonal antibody, or antigen-binding portion thereof, comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences comprising amino acid sequences selected from the group consisting of SEQ ID NOs: 41-45 and 469; SEQ ID NOs: 46-52, 122-125, 413, 415, and 470; and SEQ ID NOs: 53-59, 126-129, 414, 416, and 471; respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences comprising amino acid sequences selected from the group consisting of SEQ ID NOs: 64-65 and 472; SEQ ID NOs: 66-67 and 473; and SEQ ID NOs: 68-71, 419, and 474; respectively. Thus, such antibodies comprise the VH and VL CDR sequences of monoclonal antibodies 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3 or any of TIM3.2 to TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24 or TIM3.25, yet may comprise different framework sequences from these antibodies.

[0372] Such framework sequences can be obtained from public databases or published literature containing germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes are available from the "VBase" human germline sequence database (available on the Internet at www.mrc-cpe.cam.ac.uk / vbase), as well as Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, IM, et al. (1992) "The Repertoire of Human Germline V H Sequences Reveals about Fifty Groups of V HSegments with Different Hypervariable Loops" / . Mol. Biol. 227:776-798; and Cox, JPL et al. (1994) "A Directory of Human Germ-line V H "Segments Reveal a Strong Bias in Their Usage," Eur. J. Immunol. 24:827-836, the contents of each of which are expressly incorporated herein in their entirety.

[0373] In some embodiments, framework sequences for use in the anti-TIM3 antibodies described herein are structurally similar to the framework sequences used by the anti-TIM3 antibodies described herein. The VH CDR1, 2, and 3 sequences and the VL CDR1, 2, and 3 sequences can be grafted into framework regions having the same sequences as found in the germline immunoglobulin gene from which the framework sequences are derived, or the CDR sequences can be grafted into framework regions containing one or more mutations compared to the germline sequences. For example, in some instances, it has been found advantageous to mutate residues within framework regions to maintain or enhance the antigen-binding ability of an antibody (see, e.g., U.S. Patents 5,530,101; 5,585,089; 5,693,762; and 6,180,370 to Queen et al.).

[0374] The engineered anti-TIM3 antibodies described herein include those in which framework residues within the VH and / or VL have been modified to improve the antibody's properties, e.g., a mutation at amino acid 107 of 9F6. Generally, such framework modifications are made to reduce the antibody's immunogenicity. For example, one method is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, antibodies that have undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody is derived. To return the framework region sequences to their germline configuration, the somatic mutations are "backmutated" to the germline sequence, e.g., by site-directed mutagenesis or PCR-mediated mutagenesis. Such "backmutated" antibodies are also intended to be encompassed. Another type of framework modification involves mutating one or more residues within the framework region or one or more CDR regions to remove a T cell epitope, thereby reducing the potential immunogenicity of the antibody. This method is also referred to as "deimmunization" and is described in US Patent Publication 20030153043 by Carr et al.

[0375] Another type of variable region modification is to mutate amino acid residues within the VH and / or VL CDR1, CDR2, and / or CDR3 regions, thereby improving one or more binding properties (e.g., affinity) of the antibody of interest. Mutations can be introduced using site-directed mutagenesis or PCR-mediated mutagenesis, and their effect on antibody binding or other functional properties of interest can be assessed using in vitro or in vivo assays described herein and provided in the Examples. In some embodiments, conservative modifications (described above) are introduced. Mutations can be amino acid substitutions, additions, or deletions. Furthermore, generally, no more than one, two, three, four, or five residues within the CDR regions are altered.

[0376] Therefore, it is further provided that (a) a VH CDR1 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 41 to 45 and 469, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions relative to SEQ ID NOs: 41 to 45 and 469; (b) a VH CDR2 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 46 to 52, 122 to 125, 413, 415, and 470, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to SEQ ID NOs: 46 to 52, 122 to 125, 413, 415, and 470; (c) a VH CDR3 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 59, 126 to 129, 414, 416, and 471, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to SEQ ID NOs: 53 to 59, 126 to 129, 414, 416, and 471; (d) a VL CDR1 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 64-65 and 472, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions relative to SEQ ID NOs: 64-65 and 472; (e) a VL CDR2 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66 to 67 and 473, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions relative to SEQ ID NOs: 66 to 67 and 473; and (f) a VL CDR3 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 68 to 71, 419, and 474, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to SEQ ID NOs: 68 to 71, 419, and 474; and a heavy chain variable region comprising:

[0377] Methionine residues in the CDRs of an antibody can be oxidized, leading to potential chemical degradation and resulting reduction in antibody potency. Accordingly, also provided are anti-TIM3 antibodies in which one or more methionine residues in the heavy and / or light chain CDRs are substituted with amino acid residues that are not susceptible to oxidative degradation. In certain embodiments, the methionine residues in the CDRs of antibodies 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3, or any of TIM3.2 to TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24, and TIM3.25 are substituted with amino acid residues that are not susceptible to oxidative degradation.

[0378] Similarly, deamidation sites can be removed from anti-TIM3 antibodies, particularly the CDRs.

[0379] The anti-TIM3 variable regions described herein are Fc, e.g., IgG1, IgG2, IgG3, or IgG4 The Fc can be linked (e.g., covalently linked or fused) to any allotype or isoallotype, e.g., for IgG1: G1m, G1m1(a), G1m2(x), G1m3(f), G1m17(z); for IgG2: G2m, G2m23(n); for IgG3: G3m, G3m21(g1), G3m28(g5), G3m11(b0), G3m5(b1), G3m13(b3), G3m14(b4), G3m10(b5), G3m15(s), G3m16(t), G3m6(c3), G3m24(c5), G3m26(u), G3m27(v); and for K: Km, Km1, Km2, Km3 (see, e.g., Jefferies et al. (2009) mAbs 1:1).

[0380] In certain embodiments, the anti-TIM3 variable regions described herein are linked to an effectorless or mostly effectorless Fc, e.g., IgG1.

[0381] Generally, the variable regions described herein can be attached to an Fc region that contains one or more modifications, generally to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. Additionally, the antibodies described herein can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or modified to alter its glycosylation, to alter one or more functional properties of the antibody. Each of these embodiments is described in further detail below. The numbering of residues in the Fc region is that of the EU index of Kabat.

[0382] The Fc region encompasses domains derived from the constant region of an immunoglobulin, including fragments, analogs, variants, mutants, or derivatives of the constant region. Suitable immunoglobulin classes include IgG1, IgG2, IgG3, IgG4, and other classes such as IgA, IgD, IgE, and IgM, where the constant region of an immunoglobulin is defined as a naturally occurring or synthetically produced polypeptide that is homologous to the C-terminal region of an immunoglobulin and includes the CH1 domain, hinge, CH2 domain, CH3 domain, or CH4 domain, either separately or in combination.

[0383] Ig molecules interact with multiple classes of cell receptors. For example, IgG molecules interact with three classes of Fcγ receptors (FcγR), namely FcγRI, FcγRII, and FcγRIII, which are specific for IgG class antibodies. It has been reported that the sequences important for IgG binding to FcγR receptors are located in the CH2 and CH3 domains. The serum half-life of an antibody is affected by its ability to bind to Fc receptors (FcRs).

[0384] In certain embodiments, the Fc region is a variant Fc region, e.g., an Fc sequence that has been modified (e.g., by amino acid substitution, deletion, and / or insertion) relative to a parent Fc sequence (e.g., an unmodified Fc polypeptide that is subsequently modified to produce the variant), to provide desired structural properties and / or biological activity.

[0385] Generally, a variant of a constant region or portion thereof, such as a CH1, CL, hinge, CH2, or CH3 domain, contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations and / or up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutation, or 1-10 or 1-5 mutations, or comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding wild-type region or domain (CH1, CL, hinge, CH2, or CH3 domain, respectively), provided that the heavy chain constant region containing the particular variant retains the required biological activity.

[0386] For example, modifications can be made to the Fc region to create Fc variants that, relative to the parent Fc, have (a) increased or decreased antibody-dependent cellular cytotoxicity (ADCC), (b) increased or decreased complement-dependent cytotoxicity (CDC), (c) increased or decreased affinity for C1q, and / or (d) increased or decreased affinity for Fc receptors. Such Fc region variants generally contain at least one amino acid modification in the Fc region. Combination amino acid modifications may be particularly desirable. For example, variant Fc regions may contain substitutions therein, e.g., two, three, four, five, or the like, at specific Fc region positions identified herein.

[0387] The variant Fc region can also contain sequence modifications in which amino acids involved in disulfide bond formation are removed or replaced with other amino acids. Such removal can avoid reaction with other cysteine-containing proteins present in the host cells used to produce the anti-TIM3 antibodies described herein. Even when cysteine ​​residues are removed, the single-chain Fc domains can still form a dimeric Fc domain that is non-covalently bound together. In certain embodiments, the Fc region can be modified to make it more compatible with a selected host cell. For example, the PA sequence near the N-terminus of a typical native Fc region, which can be recognized by digestive enzymes such as E. coli proline iminopeptidase, can be removed. In certain embodiments, one or more glycosylation sites within the Fc domain can be removed. Residues that are commonly glycosylated (e.g., asparagine) can contribute to cytolytic responses. Such residues can be deleted or substituted with non-glycosylated residues (e.g., alanine). In certain embodiments, sites involved in interaction with complement, such as the C1q binding site, can be removed from the Fc region. For example, the EKK sequence of human IgG1 can be deleted or replaced. In certain embodiments, sites that affect binding to Fc receptors, preferably sites other than the salvage receptor binding site, can be removed. In certain embodiments, the Fc region can be modified to remove ADCC sites. ADCC sites are known in the art; see, for example, Molec. Immunol. 29 (5): 633-9 (1992) for the ADCC site of IgG1. Specific examples of variant Fc domains are disclosed, for example, in WO97 / 34631 and WO96 / 32478.

[0388] In some embodiments, the hinge region of an Fc is modified so that the number of cysteine ​​residues in the hinge region is altered, e.g., increased or decreased. This method is further described in U.S. Patent 5,677,425 to Bodmer et al. The number of cysteine ​​residues in the hinge region of an Fc is altered, for example, to facilitate light and heavy chain assembly or to increase or decrease the stability of the antibody. In some embodiments, the Fc hinge region of an antibody is mutated to reduce the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment so that the antibody exhibits impaired Staphylococcus protein A (SpA) binding compared to native Fc-hinge domain pA binding. This method is further described in U.S. Patent 6,165,745 to Ward et al.

[0389] In some embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue 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, 322, 330, and / or 331 can be replaced with a different amino acid residue so that the antibody has altered affinity for an effector ligand while retaining the antigen-binding ability of the parent antibody. The effector ligand for which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This method is further described in U.S. Patents 5,624,821 and 5,648,260, both to Winter et al.

[0390] In another example, one or more amino acids selected from amino acid residues 329, 331, and 322 can be replaced with a different amino acid residue such that C1q binding of the antibody is altered and / or complement dependent cytotoxicity (CDC) is reduced or eliminated, as described further in U.S. Patent 6,194,551 to Idusogie et al.

[0391] In another example, one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement. This method is further detailed in PCT Publication WO 94 / 29351 by Bodmer et al.

[0392] In yet another example, the Fc region may be selected from the following positions: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 247, 248, 249, 252, 254, 255, 256, 258, 262, 263, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 299, 301, 303, 305, 307, 309, 312, 313 , 315, 320, 322, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 433, 434, 435, 436, 437, 438, or 439 may be modified to reduce antibody-dependent cellular cytotoxicity (ADCC) and / or reduce affinity for Fcγ receptors. Exemplary substitutions include 236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D, and 332E. Examples of variants include 239D / 332E, 236A / 332E, 236A / 239D / 332E, 268F / 324T, 267E / 268F, 267E / 324T, and 267E / 268F7324T. Other modifications to enhance FcγR and complement interactions include, but are not limited to, the substitutions 298A, 333A, 334A, 326A, 2471, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 3051, and 396L. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691.

[0393] Fc modifications that increase binding to Fcγ receptors are identified at amino acid positions 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 279, 280, 283, 285, 298, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 312, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 38 and any one or more amino acid modifications of any of 4, 327, 329, 330, 335, 337, 338, 340, 360, 373, 376, 379, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439, wherein the numbering of residues in the Fc region is that of the EU index in Kabat (WO 00 / 42072).

[0394] Other Fc modifications that can be made to the Fc are those that reduce or eliminate binding to FcγR and / or complement proteins, thereby reducing or eliminating Fc-mediated effector functions such as ADCC, ADCP, and CDC. Exemplary modifications include, but are not limited to, substitutions, insertions, and deletions at positions 234, 235, 236, 237, 267, 269, 325, 328, 330, and / or 331 (e.g., 330 and 331), where numbering is according to the EU index. Exemplary substitutions include, but are not limited to, 234A, 235E, 236R, 237A, 267R, 269R, 325L, 328R, 330S, and 331S (e.g., 330S and 331S), where numbering is according to the EU index. An Fc variant can include 236R / 328R. Other modifications that reduce FcγR and complement interactions include substitutions 297A, 234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 331S, 220S, 226S, 229S, 238S, 233P, and 234V, and removal of glycosylation at position 297 by mutational or enzymatic means or by production in organisms such as bacteria that do not glycosylate proteins. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691.

[0395] Optionally, the Fc region can include non-naturally occurring amino acid residues at additional and / or alternative positions known to those of skill in the art (e.g., U.S. Patents 5,624,821; 6,277,375; 6,737,056; 6,194,551; 7,317,091; 8,101,720; PCT Patent Publication WO 00 / 42072; WO 01 / 58957; WO02 / 06919; WO04 / 016750; WO04 / 029207; WO04 / 035752; WO04 / 074455; WO04 / 099249; WO04 / 063351; WO05 / 070963; WO05 / 040217, WO05 / 092925 and WO06 / 020114).

[0396] Fc variants that enhance affinity for the inhibitory receptor FcγRIIb can also be used. Such variants can provide Fc fusion proteins with immunomodulatory activity associated with FcγRIIb cells, including, for example, B cells and monocytes. In certain embodiments, the Fc variants selectively enhance affinity for FcγRIIb over one or more activating receptors. Modifications that alter binding to FcγRIIb include one or more modifications at positions selected from the group consisting of 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327, 328, 330, 331, and 332 according to the EU index. Examples of substitutions that enhance FcγRIIb affinity include, but are not limited to, 234A, 234D, 234E, 234F, 234W, 235D, 235E, 235F, 235R, 235Y, 236D, 236N, 237A, 237D, 237N, 239D, 239E, 266M, 267D, 267E, 268D, 268E, 327D, 327E, 328F, 328W, 328Y, 330S, 331S, and 332E. Examples of substitutions include 235Y, 236D, 239D, 266M, 267E, 268D, 268E, 328F, 328W, and 328Y. Other Fc variants that enhance binding to FcγRIIb include 235Y / 267E, 236D / 267E, 239D / 268D, 239D / 267E, 267E / 268D, 267E / 268E, and 267E / 328F.

[0397] The affinity and binding of an Fc region to its ligand can be determined by a number of in vitro assay methods (biochemical or immunological-based assays) known in the art, including, but not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)) or kinetic studies (e.g., BIACORE analysis) and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods can utilize one or more labels of the component being tested and / or employ a variety of detection methods, including, but not limited to, chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinity and kinetic studies can be found in Paul, W.E., ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which discusses antibody-immunogen interactions.

[0398] In some embodiments, antibodies are modified to increase their biological half-life. Various methods are possible. For example, this can be achieved by increasing the binding affinity of the Fc region to FcRn. For example, one or more of the following residues can be mutated: 252, 254, 256, 433, 435, and 436, as described in U.S. Patent 6,277,375. Specific examples of substitutions include one or more of the following: T252L, T254S, and / or T256F. Alternatively, to increase biological half-life, antibodies can be engineered within the CH1 or CL regions to contain salvage receptor binding epitopes taken from two loops of the CH2 domain of the Fc region of IgG, as described in U.S. Patents 5,869,046 and 6,121,022 to Presta et al. Other exemplary variants that increase binding to FcRn and / or improve pharmacokinetic properties contain substitutions at positions 259, 308, 428 and 434, including, for example, 2591, 308F, 428L, 428M, 434S, 434L, 434F, 434Y and 434X1.Other variants that increase Fc binding to FcRn include 250E, 250Q, 428L, 428F, 250Q / 428L (Hinton et al. 2004, J. Biol. Chem. 279(8): 6213-6216, Hinton et al. 2006 Journal of Immunology 176:346-356), 256A, 272A, 286A, 305A, 307A, 307Q, 311A, 312A, 376A, 378Q, 380A, 382A, and 434A (Shields et al., Journal of Biological Chemistry, 2001). 276(9):6591-6604), 252F, 252T, 252Y, 252W, 254T, 256S, 256R, 256Q, 256E, 256D, 256T, 309P, 311S, 43 3R, 433S, 4331, 433P, 433Q, 434H, 434F, 434Y, 252Y / 254T / 256E, 433K / 434F / 436H, 308T / 309P / 311SDall Acqua et al. Journal of Immunology, 2002, 169:5171-5180, Dall'Acqua et al., 2006, Journal of Biological Chemistry 281:23514-23524). Other modifications that modulate FcRn binding are described in Yeung et al., 2010, J Immunol, 182:7663-7671.

[0399] In some embodiments, hybrid IgG isotypes with specific biological characteristics can be used. For example, IgG1 / IgG3 hybrid variants can be constructed by replacing IgG1 positions in the CH2 and / or CH3 regions with amino acids from IgG3 at positions where the two isotypes differ. Thus, hybrid variant IgG antibodies can be constructed that contain one or more substitutions, such as 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 4221, 435R, and 436F. In some embodiments described herein, IgG1 / IgG2 hybrid variants can be constructed by replacing IgG2 positions in the CH2 and / or CH3 regions with amino acids from IgG1 at positions where the two isotypes differ. Thus, hybrid variant IgG antibodies can be constructed that contain one or more substitutions, for example, one or more of the following amino acid substitutions: 233E, 234L, 235L, -236G (referring to the insertion of glycine at position 236), and 327A.

[0400] Furthermore, the binding sites of human IgG1 for FcγRI, FcγRII, FcγRIII, and FcR have been mapped, and variants with improved binding have been described (see Shields, RL et al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334, and 339 have been shown to improve binding to FcγRIII. Furthermore, the following combination mutants have been shown to improve FcγRIII binding, which has been shown to enhance FcγRIIIa binding and ADCC activity (Shields et al., 2001). Other IgG1 variants with strongly enhanced binding to FcγRIIIa have been identified, including variants with S239D / I332E and S239D / I332E / A330L mutations that showed the greatest increase in affinity for FcγRIIIa, reduced FcγRIIb binding, and potent cytotoxic activity in cynomolgus monkeys (Lazar et al., 2006). Triple mutations in antibodies such as alemtuzumab (CD52-specific), trastuzumab (HER2 / neu-specific), rituximab (CD20-specific), and cetuximab (EGFR-specific) and the S239D / I332E variant showed enhanced capacity for B cell depletion in monkeys, as reflected by greatly enhanced ADCC activity in vitro (Lazar et al., 2006). Furthermore, IgG1 mutants containing L235V, F243L, R292P, Y300L, and P396L mutations have been identified that showed enhanced binding to FcγRIIIa and concomitant enhanced ADCC activity in human FcγRIIIa-expressing transgenic mice, models of B-cell malignancies and breast cancer (Stavenhagen et al., 2007; Nordstrom et al., 2011). Other Fc mutants that can be used include S298A / E333A / L334A, S239D / I332E, S239D / I332E / A330L, L235V / F243L / R292P / Y300L / P396L, and M428L / N434S.

[0401] In one embodiment, the Fc is selected to have reduced binding to FcγR. An example of an Fc with reduced FcγR binding, e.g., an IgG1 Fc, contains the following three amino acid substitutions: L234A, L235E, and G237A.

[0402] In one embodiment, the Fc is selected to have reduced complement binding. An example of an Fc with reduced complement binding, e.g., an IgG1 Fc, contains the following two amino acid substitutions: A330S and P331S.

[0403] In one embodiment, the Fc is selected to have essentially no effector fun...

Claims

1. An isolated antibody that binds to human T-cell immunoglobulin and mucin domain containing 3 (TIM3) and comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3, wherein the heavy chain CDR3 comprises SEQ ID NO:414 or SEQ ID NO:

416.

2. The antibody of claim 1, wherein the heavy chain CDR1 comprises sequence number 45.

3. 3. The antibody of claim 1 or 2, wherein the heavy chain CDR2 comprises SEQ ID NO: 413 or SEQ ID NO:

415.

4. The antibody of any one of claims 1 to 3, wherein the light chain CDR1 comprises SEQ ID NO:

64.

5. The antibody of any one of claims 1 to 4, wherein the light chain CDR2 comprises SEQ ID NO:

66.

6. The antibody of any one of claims 1 to 5, wherein the light chain CDR3 comprises SEQ ID NO: 69, SEQ ID NO: 68 or SEQ ID NO:

419.

7. 1. An isolated antibody that binds to human TIM3, comprising a heavy chain CDR1, CDR2, and CDR3 and a light chain CDR1, CDR2, and CDR3, wherein (a) the heavy chain CDR1 comprises SEQ ID NO:45; (b) the heavy chain CDR2 comprises SEQ ID NO:413 or SEQ ID NO:415; (c) the heavy chain CDR3 comprises SEQ ID NO:414 or SEQ ID NO:416; (d) the light chain CDR1 comprises SEQ ID NO: 64; (e) the light chain CDR2 comprises SEQ ID NO: 66; and (f) the light chain CDR3 comprises SEQ ID NO:69, SEQ ID NO:68, or SEQ ID NO:419; antibody.

8. (a) heavy chain CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 45, 413, and 414, respectively, and light chain CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 69, respectively; (b) heavy chain CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 45, 415, and 416, respectively, and light chain CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 68, respectively; or (c) heavy chain CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 45, 415, and 416, respectively, and light chain CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 66, and 419, respectively; The antibody according to any one of claims 1 to 7.

9. The antibody of claim 8, wherein the heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 45, 413 and 414, respectively, and the light chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 64, 66 and 69, respectively.

10. The antibody of claim 8, wherein the heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 45, 415 and 416, respectively, and the light chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 64, 66 and 68, respectively.

11. The antibody of claim 8, wherein the heavy chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 45, 415 and 416, respectively, and the light chain CDR1, CDR2 and CDR3 comprise SEQ ID NOs: 64, 66 and 419, respectively.

12. An isolated antibody that binds to human TIM3, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO:

410.

13. An isolated antibody that binds to human TIM3, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VL comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO:

411.

14. An isolated antibody that binds to human TIM3, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VL comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO:

412.

15. binds to human TIM3 and cross-competes with a control antibody for TIM3 binding; (a) a heavy chain variable region (VH) comprising SEQ ID NO: 410 and a light chain variable region (VL) comprising SEQ ID NO: 417; (b) a heavy chain variable region (VH) comprising SEQ ID NO: 411 and a light chain variable region (VL) comprising SEQ ID NO: 60; (c) a heavy chain variable region (VH) comprising SEQ ID NO: 411 and a light chain variable region (VL) comprising SEQ ID NO: 418; or (d) a heavy chain variable region (VH) comprising SEQ ID NO: 412 and a light chain variable region (VL) comprising SEQ ID NO: 60 1. An isolated antibody comprising:

16. (a) a heavy chain CDR1, CDR2, and CDR3 of a heavy chain variable region (VH) comprising SEQ ID NO: 410 and a light chain CDR1, CDR2, and CDR3 of a light chain variable region (VL) comprising SEQ ID NO: 417; (b) a heavy chain CDR1, CDR2, and CDR3 of a heavy chain variable region (VH) comprising SEQ ID NO: 411 and a light chain CDR1, CDR2, and CDR3 of a light chain variable region (VL) comprising SEQ ID NO: 60; (c) a heavy chain CDR1, CDR2, and CDR3 of a heavy chain variable region (VH) comprising SEQ ID NO: 411 and a light chain CDR1, CDR2, and CDR3 of a light chain variable region (VL) comprising SEQ ID NO: 418; or (d) a heavy chain CDR1, CDR2, and CDR3 of a heavy chain variable region (VH) comprising SEQ ID NO: 412 and a light chain CDR1, CDR2, and CDR3 of a light chain variable region (VL) comprising SEQ ID NO: 60; The antibody described in claim 15.

17. binds to human TIM3 and binds to the same epitope as the control antibody, as determined by HDX; (a) a heavy chain variable region (VH) comprising SEQ ID NO: 410 and a light chain variable region (VL) comprising SEQ ID NO: 417; (b) a heavy chain variable region (VH) comprising SEQ ID NO: 411 and a light chain variable region (VL) comprising SEQ ID NO: 60; (c) a heavy chain variable region (VH) comprising SEQ ID NO: 411 and a light chain variable region (VL) comprising SEQ ID NO: 418; or (d) a heavy chain variable region (VH) comprising SEQ ID NO: 412 and a light chain variable region (VL) comprising SEQ ID NO: 60 1. An isolated antibody comprising:

18. (a) a heavy chain CDR1, CDR2, and CDR3 of a heavy chain variable region (VH) comprising SEQ ID NO: 410 and a light chain CDR1, CDR2, and CDR3 of a light chain variable region (VL) comprising SEQ ID NO: 417; (b) a heavy chain CDR1, CDR2, and CDR3 of a heavy chain variable region (VH) comprising SEQ ID NO: 411 and a light chain CDR1, CDR2, and CDR3 of a light chain variable region (VL) comprising SEQ ID NO: 60; (c) a heavy chain CDR1, CDR2, and CDR3 of a heavy chain variable region (VH) comprising SEQ ID NO: 411 and a light chain CDR1, CDR2, and CDR3 of a light chain variable region (VL) comprising SEQ ID NO: 418; or (d) the antibody of claim 17, comprising a heavy chain CDR1, CDR2 and CDR3 of a heavy chain variable region (VH) comprising SEQ ID NO: 412 and a light chain CDR1, CDR2 and CDR3 of a light chain variable region (VL) comprising SEQ ID NO:

60.

19. An antibody described in any of claims 12 and 15 to 18, wherein the VH comprises SEQ ID NO: 410 and the VL comprises SEQ ID NO:

417.

20. The antibody of any one of claims 13 to 18, wherein VH comprises SEQ ID NO: 411 and VL comprises SEQ ID NO:

60.

21. The antibody of any one of claims 13 to 18, wherein the VH comprises SEQ ID NO: 411 and the VL comprises SEQ ID NO:

418.

22. The antibody of any one of claims 13 to 18, wherein VH comprises SEQ ID NO: 412 and VL comprises SEQ ID NO:

60.

23. 1. An isolated antibody that binds to human TIM3, comprising a heavy chain CDR1, CDR2, and CDR3 and a light chain CDR1, CDR2, and CDR3, wherein (a) the heavy chain CDR1 comprises SEQ ID NO:469; (b) the heavy chain CDR2 comprises SEQ ID NO:470; and (c) the heavy chain CDR3 comprises SEQ ID NO:

471.

24. (a) the light chain CDR1 comprises SEQ ID NO:472; (b) the light chain CDR2 comprises SEQ ID NO:473; and (c) the light chain CDR3 comprises SEQ ID NO:

474. The antibody of claim 23.

25. comprising a heavy chain variable region (VH) and a light chain variable region (VL), (a) the VH comprises SEQ ID NO: 449 or SEQ ID NO: 456; and (b) the VL comprises SEQ ID NO: 450; An antibody described in claim 23 or 24.

26. The antibody of any one of claims 1 to 25, wherein the antibody is selected from the group consisting of IgG1, IgG2, IgG3, IgG4 and variants thereof.

27. 27. The antibody of claim 26, wherein the antibody is an IgG1 antibody.

28. 28. The antibody of claim 27, comprising an effectorless IgG1 Fc.

29. 1. An isolated antibody that binds to human TIM3, comprising: (a1) heavy chain and light chain sequences comprising SEQ ID NOs: 386 (or 387) and 408, respectively; (a2) heavy chain and light chain sequences comprising SEQ ID NOs: 388 (or 389) and 408, respectively; (a3) heavy chain and light chain sequences comprising SEQ ID NOs: 390 (or 391) and 408, respectively; (a4) heavy chain and light chain sequences comprising SEQ ID NOs: 392 (or 393) and 408, respectively; (b1) heavy and light chain sequences comprising SEQ ID NOs: 394 (or 395) and 29, respectively; (b2) heavy and light chain sequences comprising SEQ ID NOs: 394 (or 395) and 409, respectively; (b3) heavy chain and light chain sequences comprising SEQ ID NOs: 396 (or 397) and 29, respectively; (b4) heavy and light chain sequences comprising SEQ ID NOs: 398 (or 399) and 29, respectively; (b5) heavy and light chain sequences comprising SEQ ID NOs: 400 (or 401) and 29, respectively; (b6) heavy and light chain sequences comprising SEQ ID NOs: 402 (or 403) and 29, respectively; (b7) heavy and light chain sequences comprising SEQ ID NOs: 404 (or 405) and 29, respectively; (b8) heavy and light chain sequences comprising SEQ ID NOs: 406 (or 407) and 29, respectively; (c1) heavy and light chain sequences comprising SEQ ID NOs: 451 (or 461) and 453, respectively; (c2) heavy and light chain sequences comprising SEQ ID NOs: 452 (or 462) and 453, respectively; (d1) heavy chain and light chain sequences comprising SEQ ID NOs: 457 (or 465) and 453, respectively; or (d2) heavy chain and light chain sequences comprising SEQ ID NOs: 458 (or 466) and 453, respectively 1. An isolated antibody comprising:

30. 30. The antibody of any one of claims 1 to 29, having one or more of the following properties: (1) For example, an EC2 of 1 μg / mL or less (e.g., 0.01 μg / mL to 0.05 μg / mL) for membrane-bound human TIM3, as measured by flow cytometry. ５０ Combine with; (2) For example, membrane-bound cynomolgus monkey TIM3, e.g., as measured by flow cytometry; (3) 5 × 10 hTIM3-IgV was detected by the method described in Example 22. －8 M, 2 x 10 －8 M, 10 －8 M or 5 x 10 －9 K below M Ｄ Combine with; (4) hTIM3-IgV was measured by the method described in Example 22. －7 M, 2 x 10 －8 M or 10 －8 K below M Ｄ Combine with; (5) 5 × 10 TIM3-ECD in cynomolgus monkeys was measured by the method described in Example 22. －7 M, 2 x 10 －8 M or 10 －8 K below M Ｄ Combine with; (6) 8 × 10 hTIM3-ECD was detected by the method described in Example 22. －8 M, 5 x 10 －8 M or 10 －8 K below M Ｄ Combine with; (7) induce or enhance T cell activation (e.g., by blocking or reducing the inhibitory effect of TIM3), as evidenced by increased proliferation of TIM3-expressing T cells (e.g., Th1 cells or TILs); and / or (8) Competes in either or both directions for binding to human TIM3 with an antibody comprising the VH and VL of any of 14H7, 23B3, 13A3, TIM3.7, TIM3.10, TIM3.11, TIM3.12, TIM3.13, TIM3.14, TIM3.15, TIM3.16, TIM3.17, TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24, TIM3.25, 17C3, 9F6, TIM3.7, 3G4, and 17C8.

31. 1. An isolated antibody that binds to human TIM3 or an antigen-binding fragment thereof, (i) a VH CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 45, 415, and 416, respectively, and a VL CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 64, 66, and 68, respectively; or (ii) a VH comprising SEQ ID NO: 412 and a VL comprising SEQ ID NO: 60 and human TIM3-IgV at 5 × 10 －9 K below M Ｄ cynomolgus monkey TIM3-IgV binds at 10 －7 K below M Ｄ and / or 5×10 to cynomolgus TIM3-ECD －6 K below M Ｄ An antibody that binds to

32. 1. An isolated antibody that binds to human TIM3 or an antigen-binding fragment thereof, (i) a VH CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 45, 413, and 414, respectively, and a VL CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 64, 66, and 69, respectively; or (ii) a VH comprising SEQ ID NO: 410 and a VL comprising SEQ ID NO: 417 and human TIM3-IgV at 5 × 10 －8 K below M Ｄ 5 × 10 to cynomolgus monkey TIM3-IgV －9 K below M Ｄ and / or 5×10 to cynomolgus TIM3-ECD －9 K below M Ｄ An antibody that binds to

33. 33. The antibody of any one of claims 1 to 32, wherein the heavy chain comprises or lacks a C-terminal lysine.

34. 34. The antibody of any one of claims 1 to 33, wherein the heavy and light chains are full-length heavy and light chains, respectively (full-length antibody) or the heavy and light chains are antigen-binding heavy and light chain fragments, respectively (antigen-binding antibody fragment or portion).

35. A bispecific molecule comprising the antibody of claims 1 to 34 conjugated to a molecule with a second binding specificity.

36. A nucleic acid encoding the VH and / or VL of an antibody according to any one of claims 1 to 34.

37. 37. An expression vector comprising the nucleic acid of claim 36.

38. A cell transformed with the expression vector of claim 37.

39. An immunoconjugate comprising an antibody according to any one of claims 1 to 34 conjugated to a drug.

40. A composition comprising an antibody according to any one of claims 1 to 34, a bispecific molecule according to claim 35, a nucleic acid according to claim 36, a vector according to claim 37, a cell according to claim 38 or an immunoconjugate according to claim 39 and a carrier.

41. 40. A kit comprising an antibody according to any one of claims 1 to 34, a bispecific molecule according to claim 35, a nucleic acid according to claim 36, a vector according to claim 37, a cell according to claim 38 or an immunoconjugate according to claim 39 and instructions for use.

42. 39. A method of producing an anti-TIM3 antibody, comprising expressing the antibody in a cell of claim 38 and isolating the antibody from the cell.

43. 40. A method of stimulating an antigen-specific T cell response, comprising contacting a T cell with the antibody of any of claims 1 to 34, the bispecific molecule of claim 35, the nucleic acid of claim 36, the vector of claim 37, the cell of claim 38 or the immunoconjugate of claim 39, such that an antigen-specific T cell response is stimulated.

44. 40. A method of activating or co-stimulating an effector T cell, comprising contacting an effector T cell with the antibody of any of claims 1 to 34, the bispecific molecule of claim 35, the nucleic acid of claim 36, the vector of claim 37, the cell of claim 38 or the immunoconjugate of claim 39 and CD3, wherein the effector T cell is activated or co-stimulated.

45. 40. A method of increasing IFN-γ production in T cells, comprising contacting a T cell with an effective amount of the antibody of any of claims 1 to 34, the bispecific molecule of claim 35, the nucleic acid of claim 36, the vector of claim 37, the cell of claim 38 or the immunoconjugate of claim 39, wherein IFN-γ production from the T cell is increased.

46. 40. A method of increasing T cell proliferation, comprising contacting a T cell with an effective amount of the antibody of any of claims 1 to 34, the bispecific molecule of claim 35, the nucleic acid of claim 36, the vector of claim 37, the cell of claim 38, or the immunoconjugate of claim 39.

47. 40. A method for increasing IFN-γ production from T cells in a subject, comprising administering an effective amount of the antibody of any of claims 1 to 34, the bispecific molecule of claim 35, the nucleic acid of claim 36, the vector of claim 37, the cell of claim 38 or the immunoconjugate of claim 39, wherein IFN-γ production from T cells of the subject is increased.

48. 40. A method of stimulating TIL activity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody of any of claims 1 to 34, the bispecific molecule of claim 35, the nucleic acid of claim 36, the vector of claim 37, the cell of claim 38 or the immunoconjugate of claim 39, such that TIL activity is stimulated.

49. 40. A method of stimulating an immune response in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody of any of claims 1 to 34, the bispecific molecule of claim 35, the nucleic acid of claim 36, the vector of claim 37, the cell of claim 38 or the immunoconjugate of claim 39, such that an immune response in the subject is stimulated.

50. 50. The method of claim 49, wherein the subject has a tumor and an immune response against the tumor is stimulated.

51. 40. A method of inhibiting tumor growth or reducing tumor size in a subject in need thereof comprising administering to the subject the antibody of any of claims 1 to 34, the bispecific molecule of claim 35, the nucleic acid of claim 36, the vector of claim 37, the cell of claim 38 or the immunoconjugate of claim 39, such that tumor growth is inhibited or tumor size is reduced in the subject.

52. 40. A method of treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of the antibody of any of claims 1 to 34, the bispecific molecule of claim 35, the nucleic acid of claim 36, the vector of claim 37, the cell of claim 38, or the immunoconjugate of claim 39.

53. 53. The method of claim 52, wherein the cancer is selected from the group consisting of bladder cancer, breast cancer, uterine / cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, colon cancer, kidney cancer, head and neck cancer, lung cancer, gastric cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, lymphoma, leukemia, myeloma, sarcoma, virus-associated cancer, and any combination thereof.

54. 54. The method of claim 52 or 53, wherein the cancer is metastatic cancer, refractory cancer, or recurrent cancer.

55. 55. The method of any of claims 47 to 54, further comprising administering one or more additional therapeutic agents.

56. 56. The method of claim 55, wherein the one or more additional therapeutic agents are selected from the group consisting of an anti-PD-1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, an anti-GITR antibody, an anti-PD-L1 antibody, and any combination thereof.

57. A method for detecting the presence of T-cell immunoglobulin and mucin domain-containing 3 (TIM3) in a sample, comprising: (i) contacting the sample with an antibody of any one of claims 1 to 34 under conditions that allow the formation of a complex between the antibody and TIM3; and (ii) detecting the formation of the complex.

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