Anti-TIM3 antibodies and uses thereof

Abstract

Provided herein are antibodies, or antigen-binding portions thereof, that bind to a T cell immunoglobulin and a mucin domain-containing protein 3 (TIM3) protein. Also provided are uses of these antibodies, or antigen-binding portions thereof, in therapeutic applications, such as for the treatment of cancer. Also provided are cells producing 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.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201980018622.8. The filing date of the original application is January 11, 2019, and the invention title is "Anti-TIM3 Antibodies and Their Uses".

[0002] Reference to a Sequence Listing Submitted Electronically via EFS-WEB

[0003] The content of the sequence listing in the electronically submitted ASCII text file (name: 3338_098PC01_SequenceListing.txt; size: 912,981 bytes; creation date: January 11, 2019) submitted together with this application is hereby incorporated by reference in its entirety. BACKGROUND OF THE INVENTION

[0005] 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 the immune response. TIM3 was initially identified on activated IFN-γ-producing T cells (e.g., type 1 helper CD4 + T cells and cytotoxic CD8 + T cells) and was shown to induce T cell death or exhaustion upon binding to galectin-9. More recent studies have shown that TIM3 expression is also 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).

[0006] Like many inhibitory receptors (e.g., PD-1 and CTLA-4), TIM3 expression is associated with multiple types of chronic diseases, including cancer. TIM3 + T cells have been detected in patients with advanced melanoma, non-small cell lung cancer, or follicular B cell non-Hodgkin lymphoma. Also, the presence of TIM3 + regulatory T cells has been described as a valid indicator of lung cancer progression. See Anderson AC, Cancer Immunol Res. 2:393-8 (2014).

[0007] Several potential ligands of TIM3 have been identified: galectin-9, HMGB1, semaphorin-4A, CEACAM-1, ILT-4, and phosphatidylserine (PtdSer or PS). PS is an important cell membrane component and is normally located in the inner leaflet of the cell membrane. However, during apoptosis, PS is redistributed and exposed on the outer membrane. This redistribution has also been observed in many tumor cell lines. See Riedl S et al., Biochim Biophys Acta. 1808:2638-2645 (2011). The binding of TIM3 to PS may be crucial for phagocytosis and cross-presentation. See Nakayama M et al., Blood. 113:3821-30 (2009).

[0008] 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 show more dysfunctional phenotypes compared to T cells expressing only PD-1 or TIM3. See Fourcade J et al., J Exp Med. 207:2175-2186 (2010).

[0009] Therefore, there is a great need for agents that target TIM3 and methods of using such agents in the design of new cancer immunotherapies and the improvement of traditional cancer immunotherapies. SUMMARY OF THE INVENTION

[0011] 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 protein 3 (TIM3) ("anti-TIM3 antibodies") and comprise heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3. In some embodiments, the heavy chain CDR3 comprises SEQ ID NO: 414 or SEQ ID NO: 416. In some embodiments, the heavy chain CDR1 comprises SEQ ID NO: 45. In some embodiments, the heavy chain CDR2 comprises SEQ ID NO: 413 or SEQ ID NO: 415. In some embodiments, the light chain CDR1 comprises SEQ ID NO: 64. In some embodiments, the light chain CDR2 comprises SEQ ID NO: 66. In some embodiments, the light chain CDR3 comprises SEQ ID NO: 69, SEQ ID NO: 68, or SEQ ID NO: 419.

[0012] In some 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:

[0013] (a) The heavy chain CDR1 contains SEQ ID NO:45;

[0014] (b) The heavy chain CDR2 contains SEQ ID NO:413 or SEQ ID NO:415;

[0015] (c) The heavy chain CDR3 contains SEQ ID NO:414 or SEQ ID NO:416;

[0016] (d) The light chain CDR1 contains SEQ ID NO:64;

[0017] (e) The light chain CDR2 contains SEQ ID NO:66; and

[0018] (f) The light chain CDR3 contains SEQ ID NO:69, SEQ ID NO:68 or SEQ ID NO:419.

[0019] In some embodiments, (a) the heavy chain CDR1, CDR2 and CDR3 contain SEQ ID NO:45, 413 and 414 respectively, and the light chain CDR1, CDR2 and CDR3 contain SEQ ID NO:64, 66 and 69 respectively; (b) the heavy chain CDR1, CDR2 and CDR3 contain SEQ ID NO:45, 415 and 416 respectively, and the light chain CDR1, CDR2 and CDR3 contain SEQ ID NO:64, 66 and 68 respectively; or (c) the heavy chain CDR1, CDR2 and CDR3 contain SEQ ID NO:45, 415 and 416 respectively, and the light chain CDR1, CDR2 and CDR3 contain SEQ ID NO:64, 66 and 419 respectively.

[0020] In some embodiments, the heavy chain CDR1, CDR2 and CDR3 contain SEQ ID NO:45, 413 and 414 respectively, and the light chain CDR1, CDR2 and CDR3 contain SEQ ID NO:64, 66 and 69 respectively. In some embodiments, the heavy chain CDR1, CDR2 and CDR3 contain SEQ ID NO:45, 415 and 416 respectively, and the light chain CDR1, CDR2 and CDR3 contain SEQ ID NO:64, 66 and 68 respectively. In some embodiments, the heavy chain CDR1, CDR2 and CDR3 contain SEQ ID NO:45, 415 and 416 respectively, and the light chain CDR1, CDR2 and CDR3 contain SEQ ID NO:64, 66 and 419 respectively.

[0021] In some 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.

[0022] In some 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.

[0023] In some 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.

[0024] In some embodiments, the anti-TIM3 antibody or antigen-binding portion thereof cross-competes for binding to TIM3 with a reference antibody and comprises:

[0025] (a) a heavy chain variable region (VH) containing SEQ ID NO: 410 and a light chain variable region (VL) containing SEQ ID NO: 417;

[0026] (b) a heavy chain variable region (VH) containing SEQ ID NO: 411 and a light chain variable region (VL) containing SEQ ID NO: 60;

[0027] (c) a heavy chain variable region (VH) containing SEQ ID NO: 411 and a light chain variable region (VL) containing SEQ ID NO: 418; or

[0028] (d) a heavy chain variable region (VH) containing SEQ ID NO: 412 and a light chain variable region (VL) containing SEQ ID NO: 60.

[0029] In some embodiments, the anti-TIM3 antibody or antigen-binding portion thereof comprises:

[0030] (a) The heavy chain CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) containing SEQ ID NO: 410 and the light chain CDR1, CDR2, and CDR3 of the light chain variable region (VL) containing SEQ ID NO: 417;

[0031] (b) The heavy chain CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) containing SEQ ID NO: 411 and the light chain CDR1, CDR2, and CDR3 of the light chain variable region (VL) containing SEQ ID NO: 60;

[0032] (c) The heavy chain CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) containing SEQ ID NO: 411 and the light chain CDR1, CDR2, and CDR3 of the light chain variable region (VL) containing SEQ ID NO: 418; or

[0033] (d) The heavy chain CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) containing SEQ ID NO: 412 and the light chain CDR1, CDR2, and CDR3 of the light chain variable region (VL) containing SEQ ID NO: 60.

[0034] In some embodiments, the anti-TIM3 antibody or antigen-binding portion thereof binds to the same epitope as a reference antibody, as determined by HDX, and the anti-TIM3 antibody or antigen-binding portion thereof comprises:

[0035] (a) A heavy chain variable region (VH) containing SEQ ID NO: 410 and a light chain variable region (VL) containing SEQ ID NO: 417;

[0036] (b) A heavy chain variable region (VH) containing SEQ ID NO: 411 and a light chain variable region (VL) containing SEQ ID NO: 60;

[0037] (c) A heavy chain variable region (VH) containing SEQ ID NO: 411 and a light chain variable region (VL) containing SEQ ID NO: 418; or

[0038] (d) A heavy chain variable region (VH) containing SEQ ID NO: 412 and a light chain variable region (VL) containing SEQ ID NO: 60.

[0039] In some embodiments, the anti-TIM3 antibody or antigen-binding portion thereof comprises:

[0040] (a) The heavy chain CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) containing SEQ ID NO: 410 and the light chain CDR1, CDR2, and CDR3 of the light chain variable region (VL) containing SEQ ID NO: 417;

[0041] (b) The heavy chain CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) containing SEQ ID NO: 411 and the light chain CDR1, CDR2, and CDR3 of the light chain variable region (VL) containing SEQ ID NO: 60;

[0042] (c) The heavy chain CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) containing SEQ ID NO: 411 and the light chain CDR1, CDR2, and CDR3 of the light chain variable region (VL) containing SEQ ID NO: 418; or

[0043] (d) The heavy chain CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) containing SEQ ID NO: 412 and the light chain CDR1, CDR2, and CDR3 of the light chain variable region (VL) containing SEQ ID NO: 60.

[0044] In some embodiments, the anti-TIM3 antibody or its antigen-binding portion comprises a VH and a VL, wherein the VH comprises SEQ ID NO: 410 and the VL comprises SEQ ID NO: 417. In some embodiments, the VH comprises SEQ ID NO: 411 and the VL comprises SEQ ID NO: 60. In some embodiments, the VH comprises SEQ ID NO: 411 and the VL comprises SEQ ID NO: 418. In some embodiments, the VH comprises SEQ ID NO: 412 and the VL comprises SEQ ID NO: 60.

[0045] In some embodiments, the anti-TIM3 antibody disclosed herein comprises heavy chain CDR1, CDR2, and CDR3 and 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. In certain embodiments, (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.

[0046] In some 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.

[0047] In some embodiments, the anti-TIM3 antibody or its antigen-binding portion is selected from IgG1, IgG2, IgG3, IgG4, and variants thereof. In some embodiments, the anti-TIM3 antibody or its antigen-binding portion is an IgG1 antibody. In some embodiments, the anti-TIM3 antibody or its antigen-binding portion comprises an effectorless IgG1 Fc.

[0048] In some embodiments, the anti-TIM3 antibody or its antigen-binding portion comprises

[0049] (a1) heavy and light chain sequences comprising SEQ ID NO:386 (or 387) and 408, respectively;

[0050] (a2) heavy and light chain sequences comprising SEQ ID NO:388 (or 389) and 408, respectively;

[0051] (a3) heavy and light chain sequences comprising SEQ ID NO:390 (or 391) and 408, respectively;

[0052] (a4) heavy and light chain sequences comprising SEQ ID NO:392 (or 393) and 408, respectively;

[0053] (b1) heavy and light chain sequences comprising SEQ ID NO:394 (or 395) and 29, respectively;

[0054] (b2) heavy and light chain sequences comprising SEQ ID NO:394 (or 395) and 409, respectively;

[0055] (b3) heavy and light chain sequences comprising SEQ ID NO:396 (or 397) and 29, respectively;

[0056] (b4) heavy and light chain sequences comprising SEQ ID NO:398 (or 399) and 29, respectively;

[0057] (b5) heavy and light chain sequences comprising SEQ ID NO:400 (or 401) and 29, respectively;

[0058] (b6) heavy and light chain sequences comprising SEQ ID NO:402 (or 403) and 29, respectively;

[0059] (b7) Heavy and light chain sequences containing SEQ ID NO: 404 (or 405) and 29, respectively;

[0060] (b8) Heavy and light chain sequences containing SEQ ID NO: 406 (or 407) and 29, respectively;

[0061] (c1) Heavy and light chain sequences containing SEQ ID NO: 451 (or 461) and 453, respectively;

[0062] (c2) Heavy and light chain sequences containing SEQ ID NO: 452 (or 462) and 453, respectively;

[0063] (d1) Heavy and light chain sequences containing SEQ ID NO: 457 (or 465) and 453, respectively; or

[0064] (d2) Heavy and light chain sequences containing SEQ ID NO: 458 (or 466) and 453, respectively.

[0065] In some embodiments, the anti-TIM3 antibody or its antigen-binding portion has one or more of the following properties:

[0066] (1) Binds to membrane-bound human TIM3, e.g., with an EC 50 of 1 μg / mL or less (e.g., 0.01 μg / mL to 0.05 μg / mL), e.g., as determined by flow cytometry;

[0067] (2) Binds to membrane-bound cyno TIM3, e.g., as determined by flow cytometry;

[0068] (3) Binds to hTIM3-IgV with a K D of 5x10 -8 M, 2x10 -8 M, 10 -8 M or 5x10 -9 M or less, as determined by the method described in Example 22;

[0069] (4) Binds to hTIM3-IgV with a K D of 10 -7 M, 2x10 -8 M or 10 -8 M or less, as determined by the method described in Example 22;

[0070] (5) Binds to hTIM3-IgV with a K D of 5x10 -7 M, 2x10 -8 M or 10 -8M or less binds to cyno TIM3-ECD, as determined by the method described in Example 22;

[0071] (6) With K D 8x10 -8 M, 5x10 -8 M or 10 -8 M or less binds to hTIM3-ECD, as determined by the method described in Example 22;

[0072] (7) Induce or enhance T cell activation (e.g., by blocking or reducing the inhibitory effect of TIM3), as demonstrated by enhanced proliferation of TIM3-expressing T cells (e.g., Th1 cells or TIL); and / or

[0073] (8) Compete with antibodies containing 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 for binding to human TIM3 in one or both directions.

[0074] In some embodiments, the anti-TIM3 antibody comprises: (i) VH CDR1, CDR2 and CDR3 containing SEQ ID NO:45, 415 and 416, respectively, and VL CDR1, CDR2 and CDR3 containing SEQ ID NO:64, 66 and 68, respectively; or (ii) VH containing SEQ ID NO:412 and VL containing SEQ ID NO:60; wherein the antibody binds to human TIM3-IgV with a KD of 5x10 -9 M or less; binds to cyno TIM3-IgV with a KD of 10 -7 M or less; and / or binds to cynoTIM3-ECD with a KD of 5x10 -6 M or less.

[0075] In some embodiments, the anti-TIM3 antibody comprises: (i) VH CDR1, CDR2 and CDR3 containing SEQ ID NO:45, 413 and 414, respectively, and VL CDR1, CDR2 and CDR3 containing SEQ ID NO:64, 66 and 69, respectively; or (ii) VH containing SEQ ID NO:410 and VL containing SEQ ID NO:417; wherein the antibody binds with a KD of 5x10-8 binds to human TIM3-IgV with KD of 5x10 M or less; -9 binds to cyno TIM3-IgV with KD of 5x10 M or less; and / or -9 binds to cynoTIM3-ECD with KD of 5x10 M or less.

[0076] In some embodiments, the heavy chain of the anti-TIM3 antibodies disclosed herein comprises a C-terminal lysine. In other embodiments, the heavy chain of the anti-TIM3 antibody does not comprise a C-terminal lysine.

[0077] Provided herein are bispecific molecules comprising an anti-TIM3 antibody or an antigen-binding portion thereof of the present disclosure linked to a molecule having a second binding specificity.

[0078] Provided herein are nucleic acids encoding VH and / or VL of an anti-TIM3 antibody or an antigen-binding portion thereof, expression vectors comprising the nucleic acids, and cells transformed with the expression vectors.

[0079] Provided herein are immunoconjugates comprising an anti-TIM3 antibody or an antigen-binding portion thereof described herein linked to a reagent.

[0080] Provided herein are compositions comprising an anti-TIM3 antibody or an antigen-binding portion thereof, a bispecific molecule, or an immunoconjugate described herein and a carrier. Also provided herein are kits comprising an anti-TIM3 antibody or an antigen-binding portion thereof, a bispecific molecule, or an immunoconjugate described herein, and instructions for use.

[0081] Provided herein is a method for preparing an anti-TIM3 antibody or an antigen-binding portion thereof, the method comprising expressing the anti-TIM3 antibody or an antigen-binding portion thereof in a cell and isolating the antibody or an antigen-binding portion thereof from the cell.

[0082] Provided herein is a method for stimulating an antigen-specific T cell response, the method comprising contacting a T cell with an anti-TIM3 antibody or an antigen-binding portion thereof, a bispecific molecule, or an immunoconjugate described herein, thereby stimulating an antigen-specific T cell response (e.g., by inhibiting the negative effect of TIM3 on cells such as T cells).

[0083] Provided herein are methods for activating or co-stimulating 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 an antigen-binding portion thereof, a bispecific molecule, or an immunoconjugate described herein and CD3, wherein the effector T cell is activated or co-stimulated (e.g., by inhibiting the negative effect of TIM3 on cells (e.g., T cells)).

[0084] The present disclosure provides methods for increasing IFN-γ production and / or proliferation of T cells, such as Th1 cells or TILs, which comprise contacting the T cells with an effective amount of an anti-TIM3 antibody or antigen-binding portion, bispecific molecule, or immunoconjugate described herein.

[0085] The present disclosure provides methods for increasing IFN-γ production of T cells in a subject, which comprise administering to the subject an effective amount of an anti-TIM3 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate described herein to increase IFN-γ production from T cells.

[0086] The present disclosure provides a method for stimulating TIL activity in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-TIM3 antibody or antigen-binding portion thereof described herein to cause TILs to proliferate or secrete cytokines, such as IFN-γ.

[0087] The present disclosure provides a method for stimulating NK cells and / or macrophages or other antigen-presenting cells in a subject (e.g., by increasing the cytotoxic activity of NK cells), which comprises 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 antibody described herein can increase IL-12 secretion by antigen-presenting cells in contact with the TIM3 antibody.

[0088] The present disclosure provides a method for stimulating an immune response in a subject, the method comprising administering to the subject an anti-TIM3 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate described herein, thereby stimulating the immune response of the subject. In some embodiments, the subject has a tumor, and the immune response against the tumor is stimulated.

[0089] The present disclosure provides methods for inhibiting tumor growth or reducing tumor size in a subject, which comprise administering to the subject an anti-TIM3 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate described herein, thereby inhibiting tumor growth in the subject.

[0090] The present disclosure provides methods of treating cancer, such as by immunotherapy, which include administering to a subject in need thereof a therapeutically effective amount of an anti-TIM3 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate described herein to treat cancer. In some embodiments, the cancer is selected from: 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, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system tumors, lymphoma, leukemia, myeloma, sarcoma, virus-associated cancers, 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.

[0091] In some embodiments, the methods described herein further comprise administering in combination with the anti-TIM3 antibody one or more additional therapeutic agents, such as an anti-PD-1 antibody, anti-LAG-3 antibody, anti-CTLA-4 antibody, anti-GITR antibody, and / or anti-PD-L1 antibody.

[0092] The present disclosure provides a method of detecting the presence of TIM3 protein in a sample, the method comprising contacting the sample with an anti-TIM3 antibody or antigen-binding portion thereof under conditions that permit the formation of a complex between the antibody or antigen-binding portion thereof and TIM3, and detecting the formation of the complex.

[0093] Embodiments

[0094] Embodiment 1. An isolated antibody (e.g., a human antibody) or antigen-binding portion thereof that binds human T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) and exhibits the following characteristics:

[0095] (a) binds soluble human TIM3;

[0096] (b) binds membrane-bound human TIM3;

[0097] (c) induces or enhances T cell activation; and optionally:

[0098] (d) binds soluble cynomolgus TIM3; and

[0099] (e) binds membrane cynomolgus TIM3.

[0100] Embodiment 2. The antibody or antigen-binding portion thereof according to Embodiment 1, wherein the antibody stimulates an anti-tumor immune response.

[0101] Embodiment 3. An antibody or antigen-binding portion thereof according to Embodiment 1 or 2, wherein the antibody stimulates an antigen-specific T cell response.

[0102] Embodiment 4. An antibody or antigen-binding portion thereof according to any of the preceding embodiments, wherein the antibody increases IFN-γ production in T cells expressing TIM3.

[0103] Embodiment 5. An antibody or antigen-binding portion thereof according to any of the preceding embodiments, wherein the antibody increases T cell proliferation.

[0104] Embodiment 6. An antibody or antigen-binding portion thereof according to any of the preceding embodiments, wherein the antibody does not bind to Fc receptors or wherein the antibody lacks effector functions.

[0105] Embodiment 7. An antibody or antigen-binding portion thereof according to any of the preceding embodiments, wherein the antibody binds soluble human TIM3 with a K D 10 nM or less, as measured by Biacore.

[0106] Embodiment 8. An antibody or antigen-binding portion thereof according to any of the preceding embodiments, wherein the antibody binds soluble cynomolgus monkey TIM3 with a K D 100 nM or less, as measured by Biacore.

[0107] Embodiment 9. An antibody or antigen-binding fragment thereof according to any of the preceding embodiments, wherein the antibody is an antagonistic antibody that inhibits signal transduction of negative cells (e.g., T cells) through TIM3.

[0108] Embodiment 10. An antibody or antigen-binding portion thereof according to any of the preceding embodiments, wherein the antibody binds membrane-bound human TIM3 with an EC 50 0.1 or 1 μg / mL or less, as measured by flow cytometry.

[0109] Embodiment 11. An antibody or antigen-binding portion thereof according to any of the preceding embodiments, wherein the antibody binds membrane-bound human TIM3 with a K D 1 nM or less, as measured by Scatchard analysis.

[0110] Embodiment 12. An antibody or antigen-binding portion thereof according to any of the preceding embodiments, wherein the antibody binds membrane-bound cynomolgus monkey TIM3 with an EC 50 1 μg / mL or less, as measured by flow cytometry; or wherein the antibody binds membrane-bound cyno TIM3 with a K D 1 nM or less, as measured by Scatchard analysis.

[0111] Embodiment 13. An antibody or antigen-binding portion thereof according to any one 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, and wherein the heavy chain CDR3 comprises an amino acid sequence selected from 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.

[0112] Embodiment 14. An antibody or antigen-binding portion thereof according to Embodiment 13, wherein the heavy chain CDR1 comprises X1, X2, X3, X4, Y, X5, and X6, and 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.

[0113] Embodiment 15. An antibody or antigen-binding portion thereof according to Embodiment 13 or 14, wherein the heavy chain CDR1 comprises X1, Y, Y, M, and X2, and wherein X1 is S or D and X2 is H or S.

[0114] Embodiment 16. An antibody or antigen-binding portion thereof according to Embodiment 13 or 14, wherein the heavy chain CDR1 comprises R, X1, Y, W, and X2, and wherein X1 is H or Y and X2 is N or S.

[0115] Embodiment 17. An antibody or antigen-binding portion thereof according to any one of Embodiments 13 to 16, wherein the heavy chain CDR2 comprises X1, I, X2, X3, X4, G, X5, X6, X7, X8, Y, X9, X10, X11, X12, X13, and X14, and 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, X10 is P, S, Q, or D, X11 is S or K, X12 is L, F, or V, X13 is K or Q, and X14 is S or G.

[0116] Embodiment 18. An antibody or antigen-binding portion thereof according to any one 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, and wherein X1 is S or T, and X2 is S or P.

[0117] Embodiment 19. The antibody or antigen-binding portion thereof according to any one 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, and wherein X1 is G, T, or S, X2 is G or S, and X3 is T or I.

[0118] Embodiment 20. The antibody or antigen-binding portion thereof according to any one 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.

[0119] Embodiment 21. The antibody or antigen-binding portion thereof according to any one of Embodiments 13 to 20, wherein the light chain CDR1 comprises SEQ ID NO:64 or SEQ ID NO:65.

[0120] Embodiment 22. The antibody or antigen-binding portion thereof according to any one of Embodiments 13 to 21, wherein the light chain CDR2 comprises SEQ ID NO:66 or SEQ ID NO:67.

[0121] Embodiment 23. The antibody or antigen-binding portion thereof according to any one 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.

[0122] Embodiment 24. The antibody or antigen-binding portion thereof according to any of the foregoing embodiments, wherein the antibody or antigen-binding portion comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3, wherein

[0123] (a) The heavy chain CDR1 is selected from SEQ ID NO:41, SEQ ID NO:42; SEQ ID NO:43; SEQ ID NO:44; and SEQ ID NO:45;

[0124] (b) The heavy chain CDR2 is selected from 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;

[0125] (c) The heavy chain CDR3 is selected from 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;

[0126] (d) The light chain CDR1 contains SEQ ID NO:64 or SEQ ID NO:65;

[0127] (e) The light chain CDR2 contains SEQ ID NO:66 or SEQ ID NO:67; and

[0128] (f) The light chain CDR3 contains SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70 or SEQ ID NO:71.

[0129] Embodiment 25. An isolated antibody or antigen-binding portion thereof that binds to human TIM3, comprising:

[0130] (a1) The heavy chain variable region CDR1, CDR2 and CDR3 respectively comprising SEQ ID NO:41, 46, 53, and the light chain variable region CDR1, CDR2 and CDR3 respectively comprising SEQ ID NO:64, 66, 68;

[0131] (a2) The heavy chain variable region CDR1, CDR2 and CDR3 respectively comprising SEQ ID NO:41, 122, 53, and the light chain variable region CDR1, CDR2 and CDR3 respectively comprising SEQ ID NO:64, 66, 68;

[0132] (a3) The heavy chain variable region CDR1, CDR2 and CDR3 respectively comprising SEQ ID NO:41, 123, 53, and the light chain variable region CDR1, CDR2 and CDR3 respectively comprising SEQ ID NO:64, 66, 68;

[0133] (a4) The heavy chain variable region CDR1, CDR2 and CDR3 respectively comprising SEQ ID NO:41, 124, 53, and the light chain variable region CDR1, CDR2 and CDR3 respectively comprising SEQ ID NO:64, 66, 68;

[0134] (a5) Comprising the heavy chain variable region CDR1, CDR2, and CDR3 of SEQ ID NO: 41, 46, and 126 respectively, and the light chain variable region CDR1, CDR2, and CDR3 of SEQ ID NO: 64, 66, and 68 respectively;

[0135] (a6) Comprising the heavy chain variable region CDR1, CDR2, and CDR3 of SEQ ID NO: 41, 46, and 127 respectively, and the light chain variable region CDR1, CDR2, and CDR3 of SEQ ID NO: 64, 66, and 68 respectively;

[0136] (a7) Comprising the heavy chain variable region CDR1, CDR2, and CDR3 of SEQ ID NO: 41, 46, and 128 respectively, and the light chain variable region CDR1, CDR2, and CDR3 of SEQ ID NO: 64, 66, and 68 respectively;

[0137] (a8) Comprising the heavy chain variable region CDR1, CDR2, and CDR3 of SEQ ID NO: 41, 46, and 129 respectively, and the light chain variable region CDR1, CDR2, and CDR3 of SEQ ID NO: 64, 66, and 68 respectively;

[0138] (a9) Comprising the heavy chain variable region CDR1, CDR2, and CDR3 of SEQ ID NO: 41, 122, and 128 respectively, and the light chain variable region CDR1, CDR2, and CDR3 of SEQ ID NO: 64, 66, and 68 respectively;

[0139] (a10) Comprising the heavy chain variable region CDR1, CDR2, and CDR3 of SEQ ID NO: 41, 122, and 126 respectively, and the light chain variable region CDR1, CDR2, and CDR3 of SEQ ID NO: 64, 66, and 68 respectively;

[0140] (b1) Comprising the heavy chain variable region CDR1, CDR2, and CDR3 of SEQ ID NO: 42, 47, and 54 respectively, and the light chain variable region CDR1, CDR2, and CDR3 of SEQ ID NO: 64, 66, and 69 respectively;

[0141] (b2) Comprising the heavy chain variable region CDR1, CDR2, and CDR3 of SEQ ID NO: 42, 125, and 54 respectively, and the light chain variable region CDR1, CDR2, and CDR3 of SEQ ID NO: 64, 66, and 69 respectively;

[0142] (c) The heavy chain CDR1, CDR2, and CDR3 sequences respectively containing SEQ ID NO: 43, 48, and 55, and / or the light chain CDR1, CDR2, and CDR3 sequences respectively containing SEQ ID NO: 64, 66, and 69;

[0143] (d) The heavy chain CDR1, CDR2, and CDR3 sequences respectively containing SEQ ID NO: 44, 49, and 56, and / or the light chain CDR1, CDR2, and CDR3 sequences respectively containing SEQ ID NO: 64, 66, and 68;

[0144] (e) The heavy chain CDR1, CDR2, and CDR3 sequences respectively containing SEQ ID NO: 45, 50, and 57, and / or the light chain CDR1, CDR2, and CDR3 sequences respectively containing SEQ ID NO: 64, 66, and 69;

[0145] (f) The heavy chain CDR1, CDR2, and CDR3 sequences respectively containing SEQ ID NO: 45, 50, and 57, and / or the light chain CDR1, CDR2, and CDR3 sequences respectively containing SEQ ID NO: 64, 66, and 71;

[0146] (g1) The heavy chain CDR1, CDR2, and CDR3 sequences respectively containing SEQ ID NO: 45, 50, and 57, and / or the light chain CDR1, CDR2, and CDR3 sequences respectively containing SEQ ID NO: 65, 67, and 70;

[0147] (g2) The heavy chain variable region CDR1, CDR2, and CDR3 respectively containing SEQ ID NO: 45, 50, 57, and the light chain variable region CDR1, CDR2, and CDR3 respectively containing SEQ ID NO: 64, 66, 71;

[0148] (g3) The heavy chain variable region CDR1, CDR2, and CDR3 respectively containing SEQ ID NO: 45, 50, 57, and the light chain variable region CDR1, CDR2, and CDR3 respectively containing SEQ ID NO: 64, 66, 69;

[0149] (h) The heavy chain CDR1, CDR2, and CDR3 sequences respectively containing SEQ ID NO: 45, 51, and 58, and / or the light chain CDR1, CDR2, and CDR3 sequences respectively containing SEQ ID NO: 64, 66, and 68;

[0150] (i) The heavy chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO: 45, 52, and 59, and / or the light chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO: 64, 66, and 69.

[0151] Embodiment 26. The antibody or antigen-binding portion thereof according to Embodiment 25, wherein the antibody comprises the heavy chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO: 41, 46, and 53, and / or the light chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO: 64, 66, and 68.

[0152] Embodiment 27. The antibody or antigen-binding portion thereof according to Embodiment 25, wherein the antibody comprises the heavy chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO: 42, 47, and 54, and / or the light chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO: 64, 66, and 69.

[0153] Embodiment 28. The antibody or antigen-binding portion thereof according to Embodiment 25, wherein the antibody comprises the heavy chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO: 43, 48, and 55, and / or the light chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO: 64, 66, and 69.

[0154] Embodiment 29. The antibody or antigen-binding portion thereof according to Embodiment 25, wherein the antibody comprises the heavy chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO: 44, 49, and 56, and / or the light chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO: 64, 66, and 68.

[0155] Embodiment 30. The antibody or antigen-binding portion thereof according to Embodiment 25, wherein the antibody comprises the heavy chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO: 45, 50, and 57, and / or the light chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO: 64, 66, and 69.

[0156] Embodiment 31. The antibody or antigen-binding portion thereof according to Embodiment 25, wherein the antibody comprises the heavy chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO: 45, 50, and 57, and / or the light chain CDR1, CDR2, and CDR3 sequences respectively comprising SEQ ID NO: 64, 66, and 71.

[0157] Embodiment 32. The antibody or antigen-binding portion thereof according to 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.

[0158] Embodiment 33. The antibody or antigen-binding portion thereof according to 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.

[0159] Embodiment 34. The antibody or antigen-binding portion thereof according to 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.

[0160] Embodiment 35. An isolated antibody or antigen-binding portion thereof that binds to human TIM3 and comprises a heavy chain variable region and a light chain variable region, 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 SEQ ID NOs: 34, 35, 36, 37, 38, 39, 40, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, and 364.

[0161] Embodiment 36. An isolated antibody or antigen-binding portion thereof that binds to human TIM3 and comprises a heavy chain variable region 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 SEQ ID NOs: 60, 61, 62, and 63.

[0162] 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 reference antibody, the reference antibody comprising VH and VL, wherein the VH and the VL are selected from:

[0163] (a) VH comprising the amino acid sequence shown in SEQ ID NO: 34 and VL comprising the amino acid sequence shown in SEQ ID NO: 60;

[0164] (b) VH comprising the amino acid sequence shown in SEQ ID NO: 35 and VL comprising the amino acid sequence shown in SEQ ID NO: 61;

[0165] (c) VH comprising the amino acid sequence shown in SEQ ID NO: 36 and VL comprising the amino acid sequence shown in SEQ ID NO: 61;

[0166] (d) VH comprising the amino acid sequence shown in SEQ ID NO: 37 and VL comprising the amino acid sequence shown in SEQ ID NO: 60;

[0167] (e) VH comprising the amino acid sequence shown in SEQ ID NO: 38 and VL comprising the amino acid sequence shown in SEQ ID NO: 61;

[0168] (f) VH comprising the amino acid sequence shown in SEQ ID NO: 38 and VL comprising the amino acid sequence shown in SEQ ID NO: 62;

[0169] (g) VH comprising the amino acid sequence shown in SEQ ID NO: 38 and VL comprising the amino acid sequence shown in SEQ ID NO: 63;

[0170] (h) VH comprising the amino acid sequence shown in SEQ ID NO: 39 and VL comprising the amino acid sequence shown in SEQ ID NO: 60;

[0171] (i) VH comprising the amino acid sequence shown in SEQ ID NO: 40 and VL comprising the amino acid sequence shown in SEQ ID NO: 61.

[0172] (j) VH comprising the amino acid sequence shown in SEQ ID NO: 121 and VL comprising the amino acid sequence shown in SEQ ID NO: 63, respectively;

[0173] (k) VH comprising the amino acid sequence shown in SEQ ID NO: 120 and VL comprising the amino acid sequence shown in SEQ ID NO: 61, respectively;

[0174] (l) VH comprising the amino acid sequence shown in SEQ ID NO: 112 and VL comprising the amino acid sequence shown in SEQ ID NO: 60, respectively;

[0175] (m) a VH comprising the amino acid sequence shown in SEQ ID NO: 113 and a VL comprising the amino acid sequence shown in SEQ ID NO: 60;

[0176] (n) a VH comprising the amino acid sequence shown in SEQ ID NO: 114 and a VL comprising the amino acid sequence shown in SEQ ID NO: 60;

[0177] (o) a VH comprising the amino acid sequence shown in SEQ ID NO: 115 and a VL comprising the amino acid sequence shown in SEQ ID NO: 60;

[0178] (p) a VH comprising the amino acid sequence shown in SEQ ID NO: 116 and a VL comprising the amino acid sequence shown in SEQ ID NO: 60;

[0179] (q) a VH comprising the amino acid sequence shown in SEQ ID NO: 117 and a VL comprising the amino acid sequence shown in SEQ ID NO: 60;

[0180] (r) a VH comprising the amino acid sequence shown in SEQ ID NO: 118 and a VL comprising the amino acid sequence shown in SEQ ID NO: 60;

[0181] (s) a VH comprising the amino acid sequence shown in SEQ ID NO: 119 and a VL comprising the amino acid sequence shown in SEQ ID NO: 60; and

[0182] (t) a VH comprising the amino acid sequence shown in SEQ ID NO: 364 and a VL comprising the amino acid sequence shown in SEQ ID NO: 60.

[0183] Embodiment 38. The antibody or antigen-binding portion thereof according to Embodiment 37, which binds TIM3 at the same epitope as the reference antibody, as determined, for example, by one or more methods provided herein.

[0184] Embodiment 39. The antibody or antigen-binding portion thereof according to Embodiment 37 or 38, comprising a VH and a VL selected from:

[0185] (a) a VH comprising the amino acid sequence shown in SEQ ID NO: 34 and a VL comprising the amino acid sequence shown in SEQ ID NO: 60;

[0186] (b) a VH comprising the amino acid sequence shown in SEQ ID NO: 35 and a VL comprising the amino acid sequence shown in SEQ ID NO: 61;

[0187] (c) a VH comprising the amino acid sequence shown in SEQ ID NO:36 and a VL comprising the amino acid sequence shown in SEQ ID NO:61;

[0188] (d) a VH comprising the amino acid sequence shown in SEQ ID NO:37 and a VL comprising the amino acid sequence shown in SEQ ID NO:60;

[0189] (e) a VH comprising the amino acid sequence shown in SEQ ID NO:38 and a VL comprising the amino acid sequence shown in SEQ ID NO:61;

[0190] (f) a VH comprising the amino acid sequence shown in SEQ ID NO:38 and a VL comprising the amino acid sequence shown in SEQ ID NO:62;

[0191] (g) a VH comprising the amino acid sequence shown in SEQ ID NO:38 and a VL comprising the amino acid sequence shown in SEQ ID NO:63;

[0192] (h) a VH comprising the amino acid sequence shown in SEQ ID NO:39 and a VL comprising the amino acid sequence shown in SEQ ID NO:60;

[0193] (i) a VH comprising the amino acid sequence shown in SEQ ID NO:40 and a VL comprising the amino acid sequence shown in SEQ ID NO:61;

[0194] (j) a VH comprising the amino acid sequence shown in SEQ ID NO:121 and a VL comprising the amino acid sequence shown in SEQ ID NO:63;

[0195] (k) a VH comprising the amino acid sequence shown in SEQ ID NO:120 and a VL comprising the amino acid sequence shown in SEQ ID NO:61;

[0196] (l) a VH comprising the amino acid sequence shown in SEQ ID NO:112 and a VL comprising the amino acid sequence shown in SEQ ID NO:60;

[0197] (m) a VH comprising the amino acid sequence shown in SEQ ID NO:113 and a VL comprising the amino acid sequence shown in SEQ ID NO:60;

[0198] (n) a VH comprising the amino acid sequence shown in SEQ ID NO:114 and a VL comprising the amino acid sequence shown in SEQ ID NO:60;

[0199] (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;

[0200] (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;

[0201] (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;

[0202] (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;

[0203] (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

[0204] (t) a VH comprising the amino acid sequence set forth in SEQ ID NO:364 and a VL comprising the amino acid sequence set forth in SEQ ID NO:60.

[0205] Embodiment 40. An antibody or antigen-binding portion thereof according to Embodiment 37 or 38, comprising a VH comprising an amino acid sequence selected from 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.

[0206] Embodiment 41. An antibody or antigen-binding portion thereof according to 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.

[0207] Embodiment 42. An antibody or antigen-binding portion thereof according to Embodiment 37 or 38, comprising 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.

[0208] Embodiment 43. The antibody or antigen-binding portion thereof according to Embodiment 37 or 38 comprises a VH containing the amino acid sequence shown in SEQ ID NO: 37 and a VL containing the amino acid sequence shown in SEQ ID NO: 60.

[0209] Embodiment 44. The antibody or antigen-binding portion thereof according to Embodiment 37 or 38 comprises a VH containing the amino acid sequence shown in SEQ ID NO: 38 or SEQ ID NO: 121 and a VL containing the amino acid sequence shown in SEQ ID NO: 61, SEQ ID NO: 63 or SEQ ID NO: 62.

[0210] Embodiment 45. The antibody or antigen-binding portion thereof according to Embodiment 37 or 38 comprises a VH containing the amino acid sequence shown in SEQ ID NO: 39 and a VL containing the amino acid sequence shown in SEQ ID NO: 60.

[0211] Embodiment 46. The antibody or antigen-binding portion thereof according to Embodiment 37 or 38 comprises a VH containing the amino acid sequence shown in SEQ ID NO: 40 and a VL containing the amino acid sequence shown in SEQ ID NO: 61.

[0212] Embodiment 47. The antibody or antigen-binding portion thereof according to any one of the preceding embodiments, wherein the antibody is selected from IgG1, IgG2, IgG3, IgG4 or variants thereof.

[0213] Embodiment 48. The antibody or antigen-binding portion thereof according to Embodiment 47, wherein the antibody is an IgG1 antibody.

[0214] Embodiment 49. The antibody or antigen-binding portion thereof according to Embodiment 47, wherein the antibody comprises an IgG1 Fc with no effector function.

[0215] Embodiment 50. The antibody or antigen-binding portion thereof according to Embodiment 49, wherein the antibody or antigen-binding portion comprises an IgG1 Fc with no effector function containing the following mutations: L234A, L235E, G237A and optionally A330S and P331S.

[0216] Embodiment 51. The antibody or antigen-binding portion thereof according to any one of the preceding embodiments comprises a heavy chain constant region containing an amino acid sequence selected from SEQ ID NO: 263 - 266.

[0217] Embodiment 52. The antibody or antigen-binding portion thereof according to any one of Embodiments 1 - 51, wherein the antibody or antigen-binding portion is a human antibody or a humanized antibody.

[0218] Embodiment 53. The antibody according to any one of Embodiments 1-52, wherein the antibody comprises:

[0219] (a1) heavy and light chain sequences containing SEQ ID NO: 301 (or 302) and 29, respectively;

[0220] (a2) heavy and light chain sequences containing SEQ ID NO: 1 (or 8) and 29, respectively;

[0221] (a3) heavy and light chain sequences containing SEQ ID NO: 15 (or 22) and 29, respectively;

[0222] (a4) heavy and light chain sequences containing SEQ ID NO: 303 (or 304) and 29, respectively;

[0223] (a5) heavy and light chain sequences containing SEQ ID NO: 72 (or 82) and 29, respectively;

[0224] (a6) heavy and light chain sequences containing SEQ ID NO: 92 (or 102) and 29, respectively;

[0225] (a7) heavy and light chain sequences containing SEQ ID NO: 305 (or 306) and 29, respectively;

[0226] (a8) heavy and light chain sequences containing SEQ ID NO: 73 (or 83) and 29, respectively;

[0227] (a9) heavy and light chain sequences containing SEQ ID NO: 93 (or 103) and 29, respectively;

[0228] (a10) heavy and light chain sequences containing SEQ ID NO: 307 (or 308) and 29, respectively;

[0229] (a11) heavy and light chain sequences containing SEQ ID NO: 74 (or 84) and 29, respectively;

[0230] (a12) heavy and light chain sequences containing SEQ ID NO: 94 (or 104) and 29, respectively;

[0231] (a13) heavy and light chain sequences containing SEQ ID NO: 309 (or 310) and 29, respectively;

[0232] (a14) heavy and light chain sequences containing SEQ ID NO: 75 (or 85) and 29, respectively;

[0233] (a15) Heavy and light chain sequences containing SEQ ID NO:95 (or 105) and 29 respectively;

[0234] (a16) Heavy and light chain sequences containing SEQ ID NO:311 (or 312) and 29 respectively;

[0235] (a17) Heavy and light chain sequences containing SEQ ID NO:76 (or 86) and 29 respectively;

[0236] (a18) Heavy and light chain sequences containing SEQ ID NO:96 (or 106) and 29 respectively;

[0237] (a19) Heavy and light chain sequences containing SEQ ID NO:313 (or 314) and 29 respectively;

[0238] (a20) Heavy and light chain sequences containing SEQ ID NO:77 (or 87) and 29 respectively;

[0239] (a21) Heavy and light chain sequences containing SEQ ID NO:97 (or 107) and 29 respectively;

[0240] (a22) Heavy and light chain sequences containing SEQ ID NO:315 (or 316) and 29 respectively;

[0241] (a23) Heavy and light chain sequences containing SEQ ID NO:78 (or 88) and 29 respectively;

[0242] (a24) Heavy and light chain sequences containing SEQ ID NO:98 (or 108) and 29 respectively;

[0243] (a25) Heavy and light chain sequences containing SEQ ID NO:317 (or 318) and 29 respectively;

[0244] (a26) Heavy and light chain sequences containing SEQ ID NO:79 (or 89) and 29 respectively;

[0245] (a27) Heavy and light chain sequences containing SEQ ID NO:99 (or 109) and 29 respectively;

[0246] (a28) Heavy and light chain sequences containing SEQ ID NO:319 (or 320) and 29 respectively;

[0247] (a29) Heavy and light chain sequences containing SEQ ID NO:349 (or 350) and 29 respectively;

[0248] (a30) Heavy and light chain sequences containing SEQ ID NO: 351 (or 352) and 29, respectively;

[0249] (a31) Heavy and light chain sequences containing SEQ ID NO: 353 (or 354) and 29, respectively;

[0250] (b1) Heavy and light chain sequences containing SEQ ID NO: 321 (or 322) and 30, respectively;

[0251] (b2) Heavy and light chain sequences containing SEQ ID NO: 2 (or 9) and 30, respectively;

[0252] (b3) Heavy and light chain sequences containing SEQ ID NO: 16 (or 23) and 30, respectively;

[0253] (b4) Heavy and light chain sequences containing SEQ ID NO: 323 (or 324) and 30, respectively;

[0254] (b5) Heavy and light chain sequences containing SEQ ID NO: 80 (or 90) and 30, respectively;

[0255] (b6) Heavy and light chain sequences containing SEQ ID NO: 100 (or 110) and 30, respectively;

[0256] (b7) Heavy and light chain sequences containing SEQ ID NO: 325 (or 326) and 30, respectively;

[0257] (c1) Heavy and light chain sequences containing SEQ ID NO: 327 (or 328) and 30, respectively;

[0258] (c2) Heavy and light chain sequences containing SEQ ID NO: 3 (or 10) and 30, respectively;

[0259] (c3) Heavy and light chain sequences containing SEQ ID NO: 17 (or 24) and 30, respectively;

[0260] (c4) Heavy and light chain sequences containing SEQ ID NO: 329 (or 330) and 30, respectively;

[0261] (d1) Heavy and light chain sequences containing SEQ ID NO: 331 (or 332) and 29, respectively;

[0262] (d2) Heavy and light chain sequences containing SEQ ID NO: 4 (or 11) and 29, respectively;

[0263] (d3) Heavy and light chain sequences containing SEQ ID NO: 18 (or 25) and 29, respectively;

[0264] (d4) Heavy and light chain sequences containing SEQ ID NO: 333 (or 334) and 29, respectively;

[0265] (e1.1) Heavy and light chain sequences containing SEQ ID NO: 335 (or 336) and 32, respectively;

[0266] (e1.2) Heavy and light chain sequences containing SEQ ID NO: 335 (or 336) and 33, respectively;

[0267] (e1.3) Heavy and light chain sequences containing SEQ ID NO: 335 (or 336) and 31, respectively;

[0268] (e2) Heavy and light chain sequences containing SEQ ID NO: 5 (or 12) and 33, respectively;

[0269] (e3) Heavy and light chain sequences containing SEQ ID NO: 19 (or 26) and 33, respectively;

[0270] (e4) Heavy and light chain sequences containing SEQ ID NO: 337 (or 338) and 33, respectively;

[0271] (e5) Heavy and light chain sequences containing SEQ ID NO: 81 (or 91) and 33, respectively;

[0272] (e6) Heavy and light chain sequences containing SEQ ID NO: 101 (or 111) and 33, respectively;

[0273] (e7) Heavy and light chain sequences containing SEQ ID NO: 339 (or 340) and 33, respectively;

[0274] (f1) Heavy and light chain sequences containing SEQ ID NO: 341 (or 342) and 29, respectively;

[0275] (f2) Heavy and light chain sequences containing SEQ ID NO: 6 (or 13) and 29, respectively;

[0276] (f3) Heavy and light chain sequences containing SEQ ID NO: 20 (or 27) and 29, respectively;

[0277] (f4) Heavy and light chain sequences containing SEQ ID NO: 343 (or 344) and 29, respectively;

[0278] (g1) Heavy and light chain sequences containing SEQ ID NO: 345 (or 346) and 29, respectively;

[0279] (g2) heavy and light chain sequences containing SEQ ID NO:7 (or 43) and 30, respectively;

[0280] (g3) heavy and light chain sequences containing SEQ ID NO:21 (or 28) and 30, respectively; or

[0281] (g4) heavy and light chain sequences containing SEQ ID NO:347 (or 348) and 30, respectively;

[0282] wherein the antibody specifically binds to human TIM3.

[0283] Embodiment 54. The antibody or antigen-binding portion thereof according to any one of Embodiments 1-53, wherein the antibody or antigen-binding portion thereof has one or more of the following characteristics:

[0284] (1) Binds to soluble human TIM3, for example, binds with a KD of 10 nM or less (e.g., 0.01 nM to 10 nM), for example, as measured by Biacore;

[0285] (2) Binds to soluble cynomolgus TIM3, for example, binds with a KD of 100 nM or less (e.g., 0.01 nM to 100 nM), for example, as measured by Biacore;

[0286] (3) Binds to membrane-bound human TIM3, for example, binds with an EC50 of 1 μg / mL or less (e.g., 0.01 μg / mL to 1 μg / mL), for example, as measured by flow cytometry;

[0287] (4) Binds to membrane-bound human TIM3, for example, binds with a KD of 1 nM or less (e.g., 0.01 nM to 10 nM), for example, as measured by Scatchard analysis;

[0288] (5) Binds to membrane-bound cynomolgus TIM3, for example, binds with an EC50 of 20 μg / mL or less (e.g., 0.01 μg / mL to 20 μg / mL), for example, as measured by flow cytometry;

[0289] (6) Binds to membrane-bound cynomolgus TIM3, for example, binds with a KD of 1 nM or less (e.g., 0.01 nM to 10 nM), for example, as measured by Scatchard analysis;

[0290] (7) Induce or enhance T cell activation (e.g., by blocking or reducing the inhibitory effect of TIM3), as demonstrated by (i) increased IFN-γ production and / or (ii) enhanced proliferation in T cells expressing TIM3 (e.g., Th1 cells or TILs);

[0291] (8) Stimulate T cell proliferation in a mixed lymphocyte reaction (MLR) assay;

[0292] (9) Inhibit the binding of phosphatidylserine to TIM3, e.g., as measured by a PS-hTIM3 "in-tandem" blocking assay;

[0293] (10) Do not internalize or downregulate cell surface TIM3 when bound to TIM3 on the cell;

[0294] (11) Bind 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);

[0295] (12) Have reduced binding to a 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 as in SEQ ID NO: 286 Figure 30 )) are replaced by another amino acid;

[0296] (13) Compete with an antibody comprising the VH and VL domains of any of 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, or TIM3.7, TIM3.8, TIM3.10, TIM3.11, TIM3.12, TIM3.13, TIM3.14, TIM3.15, TIM3.16, TIM3.17, and TIM3.18 for binding to human TIM3 in one or both directions;

[0297] (14) binds to human TIM3 regions 49VPVCWGKGACPVFE62 (SEQ ID NO:367) and 111RIQIPGIMNDEKFNLKL127 (SEQ ID NO:368), as determined by HDX-MS;

[0298] (15) has a heavy chain variable region and / or a 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; numbered according to SEQ ID NO:286( Figure 30 )); and / or

[0299] (16)(a) has reduced binding to human TIM3 in which 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids C58, P59, F61, E62, C63, R111, D120, and optionally D104 and Q113 (numbered according to SEQ ID NO:286( Figure 30 )) are replaced by another amino acid, as compared to binding to wild-type human TIM3; (b) binds to 49VPVCWGKGACPVFE62 (SEQ ID NO:367), 111RIQIPGIMNDEKFNLKL127 (SEQ ID NO:368) and 119NDEKFNLKL127 (SEQ ID NO:373), as determined by HDX-MS, as described in the Examples; and / or (c) competes or cross-blocks the binding of 13A3 or TIM3.18.IgG1.3 to human TIM3.

[0300] Embodiment 55. A bispecific molecule comprising an antibody according to any of the preceding embodiments linked to a molecule having a second binding specificity.

[0301] Embodiment 56. A nucleic acid encoding a heavy chain variable region and / or a light chain variable region of an antibody or an antigen-binding portion thereof according to any one of embodiments 1-54.

[0302] Embodiment 57. An expression vector comprising the nucleic acid molecule of embodiment 56.

[0303] Embodiment 58. A cell transformed with the expression vector of embodiment 57.

[0304] Embodiment 59. An immunoconjugate comprising an antibody according to any one of Embodiments 1-54 conjugated to a reagent.

[0305] Embodiment 60. A composition comprising an antibody or an antigen-binding portion thereof, a bispecific molecule or an immunoconjugate according to any one of Embodiments 1-55 and 59 and a carrier.

[0306] Embodiment 61. A kit comprising an antibody or an antigen-binding portion thereof or a bispecific molecule or an immunoconjugate according to any one of Embodiments 1-55 and 59 and instructions for use.

[0307] Embodiment 62. A method for preparing an anti-TIM3 antibody or an antigen-binding portion thereof, comprising expressing an anti-TIM3 antibody or an antigen-binding portion thereof in the cells of Embodiment 58, and isolating the antibody or the antigen-binding portion thereof from the cells.

[0308] Embodiment 63. A method for stimulating an antigen-specific T cell response, the method comprising contacting T cells with an antibody or an antigen-binding portion thereof, a bispecific molecule or an immunoconjugate according to any one of Embodiments 1-55 and 59, thereby stimulating an antigen-specific T cell response.

[0309] Embodiment 64. A method for activating or co-stimulating effector T cells, comprising contacting effector T cells with an anti-TIM3 antibody or an antigen-binding portion thereof, a bispecific molecule or an immunoconjugate according to any one of Embodiments 1-55 and 59 and CD3, wherein the effector T cells are activated or co-stimulated.

[0310] Embodiment 65. A method for increasing IFN-γ production in T cells, which comprises contacting T cells with an effective amount of an antibody or an antigen-binding portion thereof, a bispecific molecule or an immunoconjugate according to any one of Embodiments 1-55 and 59.

[0311] Embodiment 66. A method for increasing T cell proliferation, comprising contacting the cells with an effective amount of an antibody or an antigen-binding portion thereof, a bispecific molecule or an immunoconjugate according to any one of Embodiments 1-55 and 59.

[0312] Embodiment 67. A method for increasing IFN-γ production by T cells in a subject, which comprises administering an effective amount of an antibody or an antigen-binding portion thereof, a bispecific molecule or an immunoconjugate according to any one of Embodiments 1-55 and 59 to increase IFN-γ production from T cells.

[0313] Embodiment 68. A method for stimulating TIL activity in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-TIM3 antibody according to any one of Embodiments 1-54.

[0314] Embodiment 69. A method of stimulating an immune response in a subject, the method comprising administering to the subject an antibody or an antigen-binding portion thereof, a bispecific molecule, or an immunoconjugate according to any one of Embodiments 1-55 and 59, thereby stimulating the immune response of the subject.

[0315] Embodiment 70. The method according to Embodiment 69, wherein the subject has a tumor and an immune response against the tumor is stimulated.

[0316] Embodiment 71. A method of inhibiting tumor growth or reducing tumor size in a subject, which comprises administering to the subject an antibody or an antigen-binding portion thereof, a bispecific molecule, or an immunoconjugate according to any one of Embodiments 1-55 and 59, thereby inhibiting the tumor growth of the subject.

[0317] Embodiment 72. A method of treating cancer, which comprises administering to a subject in need thereof a therapeutically effective amount of an antibody or an antigen-binding portion thereof, a bispecific molecule, or an immunoconjugate according to any one of Embodiments 1-55 and 59 to treat cancer.

[0318] Embodiment 73. The method of Embodiment 72, wherein the cancer is selected from: 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 tumors, lymphoma, leukemia, myeloma, sarcoma, and virus-related cancers.

[0319] Embodiment 74. The method of Embodiment 72 or 73, wherein the cancer is metastatic cancer, refractory cancer, or recurrent cancer.

[0320] Embodiment 75. The method according to any one of Embodiments 67-74, further comprising administering one or more additional therapeutic agents.

[0321] Embodiment 76. The method according to Embodiment 75, wherein the additional therapy is an anti-PD-l antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, an anti-GITR antibody, or an anti-PD-L1 antibody.

[0322] Embodiment 77. A method of detecting the presence of T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) in a sample, the method comprising contacting the sample with an antibody or an antigen-binding portion thereof according to any one of Embodiments 1-54 under conditions that permit the formation of a complex between the antibody or its antigen-binding portion and TIM3, and detecting the formation of the complex. Brief Description of the Drawings

[0324] Figure 1A Disclosed is 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. CDR1 (SEQ ID NO: 41), CDR2 (SEQ ID NO: 46) and CDR3 (SEQ ID NO: 53) are drawn, and the V, D and J germline derivatives are indicated.

[0325] Figure 1B Disclosed is 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. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66) and CDR3 (SEQ ID NO: 68) are drawn, and the V and J germline derivatives are indicated.

[0326] Figure 1C Disclosed are the nucleotide sequence (SEQ ID NO: 167) and amino acid sequence (SEQ ID NO: 34) of the VH region of the heavy chain of anti-TIM3 monoclonal antibody 13A3 having a signal sequence (nucleotide sequence and amino acid sequence are SEQ ID NO: 274 and 269 respectively), and the nucleotide sequence (SEQ ID NO: 193) and amino acid sequence (SEQ ID NO: 60) of the VL region of the light chain of anti-TIM3 monoclonal antibody 13A3 having a signal sequence (nucleotide sequence and amino acid sequence are SEQ ID NO: 273 and 268 respectively).

[0327] Figure 2A Disclosed is 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. CDR1 (SEQ ID NO: 42), CDR2 (SEQ ID NO: 47) and CDR3 (SEQ ID NO: 54) are drawn, and the V, D and J germline derivatives are indicated.

[0328] Figure 2B Disclosed is 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. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66) and CDR3 (SEQ ID NO: 69) are drawn, and the V and J germline derivatives are indicated.

[0329] Figure 2C Disclosed is the nucleotide sequence (SEQ ID NO: 168) and amino acid sequence (SEQ ID NO: 35) of the heavy chain VH region of anti-TIM3 monoclonal antibody 8B9 having a signal sequence (the nucleotide sequence and amino acid sequence are SEQ ID NO: 274 and 269, respectively), and the nucleotide sequence (SEQ ID NO: 194) and amino acid sequence (SEQ ID NO: 61) of the light chain VL region of anti-TIM3 monoclonal antibody 8B9 having a signal sequence (the nucleotide sequence and amino acid sequence are SEQ ID NO: 273 and 268, respectively).

[0330] Figure 3A Disclosed is 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. CDR1 (SEQ ID NO: 43), CDR2 (SEQ ID NO: 48) and CDR3 (SEQ ID NO: 55) are drawn, and the V, D and J germline derivatives are indicated.

[0331] Figure 3B Disclosed is 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. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66) and CDR3 (SEQ ID NO: 69) are drawn, and the V and J germline derivatives are indicated.

[0332] Figure 3C Disclosed is the nucleotide sequence (SEQ ID NO: 169) and amino acid sequence (SEQ ID NO: 36) of the heavy chain VH region of anti-TIM3 monoclonal antibody 8C4 having a signal sequence (the nucleotide sequence and amino acid sequence are SEQ ID NO: 274 and 269, respectively), and the nucleotide sequence (SEQ ID NO: 194) and amino acid sequence (SEQ ID NO: 61) of the light chain VL region of anti-TIM3 monoclonal antibody 8C4 having a signal sequence (the nucleotide sequence and amino acid sequence are SEQ ID NO: 273 and 268, respectively).

[0333] Figure 4ADisclosed is 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. CDR1 (SEQ ID NO: 44), CDR2 (SEQ ID NO: 49) and CDR3 (SEQ ID NO: 56) are drawn, and the V, D and J germline derivatives are indicated.

[0334] Figure 4B Disclosed is 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. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66) and CDR3 (SEQ ID NO: 68) are drawn, and the V and J germline derivatives are indicated.

[0335] Figure 4C Disclosed are the nucleotide sequence (SEQ ID NO: 170) and amino acid sequence (SEQ ID NO: 37) of the VH region of the heavy chain of anti-TIM3 monoclonal antibody 17C3 having a signal sequence (the nucleotide sequence and amino acid sequence are SEQ ID NO: 272 and 267 respectively), and the nucleotide sequence (SEQ ID NO: 193) and amino acid sequence (SEQ ID NO: 60) of the VL region of the light chain of anti-TIM3 monoclonal antibody 17C3 having a signal sequence (the nucleotide sequence and amino acid sequence are SEQ ID NO: 273 and 268 respectively).

[0336] Figure 5A Disclosed is 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. CDR1 (SEQ ID NO: 45), CDR2 (SEQ ID NO: 50) and CDR3 (SEQ ID NO: 57) are drawn, and the V, D and J germline derivatives are indicated.

[0337] Figure 5B Disclosed is 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. CDR1 (SEQ ID NO: 65), CDR2 (SEQ ID NO: 67) and CDR3 (SEQ ID NO: 70) are drawn, and the V and J germline derivatives are indicated.

[0338] Figure 5CDisclosed is 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 the anti-TIM3 monoclonal antibody 9F6. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66) and CDR3 (SEQ ID NO: 71) are drawn, and the V and J germline derivatives are indicated.

[0339] Figure 5D Disclosed is 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 the anti-TIM3 monoclonal antibody 9F6. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66) and CDR3 (SEQ ID NO: 69) are drawn, and the V and J germline derivatives are indicated.

[0340] Figure 5E Disclosed are the nucleotide sequence (SEQ ID NO: 171) and amino acid sequence (SEQ ID NO: 38) of the heavy chain VH region of the anti-TIM3 monoclonal antibody 9F6 having a signal sequence (the nucleotide sequence and amino acid sequence are SEQ ID NO: 275 and 270 respectively), and the nucleotide sequences (SEQ ID NO: 195, 196 and 194 respectively) and amino acid sequences (SEQ ID NO: 62, 63 and 61 respectively) of the light chain VL regions of VK1, VK2 and VK3 of the anti-TIM3 monoclonal antibody 9F6 having signal sequences (the nucleotide sequences and amino acid sequences are SEQ ID NO: 276 and 271 respectively).

[0341] Figure 6A Disclosed is the nucleotide sequence (SEQ ID NO: 172) and amino acid sequence (SEQ ID NO: 39) of the mature heavy chain variable (VH) region of the anti-TIM3 monoclonal antibody 3G4. CDR1 (SEQ ID NO: 45), CDR2 (SEQ ID NO: 51) and CDR3 (SEQ ID NO: 58) are drawn, and the V, D and J germline derivatives are indicated.

[0342] Figure 6B Disclosed is the nucleotide sequence (SEQ ID NO: 193) and amino acid sequence (SEQ ID NO: 60) of the mature light chain variable (VL) region of the anti-TIM3 monoclonal antibody 3G4. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66) and CDR3 (SEQ ID NO: 68) are drawn, and the V and J germline derivatives are indicated.

[0343] Figure 6C Disclosed are the nucleotide sequence (SEQ ID NO: 172) and amino acid sequence (SEQ ID NO: 39) of the heavy chain VH region of anti-TIM3 monoclonal antibody 3G4 having a signal sequence (nucleotide sequence and amino acid sequence are SEQ ID NO: 275 and 270 respectively), and the nucleotide sequence (SEQ ID NO: 193) and amino acid sequence (SEQ ID NO: 60) of the light chain VL region of anti-TIM3 monoclonal antibody 3G4 having a signal sequence (nucleotide sequence and amino acid sequence are SEQ ID NO: 273 and 268 respectively).

[0344] Figure 7A Disclosed are 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. CDR1 (SEQ ID NO: 45), CDR2 (SEQ ID NO: 52) and CDR3 (SEQ ID NO: 59) are drawn, and the V, D and J germline derivatives are indicated.

[0345] Figure 7B Disclosed are 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. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66) and CDR3 (SEQ ID NO: 69) are drawn, and the V and J germline derivatives are indicated.

[0346] Figure 7C Disclosed are the nucleotide sequence (SEQ ID NO: 173) and amino acid sequence (SEQ ID NO: 40) of the heavy chain VH region of anti-TIM3 monoclonal antibody 17C8 having a signal sequence (nucleotide sequence and amino acid sequence are SEQ ID NO: 275 and 270 respectively), and the nucleotide sequence (SEQ ID NO: 194) and amino acid sequence (SEQ ID NO: 61) of the light chain VL region of anti-TIM3 monoclonal antibody 17C8 having a signal sequence (nucleotide sequence and amino acid sequence are SEQ ID NO: 273 and 268 respectively).

[0347] Figure 8ADisclosed is 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. CDR1 (SEQ ID NO: 45), CDR2 (SEQ ID NO: 413), and CDR3 (SEQ ID NO: 414) are depicted, and the V, D, and J germline derivatives are indicated.

[0348] Figure 8B Disclosed is 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. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66), and CDR3 (SEQ ID NO: 69) are depicted, and the V and J germline derivatives are indicated.

[0349] Figure 8C Disclosed are the nucleotide sequence (SEQ ID NO: 420) and amino acid sequence (SEQ ID NO: 410) of the VH region of the heavy chain of anti-TIM3 monoclonal antibody 14H7 having a signal sequence (nucleotide sequence and amino acid sequence are SEQ ID NO: 362 and 361, respectively), and the nucleotide sequence (SEQ ID NO: 427) and amino acid sequence (SEQ ID NO: 417) of the VL region of the light chain of anti-TIM3 monoclonal antibody 14H7 having a signal sequence (nucleotide sequence and amino acid sequence are SEQ ID NO: 363 and 361, respectively).

[0350] Figure 9A Disclosed is 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. CDR1 (SEQ ID NO: 45), CDR2 (SEQ ID NO: 415), and CDR3 (SEQ ID NO: 416) are depicted, and the V, D, and J germline derivatives are indicated.

[0351] Figure 9B Disclosed is 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. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66), and CDR3 (SEQ ID NO: 68) are depicted, and the V and J germline derivatives are indicated.

[0352] Figure 9CDisclosed is 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. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66), and CDR3 (SEQ ID NO: 419) are drawn, and the V and J germline derivatives are indicated.

[0353] Figure 9D Disclosed are the nucleotide sequence (SEQ ID NO: 421) and amino acid sequence (SEQ ID NO: 411) of the heavy chain VH region of the anti-TIM3 monoclonal antibody 23B3 having a signal sequence (nucleotide sequence and amino acid sequence are SEQ ID NO: 362 and 361 respectively), and the nucleotide sequences (SEQ ID NO: 193 and 428 respectively) and amino acid sequences (SEQ ID NO: 60 and 418 respectively) of the light chain VL regions of VK1 and VK2 of the anti-TIM3 monoclonal antibody 23B3 having signal sequences (nucleotide sequences and amino acid sequences are SEQ ID NO: 363 and 361 respectively).

[0354] Figure 10A Disclosed is the sequence alignment of the heavy chain variable (VH) regions of the monoclonal antibodies 13A3, 8B9, 8C4, 17C3, 9F6, 3G4, 17C8, 23B3, and 14H7. The complementarity-determining regions (CDRs) are in the boxes.

[0355] Figure 10B Listed are the SEQ ID NOs of the VH regions of the antibodies, each CDR, and its mutants.

[0356] Figure 11A Disclosed is the sequence alignment of the light chain variable (VL) regions of the monoclonal antibodies 13A3, 8B9, 8C4, 17C3, 9F6_VK1, 9F6_VK2, 9F6_VK3, 3G4, 17C8, 23B3_VK1, 23B3_VK2, and 14H7. The complementarity-determining regions (CDRs) are in the boxes.

[0357] Figure 11B Listed are the SEQ ID NOs of the VL regions of the antibodies and each CDR.

[0358] Figure 12Shows the 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.

[0359] Figure 13 Shows the 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.

[0360] Figure 14 Shows the sequence alignment of the mature full-length HC of monoclonal antibody 8B9 and its exemplary variant TIM3.8 (S61P). The VH region of each heavy chain is underlined.

[0361] Figure 15 Shows the sequence alignment of the mature full-length HC of monoclonal antibody 23B3 and its exemplary variant TIM3.25 (G6E / D79Y). The VH region of each heavy chain is underlined.

[0362] Figure 16 Lists the SEQ ID NOs of 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. Also indicated are the isotypes of the heavy and light chains. "H.n." refers to the hybridoma name. Figure 16 The heavy and light chains referred to in may be derived from their components, e.g., the variable and constant regions disclosed herein. If the SEQ ID NO does not appear in the given column on the second or third page of the table, the SEQ ID NO is provided in the column on the page before it or on the page before that page.

[0363] Figure 17A and 17B Shows the binding curves and EC Figure 17A ) and activated human T cells ( Figure 17B ) of several anti-TIM3 antibodies to human TIM3-transfected CHO cells ( 50 values. In Figure 17A are shown the following anti-TIM3 antibodies: 13A3, 17C3, 17C8, 3G4, 8B9, and 9F6. InFigure 17B showed the following anti-TIM3 antibodies: 13A3, 17C3, 17C8, 3G4, 8B9, 8C4, and 9F6. In Figure 17A and 17B In these two figures, isotype control antibodies human IgG1 (hG1), IgG2 (hG2), and IgG4 (hG4) were used as negative controls.

[0364] Figure 18 showed the binding curves and EC 50 values of anti-TIM3 antibodies 14H7 and 23B3 binding to human TIM3-positive cells compared with anti-TIM3 antibody 13A3. Isotype control antibodies IgG1, IgG1.1 (hG1.1), IgG2, and IgG4 were used as negative controls.

[0365] Figure 19A and 19B 19C showed the binding analysis of anti-TIM3 antibodies to cynomolgus monkey TIM3-transfected CHO cell lines ( Figure 19A ) and activated cynomolgus monkey T cells ( Figure 19B and 19C ). In Figure 19A the binding curves and EC50 values of anti-TIM3 antibodies 13A3, 17C3, 17C8, 3G4, 8B9, and 9F6 were provided. In Figure 19B the binding curve and EC50 value of anti-TIM3 antibody 13A3 were provided. In Figure 19C 100 ug of the relevant antibody was tested for binding to activated cynomolgus monkey CD8+ T cells. The binding data was shown as mean fluorescence index (MFI). In Figure 19A - 19C the relevant isotype control antibodies (hIgG1, hIgG2, and hIgG4) or no antibody treatment (no Ab) were used as negative controls.

[0366] Figure 20 showed flow cytometry plots of anti-TIM3 antibodies 14H7 (left panel) and 23B3 (right panel) binding to CHO cells transfected with human TIM3 ("1") or cynomolgus monkey TIM3 ("2"). CHO cells transfected with isotype control antibodies were used as negative controls ("3").

[0367] Figure 21 showed anti-TIM3 activity promoting IFN-γ production by tumor-infiltrating leukocytes (TIL) in renal cell carcinoma (RCC) (at multiple antibody concentrations shown in the box). As shown, the 8 columns for each antibody represent different concentrations of the antibody. Data for anti-TIM3 antibodies 13A3, 3G4, 17C3, 17C8, 8B9, and 9F6 were shown. Isotype control antibodies were used as controls. The horizontal dashed line provided the background threshold level.

[0368] Figure 22A and 22B showed anti-TIM3 activity promoting IFN-γ production by lung cancer TILs (at multiple antibody concentrations shown in the box)( Figure 22A , IFN-γ ELISA; Figure 22B , intracellular IFN-γ staining). In Figure 22A , each bar for each antibody (13A3 and 3G4) shown represents a different antibody concentration shown. In Figure 22B , the upper panel shows CD4 + T cells and the lower panel shows CD8 + T cells. The level of TIM3 was 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.

[0369] Figure 23 showed the efficacy of anti-TIM3 antibodies (i.e., antibodies 13A3 and 3G4) in promoting IFN-γ secretion by TILs isolated from multiple tissues of patients (i.e., kidney, pancreas, lung, and thyroid) and cultured in vitro with anti-TIM3 antibody 13A3 or 3G4. IFN-γ secretion was measured in the presence of CHO-OKT3 cells. Data are shown as fold increase relative to the level of IFN-γ produced by TILs in the absence of anti-TIM3 antibody.

[0370] Figure 24A and 24B showed cross-blocking data of anti-TIM3 antibodies on activated human T cells. In Figure 24A , the binding of anti-TIM3 antibodies 17C3 (dark gray), 8B9 (black), and 13A3 (light gray) to activated human T cells that were pre-incubated with one of the following anti-TIM3 antibodies: 3G4, 8B9, 9F6, 17C3, 17C8, and 8C4. In Figure 24B , activated human T cells were pre-incubated with 14H7 or 23B3 antibody, and then the binding of 13A3 antibody was measured. In Figure 24A and 24B , the geometric mean fluorescence intensity of the binding of anti-TIM3 antibodies to activated human T cells was provided.

[0371] Figure 25 showed the amino acid residues required for the binding of anti-TIM3 monoclonal antibodies 13A3, 3G4, 17C3, and 8B9 to human TIM3. The signal sequence and transmembrane domain are underlined.

[0372] Figure 26A and 26BCertain anti-TIM3 antibodies are shown to block the interaction between human TIM3 and PS-liposomes. Figure 26A A schematic diagram of the phosphatidylserine (PS)-hTIM3 "tandem" blocking assay is shown. Figure 26B Blocking of the binding of hTIM3-Fc to PS-liposomes by certain anti-TIM3 antibodies is shown, as measured by the Figure 26A PS-hTIM3 "tandem" blocking assay shown. Data for anti-TIM3 antibodies 3G4, 13A3, 17C3, and 17C8 are shown in the upper panel. Data for anti-TIM3 antibodies 8B9, 8C4, and 9F6 are shown in the lower panel. The PS response time and whether hTIM3-Fc binds to PS-liposomes are provided in the table to the right of each graph.

[0373] Figure 27 A summary of the functional activities of multiple anti-TIM3 antibodies (e.g., TIM3.5 (13A3), TIM3.4 (3G4), TIM3.2 (17C3), TIM3.9 (17C8), 9F6, TIM3.8 (8B9 with S61P substitution in VH), and TIM3.6 (8C4)) is shown. Data for binding assays, T cell assays, TIL assays, and PS-TIM3 blocking assays are provided.

[0374] Figure 28 A list of all SEQ ID numbers is provided, and the sequences represented by the SEQ ID numbers are described.

[0375] Figure 29A and 29B The anti-tumor activity of the combined administration of anti-PD1 and anti-TIM3 antibodies in a CT26 colorectal tumor mouse model is shown. Figure 29A The tumor volumes at various time points after tumor implantation in mice (n = 10 per 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) a combination of RMT3-23 anti-TIM3 and RMP1-14 anti-PD1 antibodies (lower right panel) are shown. Figure 29B The function of the change in mean tumor volume over time (days after tumor implantation) in mice treated with (i) RMT3-23 anti-TIM3 antibody alone, (ii) AbM anti-TIM3 antibody alone, (iii) RMP1-14 anti-PD1 antibody alone, (iv) a combination of RMT3-23 anti-TIM3 and RMP1-14 anti-PD1 antibodies, (v) a combination of AbM anti-TIM3 and RMP1-14 anti-PD1 antibodies, and (vi) isotype control antibody is shown.

[0376] Figure 30 Shows a list of hTIM3 common peptides used to map the epitopes of anti-TIM3 antibodies (13A3 and 3G4) using hydrogen / deuterium exchange mass spectrometry (HDX-MS). Each bar represents a peptic peptide. Residues in circles (i.e., N99, T145, and N172) indicate glycosylation sites.

[0377] Figure 31 Shows the human TIM3 binding regions of anti-TIM3 antibodies (13A3 and 3G4) identified using HDX-MX. The upper panel shows the binding region of the 13A3 anti-TIM3 antibody. The lower panel shows the binding region of the 3G4 anti-TIM3 antibody.

[0378] Figure 32A and 32B Shows the results of Scatchard analysis of the binding of TIM3.18.IgG1.3 to CHO cells ectopically expressing human or cynomolgus TIM3. Figure 32A Shows 125 I-TIM3 Ab standard curve. Figure 32B Shows the amount of TIM3.18.IgG1.3 antibody that binds to CHO cells expressing human (left panel) and cynomolgus (right panel) TIM3. The estimated affinity values and the number of TIM3 molecules expressed on the surface of CHO cells are provided below each panel. These values were calculated as described in Example 4.

[0379] Figure 33 Shows the results of Scatchard analysis of the binding of TIM3.18.IgG1.3 to activated Th1 cells from two separate donors (left and right panels). The estimated affinity values and the number of TIM3 molecules expressed on the surface of CHO cells are provided below each panel. These values were calculated as described in Example 4.

[0380] Figure 34A and 34B Shows that in a polarized Th1 / irradiated CHO-OKT3 co-culture assay, TIM3.18.IgG1.3 and TIM3.18.IgG1.3 Fab enhanced the proliferation of Th1 T cells. Figure 34A Shows Th1 cell proliferation observed at multiple concentrations of TIM3.18.IgG1.3, 13A3 ("13A3-g4"), no antibody, or isotype control antibodies (hIgG1.1 or hIgG4). Each bar for an antibody represents a different concentration. Figure 34BShows Th1 cell proliferation observed in the presence of multiple 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.

[0381] Figure 35 Shows enhanced Th1 T cell proliferation mediated by TIM3.24 and TIM3.25 in a polarized Th1 / irradiated CHO-OKT3 co-culture assay. TIM3.18 antibody was included for comparison purposes. CHO cells untreated with any antibody ("no Ab") or treated with isotype control IgG1.3 antibody ("IgG1.3") were used as negative controls. The horizontal dashed line represents proliferation relative to the negative control.

[0382] Figure 36 Shows that the anti-TIM3 antibody TIM3.18.IgG1.3 (black circles) enhanced Th1 T cell proliferation in combination with nivolumab in a polarized Th1 / irradiated CHO-OKT3-PD-L1 co-culture assay. CHO cells untreated with any antibody (open squares) or treated with isotype control IgG1.3 antibody (gray squares) were used as negative controls.

[0383] Figure 37 Shows that the anti-TIM3 antibody TIM3.18.IgG1.3 enhanced interferon-γ secretion of 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 any antibody were used as negative controls. Each bar for the TIM3.18 antibody represents a different concentration of the antibody.

[0384] Figure 38 Shows that the anti-TIM3 antibody TIM3.18.IgG1.3 enhanced interferon-γ secretion of breast cancer TILs stimulated with irradiated CHO-OKT3 cells. Assays using no antibody or isotype control antibody were used as controls. The horizontal dashed line represents interferon-γ secretion above the negative control.

[0385] Figure 39 Shows histograms of CD163, CD206, and TIM3 expression on M0 macrophages used in the AlloMLR (mixed lymphocyte reaction) assay, the results of which are shown in Figure 40 . Each histogram shows the fluorescence intensity of (1) CD163, CD206, or TIM3, (2) isotype control antibody (i.e., negative control), and (3) background fluorescence ("unstained") (i.e., negative control).

[0386] Figure 40 Shows cell proliferation in an AlloMLR assay performed in the presence of the anti-TIM3 antibody TIM3.18.IgG1.3, an isotype control, or in the absence of antibody.

[0387] Figure 41 Is a ribbon diagram of the structure of the TIM3:TIM3.18 Fab complex determined by crystallography. The Fab fragment is shown in light gray and TIM3 is shown in dark gray.

[0388] Figure 42 Shows the structure of the TIM3:TIM3.18 Fab complex determined by crystallography. The Fab fragment is shown as a ribbon diagram. TIM3 is represented as a white surface and Fab contact residues are shown in dark gray.

[0389] Figure 43 Is a diagram of an assay for measuring the potential internalization of an anti-TIM3 antibody. The left panel provides a schematic of the assay. The right panel describes the calculation of the internalization value.

[0390] Figure 44 Shows that the anti-TIM3 antibody 13A3 (lower left panel) and certain of its variants (D101E - upper left panel; N60Q - upper right panel) do not trigger receptor (i.e., TIM3)-mediated internalization.

[0391] Figure 45A and 45B Shows a ribbon diagram depicting the epitopes of the anti-TIM3 antibodies 13A3 ( Figure 45A ) and 3G4 ( Figure 45B ). The amino acid sequences of the epitopes of each antibody are provided below the ribbon diagram. Different patterns identify specific regions of the anti-TIM3 antibodies corresponding to specific epitopes. DETAILED DESCRIPTION OF THE INVENTION

[0393] To make the present specification easier to understand, certain terms are first defined. Further definitions are given throughout the detailed specification.

[0394] Note that the term "a" or "an" entity refers to one or more of that entity; for example, "a nucleotide sequence" is understood to represent one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more" and "at least one" may be used interchangeably herein.

[0395] In addition, as used herein, "and / or" is regarded as a specific disclosure that each of the two specified features or components has or does not have the other. Thus, the term "and / or" as used herein in a phrase such as "A and / or B" is intended to include "A and B", "A or B", "(only) A", and "(only) B". Similarly, the term "and / or" as used herein in a phrase such as "A, B, and / or C" is intended to cover each of the following aspects: 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; (only) A; (only) B; and (only) C.

[0396] It should be understood that wherever an aspect is described in terms of "comprising", "including", a similar aspect described in terms of "consisting of" and / or "consisting essentially of" is also provided.

[0397] Unless otherwise defined, 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 disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd Edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd Edition, 1999, Academic Press; and the Oxford Dictionary of Biochemistry And Molecular Biology, Revised 2000, Oxford University Press, provide a general dictionary of many of the terms used in this disclosure to those skilled in the art.

[0398] Units, prefixes, and symbols are expressed in the form accepted by the Système International de Unites (SI). Numerical ranges include the numbers defining the range. Unless otherwise indicated, nucleotide sequences are written left to right in the 5' to 3' direction. Amino acid sequences are written left to right in the amino to carboxyl direction. The headings provided herein are not limitations on the various aspects of the disclosure, and the various aspects of the disclosure can be obtained by referring to the specification as a whole. Thus, the terms defined immediately below are more fully defined by reference to the entire specification.

[0399] The term "about" is used herein to mean approximately, roughly, around or in the range of. When the term "about" is used in connection with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. Generally, the term "about" can adjust a numerical value above or below the stated value by a variance of, for example, 10% higher or lower (higher or lower).

[0400] As used herein, the term "T cell immunoglobulin and mucin domain-containing protein 3" or "TIM3" refers to a receptor that is a member of the T cell immunoglobulin and mucin domain (TIM) protein family. The major ligand of TIM3 includes phosphatidylserine (TIM3-L). TIM3 is also known 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 that is naturally expressed by a cell. Thus, the antibodies described herein can cross-react with TIM3 from species other than human (e.g., cynomolgus monkey TIM3). Alternatively, the antibodies can be specific for human TIM3 and do not exhibit any cross-reactivity with other species. TIM3 or any of its variants and isoforms can be isolated from cells or tissues that naturally express them or can be recombinantly produced using techniques well known in the art and / or techniques described herein.

[0401] 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 canonical 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 domain, cytoplasmic domain, and part of the extracellular domain of TIM3. The amino acid residues 132-142 also differ from the above-described canonical sequence.

[0402] The following are the amino acid sequences of two known human TIM3 isoforms.

[0403] (A) Human TIM3 isoform 1 (Accession No. NP_116171; SEQ ID NO:286; encoded by a nucleotide sequence having Accession No. NM_032782.4; SEQ ID NO:288; Figure 25 ):

[0404]

[0405] (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):

[0406]

[0407] The signal sequences of isoforms 1 and 2 correspond to amino acids 1 - 21 (underlined). Thus, the 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 - 202 of SEQ ID NO: 286 and has the following amino acid sequence:

[0408]

[0409] The cynomolgus monkey TIM3 protein consists of the following amino acid sequence (including the signal sequence):

[0410]

[0411] In some embodiments, the term "antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains that are inter-connected 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 certain antibodies, such as naturally-occurring IgG antibodies, the heavy chain constant region comprises a hinge and three domains CH1, CH2, and CH3. In some antibodies, such as naturally-occurring IgG antibodies, each light chain comprises 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 hypervariable regions, called complementarity-determining regions (CDRs), which are interspersed with more conserved regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, which are arranged in the following order from the amino-terminus to the carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with an antigen. The constant region of the antibody can 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 of the classical complement system (C1q). The heavy chain may or may not have a C-terminal lysine. Unless otherwise specified herein, the amino acids in the variable regions are numbered using the Kabat numbering system, while the amino acids in the constant regions are numbered using the EU system.

[0412] In some embodiments, "IgG antibody" used herein, such as human IgG1, IgG2, IgG3 and IgG4 antibodies have the structure of naturally occurring IgG antibodies, i.e., they have the same number of heavy chains and light chains and disulfide bonds as natural IgG antibodies of the same subclass. For example, anti-TIM3 IgG1, IgG2, IgG3 or IgG4 antibodies are composed of two heavy chains (HC) and two light chains (LC), wherein the two HCs and LCs are connected by disulfide bonds of the same number and position that exist in naturally occurring IgG1, IgG2, IgG3 and IgG4 antibodies, respectively (unless the antibody has been subjected to a mutation that modifies the disulfide bonds).

[0413] Antibodies usually bind their corresponding antigens specifically with high affinity, as indicated by -5 Up to 10 -11 M or less dissociation constant (K D ) reflects. It is generally believed that any value greater than about 10 -4 M of K D As used herein, an antibody that "specifically binds" to an antigen refers to an antibody that binds to the antigen with high affinity and is substantially identical to the antigen, meaning that it has 10 -7 M or smaller, 10 -8 M or smaller, 5x 10 -9 M or less or within 10 -8 M and 10 -10 M or smaller K D , but does not bind to unrelated antigens with high affinity. An antigen is "substantially identical" to a given antigen if it exhibits a high degree of sequence identity to the given antigen, for example, if it exhibits at least 80%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity to the sequence of the given antigen. For example, in some embodiments, an antibody that specifically binds to human TIM3 may also have cross-reactivity with TIM3 antigens from certain primate species (e.g., cynomolgus monkey TIM3), but may not cross-react with TIM3 antigens from other species or with antigens that are not TIM3.

[0414] The immunoglobulin can be 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 IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. In some embodiments, the anti-TIM3 antibodies described herein belong to the IgG1 subtype. Immunoglobulins (such as IgG1) exist in several allotypes that differ from each other at most by a few amino acids. By way of example, "antibody" includes naturally occurring antibodies and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human antibodies and non-human antibodies, and fully synthetic antibodies.

[0415] 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 an antigen (such as 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 by the term "antigen-binding portion" of an antibody such as the anti-TIM3 antibodies described herein include (i) Fab fragments (papain-cleaved fragments) or similar monovalent fragments consisting of V L , V H , LC, and CH1 domains; (ii) F(ab')2 fragments (from pepsin-cleaved fragments) or similar divalent fragments comprising two Fab fragments linked by a disulfide bond in the hinge region; (iii) Fd fragments consisting of V H and CH1 domains; (iv) Fv fragments consisting of the V L and V H domains of a single arm of an antibody; (v) dAb fragments consisting of V H domains (Ward et al., (1989) Nature 341:544-546); (vi) isolated complementarity-determining regions (CDRs); and (vii) combinations of two or more isolated CDRs, which may optionally be joined by a synthetic linker. In addition, although the two domains V L and V H of an Fv fragment are encoded by different genes, they can be joined together by recombinant methods using a synthetic linker so that they form a single protein chain, wherein the V L region and the V HRegions pair to form monovalent molecules (referred to as single-chain Fv (scFv); 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 included in the "antigen-binding portion" of the term antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the utility of the fragments is screened in the same manner as intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0416] A "bispecific" or "bifunctional antibody" is an artificial hybrid antibody having two different heavy chain / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or ligation of Fab' fragments. See, e.g., Songsivilai &

[0417] Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

[0418] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies, i.e., each antibody contained in the population is substantially similar and binds 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 generally being present in minor amounts. The modifier "monoclonal" indicates the character of the antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. The term "human monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies that exhibits a single binding specificity and has variable and optionally constant regions derived from human germline immunoglobulin sequences. In some embodiments, human monoclonal antibodies are produced by hybridomas, including fusing B cells obtained from transgenic non-human animals (e.g., transgenic mice) to immortalized cells, wherein the transgenic non-human animals have a genome comprising a human heavy chain transgene and a light chain transgene.

[0419] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, produced or isolated by recombinant means, e.g., (a) antibodies isolated from transgenic or transchromosomal animals (e.g., mice) that have human immunoglobulin genes or from hybridomas prepared therefrom, (b) antibodies isolated from host cells transformed to express antibodies, e.g., from transfectomas, (c) antibodies isolated from recombinant combinatorial human antibody libraries, and (d) antibodies prepared, expressed, produced or isolated by any other means that involve splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies contain variable and constant regions utilizing specific human germline immunoglobulin sequences that are encoded by germline genes, but include subsequent rearrangements and mutations, such as those that occur during antibody maturation. As is known in the art (see, e.g., Lonberg (2005) Nature Biotech. 23(9):1117-1125), the variable region contains the antigen-binding domain, which is encoded by multiple genes that rearrange to form an antibody specific for a foreign antigen. In addition to rearrangement, the variable region can be further modified by multiple single amino acid changes (termed somatic mutations or hypermutations) to increase the affinity of the antibody for the foreign antigen. In a further response to antigen, the constant region will change (i.e., isotype switching). Thus, the nucleic acid molecules encoding the light and heavy chain immunoglobulin polypeptides that are rearranged and somatically mutated in response to antigen cannot have sequence identity with the original nucleic acid molecules, but are substantially identical or similar (i.e., at least 80% identity).

[0420] A "human" antibody (HuMAb) is an antibody that has a variable region in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In addition, 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 in vitro random or site-specific mutagenesis or by somatic mutations in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are grafted onto human framework region sequences. The terms "human" antibody and "fully human" antibody are used synonymously.

[0421] A "humanized" antibody refers to an antibody in which some, most, or all of the amino acids outside the CDR domains of the non-human antibody have been replaced with the corresponding amino acids derived from a human immunoglobulin. In some embodiments of the humanized form of the antibody, some, most, or all of the amino acids outside the CDR domains have been replaced with amino acids from a human immunoglobulin, while some, most, or all of the amino acids within one or more CDR regions have not been altered. Minor additions, deletions, insertions, substitutions, or modifications of the amino acids are permitted, provided that they do not eliminate the ability of the antibody to bind to a specific antigen. A "humanized" antibody retains antigen specificity similar to the original antibody.

[0422] 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 an antibody in which the variable region is derived from a murine antibody and the constant region is derived from a human antibody.

[0423] As used herein, "isotype" refers to the antibody class encoded by the heavy chain constant region gene (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies).

[0424] "Allotype" refers to naturally occurring variants within a particular isotype group that differ in some 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 the "IgG1f," "IgG1.1f," or "IgG1.3f" isotypes are the IgG1, IgG1.1 without effector function, and IgG1.3 without effector function allotypes of allotype "f," i.e., having, for example, 214R, 356E, and 358M according to the EU index as described in Kabat as shown in SEQ ID NO: 3.

[0425] The phrases "antibody that recognizes an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen."

[0426] As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other proteins and cellular material.

[0427] As used herein, an antibody that "inhibits TIM3-L binding to TIM3" is used to refer to an antibody that inhibits the binding of TIM3 to its ligand, such as phosphatidylserine, e.g., in a binding assay using human TIM3-transfected CHO cells or activated T cells expressing TIM3, having an EC 50About 1 μg / mL or less, such as about 0.9 μg / mL or less, about 0.85 μg / mL or less, 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, in methods known in the art, such as the FACS-based binding assays described herein.

[0428] "Effector function" refers to the interaction of the Fc region of an antibody with an Fc receptor or ligand or the resulting biochemical event. Exemplary "effector functions" include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, FcγR-mediated effector functions such as ADCC and antibody-dependent cell-mediated phagocytosis (ADCP), and downregulation of cell surface receptors (e.g., B cell receptor; BCR). Such effector functions generally require the Fc region to be associated with a binding domain (e.g., the antibody variable domain).

[0429] "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 and alternatively spliced forms of these receptors. The FcγR family consists of three activating receptors (FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory receptor (FcγRIIB). Multiple 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, while natural killer (NK) cells selectively express one activating Fc receptor (FcγRIII in mice, FcγRIIIA in humans), but do not express the inhibitory FcγRIIB in mice and humans. Human IgG1 binds to most human Fc receptors and is considered equivalent to murine IgG2a in terms of the type of activating Fc receptor to which it binds.

[0430] "Fc region" (fragment crystallizable region), "Fc domain", or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates the binding of immunoglobulins 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 of the classical complement system (C1q). Thus, the Fc region contains the constant regions of the antibody except for 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 two heavy chains of the antibody; IgM and IgE Fc regions contain three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. For IgG, the Fc region contains the immunoglobulin domains CH2 and CH3 and the hinge between the CH1 and CH2 domains. Although the definition of the boundaries of the immunoglobulin heavy chain Fc region may vary, as defined herein, the human IgG heavy chain Fc region is defined as extending from amino acid residue D221 of IgG1, V222 of IgG2, L221 of IgG3, and P224 of IgG4 to the carboxyl terminus of the heavy chain, where the numbering is according to the EU index in Kabat. The CH2 domain of the human IgG Fc region extends from amino acid 237 to amino acid 340, and the CH3 domain is located on the C-terminal side of the CH2 domain in the Fc region, i.e., it extends from amino acid 341 to amino acid 447 or 446 (if the C-terminal lysine residue is absent) or 445 (if both the C-terminal glycine and lysine residues are absent) of IgG. As used herein, the Fc region can be a native sequence Fc, including any allotypic variant or variant Fc (e.g., non-naturally occurring Fc). Fc can also refer to the isolated region or the region in the context of a protein polypeptide containing Fc, e.g., a "binding protein containing an Fc region" is also referred to as an "Fc fusion protein" (e.g., an antibody or an immunoadhesin).

[0431] "Native sequence Fc region" or "native sequence Fc" contains an amino acid sequence identical to the amino acid sequence of the Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc region; native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region and their naturally occurring variants. Native sequence Fc includes the various allotypes of Fc (see, e.g., Jefferis et al. (2009) mAbs 1:1).

[0432] The term "epitope" or "antigenic determinant" refers to the site on an antigen (e.g., TIM3) to which an immunoglobulin or antibody specifically binds, e.g., as defined by a particular method used to identify it. Epitopes can be formed either by contiguous amino acids (usually linear epitopes) or by non-contiguous amino acids juxtaposed by the tertiary folding of a protein (usually conformational epitopes). Epitopes formed by contiguous amino acids usually but not always remain when exposed to denaturing solvents, while epitopes formed by tertiary folding are usually lost upon treatment with denaturing solvents. Epitopes typically contain 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 epitopes a given antibody binds to (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, in which the reactivity of overlapping or contiguous peptides from a protein (e.g., from TIM3) is tested using a given antibody (e.g., an anti-TIM3 antibody). Methods for determining the spatial conformation of an epitope include techniques known in the art and described herein, such as X-ray crystallography, antigen mutagenesis analysis, two-dimensional nuclear magnetic resonance, and HDX-MS (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, edited by G.E. Morris (1996)).

[0433] The term "epitope mapping" refers to the process of identifying the molecular determinants for antibody-antigen recognition.

[0434] When referring to two or more antibodies, the term "binding the same epitope" means that the antibodies bind the same segment of amino acid residues, as determined by a given method. Techniques for determining whether an antibody binds the "same epitope of TIM3" using the antibodies described herein include, for example, epitope mapping methods such as X-ray analysis of crystals of the antigen:antibody complex, which provides atomic resolution of the epitope; and hydrogen / deuterium exchange mass spectrometry (HDX-MS). Other methods monitor the binding of an antibody to antigen fragments or mutant variants of the antigen, where loss of binding due to modification of an amino acid residue within the antigen sequence is generally considered an indication of an epitope component. Additionally, computer-based combinatorial methods for epitope mapping can also be used. These methods rely on the ability of the target antibody to affinity isolate specific short peptides from a combinatorial phage display peptide library. Antibodies with the same VH and VL or the same CDR1, 2, and 3 sequences are expected to bind the same epitope.

[0435] An "antibody that competes with another antibody for binding to a target" refers to an antibody that (partially or completely) inhibits the binding of another antibody to the target. Known competition assays can be used, such as Surface plasmon resonance (SPR) analysis determines whether two antibodies compete with each other for binding to a target, i.e., whether one antibody inhibits and to what extent the binding of another antibody to the target. In some embodiments, one antibody competes with and inhibits the binding of another antibody to the 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 incubated with the target first). The competition assay can be performed as described in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi:10.1101 / pdb.prot4277 or Chapter 11 of Ed Harlow and David Lane, "Using Antibodies", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999. If two antibodies block each other, the antibodies "cross-compete", blocking at least 50% in both ways, i.e., in the competition experiment, regardless of whether one antibody or the other antibody is contacted with the antigen first.

[0436] Competitive binding assays for determining whether two antibodies compete or cross-compete for binding include, for example, competing for binding to TIM3-expressing T cells by flow cytometry as described in the examples. Other methods include: SPR (e.g., ), 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 labeling assay, solid-phase direct labeled sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid-phase direct labeling RIA using 1-125 (see Morel et al., Mol. Immunol.

[0437] 25(1):7 (1988)); solid-phase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and direct-labeled RIA. (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

[0438] As used herein, the terms "specifically bind", "selectively bind", "selectively binds", and "specific binding" refer to an antibody binding to an epitope on a predetermined antigen. Generally, an antibody (i) binds with a binding affinity of about less than 10 -7 M, such as about less than 10 -8 M, 10 -9 M or 10 -10 M or even lower equilibrium dissociation constant (K D ), e.g., when using a predetermined antigen (e.g., recombinant human TIM3) as an analyte and the antibody as a ligand, determined by, for example, surface plasmon resonance (SPR) technology in a 2000 instrument, or by Scatchard analysis of the binding of the antibody to antigen-positive cells, and (ii) binds to the predetermined antigen with an affinity that is at least two-fold greater than the binding affinity for non-specific antigens (e.g., BSA, casein) other than the predetermined antigen or antigens closely related thereto. Thus, an "antibody that specifically binds human TIM3" refers to an antibody that binds soluble or cell-bound human TIM3 with a K -7 of 10 -8 M or less, such as about less than 10 -9 M, 10 -10 M or 10 D M or even lower. An "antibody that cross-reacts with cynomolgus monkey TIM3" refers to an antibody that binds cynomolgus monkey TIM3 with a K -7 of 10 -8 M or less, such as about less than 10 -9 M, 10 -10 M or 10 D M or even lower. In some embodiments, such antibodies that do not cross-react with TIM3 from non-human species show substantially undetectable binding to these proteins in standard binding assays.

[0439] As used herein, the term "k assoc " or "k a " refers to the association rate of a particular antibody-antigen interaction, and as used herein, the term "k dis " or "k d " refers to the dissociation rate of a particular antibody-antigen interaction. As used herein, the term "K D " refers to the dissociation constant, which is obtained from the ratio of k d to k a (i.e., k d / k a ) and is expressed as a molar concentration (M). The K D value of an antibody can be determined using methods well known in the art. Determining the antibody K DAvailable methods for values include surface plasmon resonance, biosensor systems (such as systems) or flow cytometry and Scatchard analysis.

[0440] As used herein, the term "high affinity" for an IgG antibody refers to an antibody having a K D 10 -8 M or less, 10 - 9 M or less or 10 -10 M or less for the target antigen. However, "high affinity" binding may vary for other antibody isotypes. For example, "high affinity" binding for the IgM isotype is an antibody having a K D 10 -10 M or less or 10 -8 M or less.

[0441] In the context of in vitro or in vivo assays using an antibody or an antigen-binding fragment thereof, the term "EC 50 " refers to the concentration of the antibody or its antigen-binding portion that induces a response that is 50% of the maximum response, i.e., the 50% maximum response is halfway between the maximum response and the baseline.

[0442] As used herein, the term "naturally occurring" applied to an object refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) is naturally occurring, and the polypeptide or polynucleotide sequence can be isolated from nature and has not been deliberately modified by man.

[0443] "Polypeptide" refers to a chain comprising at least two contiguous amino acid residues, and there is no upper limit on the length of the chain. 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 comprise one or more polypeptides.

[0444] As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. The nucleic acid molecule can be single-stranded or double-stranded, and can be cDNA.

[0445] "Conservative amino acid substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chain amino acids (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chain amino acids (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chain amino acids (e.g., threonine, valine, isoleucine), and aromatic side chain amino acids (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 nucleotide and amino acid conservative 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)).

[0446] For nucleic acids, the term "substantial homology" means that when two nucleic acids or their designated sequences are optimally aligned and compared, at least about 80%, at least about 90% to 95%, or at least about 98% to 99.5% of the nucleotides are identical in the two nucleic acids or their designated sequences with appropriate nucleotide insertions or deletions. Alternatively, substantial homology exists when the segment hybridizes to the complementary sequence of the strand under selected hybridization conditions.

[0447] For polypeptides, the term "substantial homology" means that when two polypeptides or their designated sequences are optimally aligned and compared, at least about 80% of the amino acids, at least about 90% to 95%, or at least about 98% to 99.5% of the amino acids are identical in the two polypeptides or their designated sequences with appropriate amino acid insertions or deletions.

[0448] 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), where the number of gaps and the length of each gap are considered, and these gaps are introduced for the optimal alignment of the two sequences. Sequence comparison between two sequences and determination of the percent identity can be accomplished using a mathematical algorithm, as described in the following non-limiting examples.

[0449] The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (obtainable from worldwideweb.gcg.com), using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide sequences 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 the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the 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 in the GCG software package (obtainable from http: / / www.gcg.com), using the Blossum 62 matrix or the PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0450] The nucleic acid and protein sequences described herein can further be used as a "query sequence" to search public databases to, for example, 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. The BLAST nucleotide search can be performed using the NBLAST program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. The BLAST protein search can be performed using the XBLAST program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When using the BLAST and gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. See worldwideweb.ncbi.nlm.nih.gov.

[0451] Nucleic acids can be present in whole cells, cell lysates, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "present as substantially pure" when it is purified from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other parts of the chromosome) or proteins, by standard techniques including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other methods well known in the art. See F. Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

[0452] Nucleic acids such as cDNA can be mutated according to 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 native V, D, J, constant regions, switches, and other such sequences described herein are contemplated (where "derived" means that one sequence is identical to or modified from another sequence).

[0453] 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 into the viral genome. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). After introduction into a host cell, other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell and thus replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes operably linked thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Usually, expression vectors useful in recombinant DNA techniques are commonly in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used form of vector. However, other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which have equivalent functions, are also included.

[0454] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell that contains a nucleic acid that is not naturally present in the cell and can be a cell into which a recombinant expression vector has been introduced. It should be understood that these terms are intended to refer not only to the particular subject cell but also to the progeny of that cell. Because certain modifications may occur in the progeny due to mutations or environmental influences, such progeny may not in fact be identical to the parental cell, but are still included within the scope of the term "host cell" as used herein.

[0455] An "immune response" is as understood in the art and generally refers to a biological response in a vertebrate against foreign substances or abnormalities such as cancer cells, which response protects the organism from these substances and the diseases caused by them. The immune response is mediated by the action of one or more cells of the immune system (such as T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells or neutrophils) and soluble macromolecules, where the soluble macromolecules are produced by any of these cells or by the liver (including antibodies, cytokines and complement), which results in the selective targeting, binding, destruction, elimination and / or clearance from the vertebrate body of invading pathogens, pathogen-infected cells or tissues, cancer cells or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, against normal human cells or tissues. The immune response includes, for example, activating or inhibiting T cells, such as effector T cells, Th cells, CD4 + cells, CD8 + T cells or Treg cells, or activating or inhibiting any other cells of the immune system, such as NK cells.

[0456] "Immunomodulator" or "immunoregulator" refers to a substance, such as a substance that targets a component of a signaling pathway, which can participate in regulating, adjusting, or modifying an immune response. "Regulating," "adjusting," or "modifying" an immune response refers to any alteration in the activity of cells of the immune system or of such cells (e.g., effector T cells, such as Th1 cells). Such regulation includes stimulation or inhibition of the immune system, which can be manifested by an increase or decrease in the number of various cell types, an increase or decrease in the activity of these cells, or any other change that can occur in the immune system. Inhibitory and stimulatory immunomodulators have been identified, some of which can have enhanced functions in the tumor microenvironment. In some embodiments, the immunomodulator targets a molecule on the surface of a T cell. An "immunomodulatory target" or "immunoregulatory target" is a molecule, such as a cell surface molecule, that is targeted by the binding of a substance, agent, moiety, compound, or molecule and whose activity is altered by such binding. Immunomodulatory targets include, for example, receptors ("immunomodulatory receptors") and receptor ligands ("immunomodulatory ligands") on the cell surface.

[0457] "Immunotherapy" refers to treating a subject having a disease, at risk of having a disease, or disease recurrence by methods including inducing, enhancing, inhibiting, or otherwise altering the immune system or immune response.

[0458] "Immunostimulatory therapy" or "immunostimulating therapy" refers to a therapy that results in an increase (induction or enhancement) of a subject's immune response to, for example, treat cancer.

[0459] "Enhancing the endogenous immune response" refers to increasing the potency or efficacy of the immune response present in a subject. For example, an increase in effectiveness and potency can be achieved by overcoming mechanisms that suppress the endogenous host immune response or by stimulating mechanisms that enhance the endogenous host immune response.

[0460] "T effector" ("T eff ") cells refer to T cells having cytolytic activity (e.g., CD4 + and CD8 + T cells) and T helper (Th) cells, such as Th1 cells, which secrete cytokines and activate and direct other immune cells, but do not include regulatory T cells (Treg cells). Certain anti-TIM3 antibodies described herein activate T eff cells, such as CD4 + and CD8 + T eff cells and Th1 cells.

[0461] Enhanced agonist activity of T cell co-stimulatory receptors and / or enhanced antagonist activity of inhibitory receptors can lead to an enhanced ability to stimulate an immune response or the immune system. The enhanced ability to stimulate an immune response or the immune system can be reflected by an increase fold or maximum activity level and proliferation in an assay measuring an immune response (e.g., an assay measuring changes in cytokine or chemokine release, cytotoxic activity (determined directly against target cells or indirectly by detecting CD107a or granzyme)). 50 The ability to stimulate an immune response or immune system activity can be increased by at least 10%, 30%, 50%, 75%, 2-fold, 3-fold, 5-fold or more.

[0462] As used herein, the term "linked" refers to the association of two or more molecules. The linkage can be covalent or non-covalent. The linkage can also be a genetic linkage (i.e., recombinant fusion). Such linkages can be achieved using a variety of techniques well known in the art, such as chemical conjugation and recombinant protein production.

[0463] As used herein, "administering" refers to physically introducing a composition comprising a therapeutic agent into a subject using a variety of methods and delivery systems known to those of skill in the art. Different routes of administration of the anti-TIM3 antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, intraspinal or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase "parenteral administration" refers to a mode 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, intratracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. Alternatively, the antibodies described herein can be administered by non-parenteral routes, such as topical, epidermal or mucosal routes of administration, such as intranasal, oral, vaginal, rectal, sublingual or topical administration. Administration can also be carried out, for example, once, multiple times and / or over one or more extended periods.

[0464] As used herein, the term "T cell-mediated response" refers to a response mediated by T cells, which include effector T cells (e.g., CD8 + cells) and helper T cells (e.g., CD4 + cells). T cell-mediated responses include, for example, the cytotoxicity and proliferation of T cells.

[0465] As used herein, the term "cytotoxic T lymphocyte (CTL) response" refers to an immune response induced by cytotoxic T cells. The CTL response is mainly mediated by CD8 + T cells.

[0466] As used herein, the terms "inhibit" or "block" (e.g., referring to inhibiting / blocking the binding of TIM3-L to TIM3 on cells) are used interchangeably and include partial and complete inhibition / blocking. In some embodiments, an anti-TIM3 antibody inhibits the binding of TIM3-L to TIM3 by at least about 50%, such as about 60%, 70%, 80%, 90%, 95%, 99% or 100%, e.g., as determined further herein. In some embodiments, an anti-TIM3 antibody inhibits the binding of TIM3-L to TIM3 by no more than 50%, such as inhibiting by about 40%, 30%, 20%, 10%, 5% or 1%, e.g., as determined further herein.

[0467] As used herein, the phrase "inhibit the growth of a tumor" includes any measurable decrease in tumor growth, e.g., inhibition of tumor growth by at least about 10%, such as 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%.

[0468] 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 invade adjacent tissues and may metastasize to distant parts of the body through the lymphatic system or the blood.

[0469] As used herein, the term "treatment" refers to any type of intervention performed on a subject or the process of administering or applying an active agent to or on a subject to reverse, alleviate, improve, inhibit or slow down or prevent the progression, development, severity or recurrence of symptoms, complications, conditions or biochemical markers associated with a disease, or to increase overall survival. Treatment can be directed to a subject having a disease or a subject without a disease (e.g., for prevention).

[0470] "Hematological malignancies" include lymphomas, leukemias, myelomas or lymphoid malignancies, as well as cancers of the spleen and lymph nodes. Exemplary lymphomas include B-cell lymphomas (B-cell hematological cancers) and T-cell lymphomas. B-cell lymphomas include Hodgkin lymphoma and most non-Hodgkin 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 (which overlaps with chronic lymphocytic leukemia), mantle cell lymphoma (MCL), Burkitt lymphoma, mediastinal large B-cell lymphoma, Waldenstrom macroglobulinemia, nodal marginal zone B-cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, 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 malignancies also include leukemias such as, but not limited to, secondary leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia and acute lymphoblastic leukemia. Hematological malignancies also include myelomas such as, but not limited to, multiple myeloma and smoldering multiple myeloma. The term hematological malignancies includes other hematological and / or B-cell or T-cell related cancers.

[0471] The term "effective amount" or "effective dose" is defined as an amount sufficient to achieve or at least partially achieve the desired effect. A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is any amount of the drug that, when administered alone or in combination with another therapeutic agent, promotes regression of the disease, where the regression of the disease is evidenced by a decrease in the severity of the disease symptoms, an increase in the frequency and duration of asymptomatic periods, or prevention of damage or disability due to the affliction of the disease. The therapeutically effective amount or dose of a drug includes a "prophylactically effective amount" or "prophylactically effective dose", which is any amount of the drug when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or at risk of disease recurrence, and which amount inhibits the occurrence or recurrence of the disease. The ability of a therapeutic agent to promote regression of the disease or inhibit the occurrence or recurrence of the disease can be evaluated using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems to predict the efficacy of the therapeutic agent in humans, or by assaying the activity of the agent in in vitro assays.

[0472] For example, an anti-cancer agent is a drug that promotes cancer regression in a subject. In some embodiments, a therapeutically effective amount of the drug promotes cancer regression to the extent of eliminating the cancer. "Promoting cancer regression" means that administering an effective amount of the drug, alone or in combination with an anti-tumor drug, results in a reduction in tumor growth or size, tumor necrosis, a decrease in the severity of at least one disease symptom, an increase in the frequency and duration of asymptomatic periods, prevention of damage or disability due to the disease affliction, or otherwise alleviation of the patient's disease symptoms. Additionally, the terms "effective" and "efficacy" with respect to treatment include pharmaceutical efficacy and physiological safety. Pharmaceutical efficacy refers to the ability of the drug to promote cancer regression in the patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (adverse effects) at the cellular, organ, and / or organism level caused by the administration of the drug.

[0473] As an example of treating a tumor, a therapeutically effective amount or dose of the drug inhibits cell growth 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 dose of the drug completely inhibits cell growth or tumor growth, i.e., inhibits cell growth or tumor growth by 100%. The ability of a compound to inhibit tumor growth can be evaluated using the assays described below. Alternatively, this property of the composition can be evaluated by examining the ability of the compound to inhibit cell growth, and this inhibition can be measured in vitro using assays known to those of skill in the art. In some embodiments described herein, tumor regression can be observed and can persist for at least about 20 days, at least about 40 days, or at least about 60 days.

[0474] The term "patient" includes humans and other mammalian subjects who receive prophylactic or therapeutic treatment.

[0475] 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 having cancer. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0476] The term "weight-based" amount or dose as used herein refers to calculating the dose administered to a patient based on the patient's weight. For example, when a patient weighing 60 kg requires an anti-TIM3 antibody at 3 mg / kg, an appropriate amount of anti-TIM3 antibody (i.e., 180 mg) can be calculated and administered.

[0477] For the methods of the present disclosure, the term "fixed dose" 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) in a single composition are present in the composition in a specific (fixed) ratio to each other. In some embodiments, the fixed dose is based on the weight of the antibody (e.g., mg). In some embodiments, the fixed dose is based on the concentration of the antibody (e.g., mg / ml). In some embodiments, the ratio of the 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 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 the first antibody (e.g., anti-TIM3 antibody) to mg of the second antibody. For example, a 2:1 ratio of anti-TIM3 antibody and a PD-1 antibody (e.g., nivolumab) can mean 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.

[0478] Regarding the methods and doses described herein, the term "flat dose" refers to a dose administered to a patient without regard to the patient's body weight or body surface area (BSA). Thus, a flat dose is not provided in a mg / kg dose, but rather in an absolute amount of the drug (e.g., anti-TIM3 antibody). For example, a 60 kg person and a 100 kg person will receive the same dose of the antibody (e.g., 480 mg of anti-TIM3 antibody).

[0479] As used herein, the terms "ug" and "uM" are used interchangeably with "μg" and "μΜ", respectively.

[0480] The various aspects described herein will be described in further detail in the following subsections.

[0481] I. Anti-human TIM3 antibody

[0482] Described herein are antibodies, such as fully human antibodies, that are characterized by specific functional features or properties. For example, the antibodies specifically bind to human TIM3, and more specifically, bind to a specific domain (e.g., a functional domain) within the extracellular domain of human TIM3. In some embodiments, the antibodies specifically bind to the site on TIM3 bound by TIM3-L. In some embodiments, the antibodies are antagonistic antibodies, i.e., they inhibit or suppress the T cell inhibitory activity of TIM3 on cells such as T cells. In some embodiments, the anti-TIM3 antibodies cross-react with TIM3 from one or more non-human primates such as cynomolgus TIM3. In some embodiments, the antibodies specifically bind to the extracellular region of human TIM3 and the extracellular region of cynomolgus TIM3. In some embodiments, the antibodies bind to human TIM3 with high affinity.

[0483] The anti-TIM3 antibodies described herein exhibit one or more of the following functional properties:

[0484] (a) Bind to soluble and / or membrane-bound human TIM3;

[0485] (b) Bind to soluble and / or membrane-bound cyno TIM3;

[0486] (c) Induce or stimulate an immune response;

[0487] (d) Induce or stimulate T cell activation, e.g., Th1 cell activation (as evidenced by enhanced cytokine secretion and / or proliferation);

[0488] (e) Induce or stimulate T cell proliferation (e.g., CD4 + , CD8 + T cells, Th1 cells or TILs), e.g., in co-culture assays such as those described in the Examples;

[0489] (f) Induce or stimulate IFN-γ production by T cells, e.g., Th1 cells or tumor infiltrating lymphocytes (TILs), such as TILs from human renal cell carcinoma, lung cancer, pancreatic cancer or breast cancer tumors, e.g., as measured in the assays described in the Examples;

[0490] (g) Block or inhibit the binding of human TIM3 to PtdSer, e.g., as measured in the assays described in the Examples;

[0491] (h) Do not internalize or down-regulate cell surface TIM3 when bound to TIM3 on cells;

[0492] (i)Bound to the extracellular domain of human TIM3: (i) CPVFECG (SEQ ID NO:296); (ii) RIQIPGIMND (SEQ ID NO:298); (iii) CPVFECG and RIQIPGIMND (SEQ ID NO:296 and 298, respectively); or (iv) WTSRYWLNGDFR (SEQ ID NO:297);

[0493] (j)Competitive or cross-blocking of the binding of an anti-TIM3 antibody as described herein (e.g., 13A3, 8B9, 8C4, 3G4, 17C3, 17C8, 9F6, 14H7, 23B3 or any of TIM3.2 to TIM3.18, TIM3.24 and TIM3.25) to human TIM3, as measured, for example, in the assays described in the Examples;

[0494] (k)Bound to human TIM3, but not bound to human TIM3 with amino acid substitutions at one or more 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, numbered as in SEQ ID NO:286 ( Figure 25 ); and

[0495] (l)Bound to the human TIM3 region 49 VPVCWGKGACPVFE 62 (SEQ ID NO:367) and 111 RIQIPGIMNDEKFNLKL 127 (SEQ ID NO:368), as determined by HDX-MS;

[0496] (m)Having a heavy chain variable region and / or a 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; and / or

[0497] (n)Competitive or cross-blocking of the binding of 13A3 or TIM3.18.IgG1.3 to human TIM3, as described, for example, in the Examples.

[0498] In some embodiments, the anti-TIM3 antibodies described herein bind to human TIM3 with high affinity, e.g., with a K D of 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 to 10 -7 M, 10 -11 M to 10 -7 M, 10 -10 M to 10 -7 M or 10 -9 M to 10 -7 M. In some embodiments, the anti-TIM3 antibodies bind soluble human TIM3, e.g., as measured by BIACORE TM (e.g., as described in the Examples), with a K D of 10 -7 M or less, 10 -8 M or less, 10 -9 M (1 nM) or less, 10 -10 M or less, 10 -12 M to 10 -7 M, 10 -11 M to 10 -7 M, 10 -10 M to 10 -7 M, 10 -9 M to 10 -7 M or 10 -8 M to 10 -7 M. In some embodiments, the anti-TIM3 antibodies bind to bound (e.g., cell membrane-bound) human TIM3, such as on activated human T cells, e.g., as measured by flow cytometry and Scatchard plot, with a K D of 10 -7 M or less, 10 -8 M or less, 10 -9 M (1 nM) or less, 5x10 -10 M or less, 10 -10 M or less, 10 -12 M to 10 -7 M, 10 -11 M to 10 -8 M, 10 -10 M to 10 -8 M, 10 -9 M to 10 -8 M, 10-11 M to 10 -9 M or 10 -10 M to 10 -9 M binds. In some embodiments, the anti-TIM3 antibody binds to membrane-bound (e.g., cell membrane-bound) human TIM3, such as on activated human T cells, e.g., as measured by flow cytometry, with an EC 50 10 ug / mL or less, 5 ug / mL or less, 1 ug / mL or less, 0.9 ug / mL or less, 0.8 ug / mL or less, 0.7 ug / mL or less, 0.6 ug / mL or less, 0.5 ug / mL or less, 0.4 ug / mL or less, 0.3 ug / mL or less, 0.2 ug / mL or less, 0.1 ug / mL or less, 0.05 ug / mL or less or 0.01 ug / mL or less. In some embodiments, the anti-TIM3 antibodies described herein bind, for example, with a K D 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 to 10 -7 M, 10 -11 M to 10 -7 M, 10 -10 M to 10 -7 M or 10 -9 M to 10 -7 M binds to cyno TIM3. In some embodiments, the anti-TIM3 antibody binds to soluble cyno TIM3, e.g., as measured by BIACORE TM (e.g., as described in the Examples) with a K D 10 -7 M or less, 10 -8 M or less, 10 -9 M (1 nM) or less, 10 -10 M or less, 10 -12 M to 10 -7 M, 10 -11 M to 10 -7 M, 10 -10 M to 10 -7 M, 10 -9 M to 10 -7 M or 10 -8 M to 10 -7 M binds. The anti-TIM3 antibody can bind to membrane-bound cynomolgus monkey TIM3, e.g., with an EC50 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 binding, e.g., as determined by flow cytometry (e.g., as described in the Examples). In some embodiments, the anti-TIM3 antibody binds bound (e.g., cell membrane-bound) cyno TIM3, such as on activated human T cells, e.g., as determined by flow cytometry and Scatchard plot, with a K D 10 -7 M or less, 10 -8 M or less, 10 -9 M (1 nM) or less, 5x10 -10 M or less, 10 -10 M or less, 10 -12 M to 10 -7 M, 10 -11 M to 10 -8 M, 10 -10 M to 10 -8 M, 10 -9 M to 10 -8 M, 10 -11 M to 10 -9 M or 10 -10 M to 10 -9 M binding.

[0499] In some embodiments, the anti-TIM3 antibodies described herein stimulate or enhance an immune response, e.g., by activating T cells, e.g., in a tumor. For example, the anti-TIM3 antibodies can activate or co-stimulate cells, as evidenced, e.g., by enhanced cytokine (e.g., IFN-γ) secretion and / or enhanced proliferation, which can be caused by inhibiting TIM3-mediated T cell inhibitory activity. In some embodiments, T cell activation or co-stimulation by the TIM3 antibody occurs in the presence of CD3 stimulation. In some embodiments, the anti-TIM3 antibody increases 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, 100-fold, as measured, e.g., on primary human T cells and / or T cells expressing human TIM3 (e.g., tumor infiltrating lymphocytes (TIL)).

[0500] In some embodiments, the anti-TIM3 antibody has an EC of 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 50Inhibit the binding of phosphatidylserine to human TIM3 on cells expressing human TIM3 such as CHO cells or activated T cells.

[0501] In some embodiments, the anti-TIM3 antibodies described herein bind to an epitope, such as a conformational epitope, in the extracellular portion of human TIM3, such as in the Ig-like domain of the extracellular region, i.e., amino acids 22 to 202 of SEQ ID NO:286( Figure 25 ). In some embodiments, the anti-TIM3 antibody binds to an epitope located within amino acids 22 to 120 of the extracellular domain of human TIM3 (SEQ ID NO: 286) or within 1-99 of mature human TIM3 (SEQ ID NO: 290) (see Examples). In some embodiments, the anti-TIM3 antibody binds to or is directed against an epitope within the region consisting of amino acids 58-64 of human TIM3 having SEQ ID NO:286, which corresponds to amino acid residues 37-43 of mature human TIM3 (CPVFECG, SEQ ID NO:296; see Figure 25 ). In some embodiments, the anti-TIM3 antibody binds to or is directed against an epitope within the region consisting of amino acids 111-120 of human TIM3 having SEQ ID NO:286, which corresponds to amino acid residues 90-99 of mature human TIM3 (RIQIPGIMND, SEQ IDNO:298; see Figure 25 ). In some embodiments, the anti-TIM3 antibody binds to or is directed against an epitope within the region consisting of amino acids 58-64 of human TIM3 having SEQ ID NO:286 (CPVFECG, SEQ ID NO:296); and binds to or is directed against an epitope within the region consisting of amino acids 111-120 of human TIM3 having SEQ ID NO:286 (RIQIPGIMND, SEQ ID NO:298; see Figure 25 ). In some embodiments, the anti-TIM3 antibody binds to or is directed against an epitope within the region consisting of amino acids 78-89 of human TIM3 having SEQ ID NO:286, which corresponds to amino acid residues 57-83 of mature human TIM3 (WTSRYWLNGDFR, SEQ ID NO:297; see Figure 25 ).

[0502] In some embodiments, the anti-TIM3 antibody binds to an epitope that is substantially the same as that of 13A3, i.e., an epitope (or region of human TIM3) comprising one or more of the amino acid residues C58, P59, F61, E62, C63, R111, and D120 of SEQ ID NO:286( Figure 25)。In some embodiments, the anti-TIM3 antibody binds to an epitope (or region of human TIM3) comprising one or more of the amino acid residues C58, P59, F61, E62, C63, D104, R111, Q113, and D120 of SEQ ID NO:286 ( Figure 25 )。In some embodiments, the anti-TIM3 antibody does not significantly bind or binds with only a significantly reduced affinity to a human TIM3 protein in which one or more of the amino acid residues C58, P59, F61, E62, C63, R111, and D120 of SEQ ID NO:286 are changed to another amino acid (e.g., by non-conservative amino acid substitution). In some embodiments, the anti-TIM3 antibody does not significantly bind or binds with only a significantly reduced affinity to a human TIM3 protein in which one or more of the amino acid residues C58, P59, F61, E62, C63, D104, R111, Q113, and D120 of SEQ ID NO:286 are changed to another amino acid (e.g., by non-conservative amino acid substitution).

[0503] In some embodiments, the anti-TIM3 antibody binds to an epitope substantially the same as 3G4, i.e., the epitope (or region of human TIM3) comprises one or more of the amino acid residues C58, P59, V60, F61, E62, C63, G116, and M118 of SEQ ID NO:286( Figure 25 )。In some embodiments, the anti-TIM3 antibody binds to an epitope (or region of human TIM3) comprising one or more of the amino acid residues C58, P59, V60, F61, E62, C63, D104, G116, and M118 of SEQ ID NO:286( Figure 25 )。In some embodiments, the anti-TIM3 antibody does not significantly bind or binds with only a significantly reduced affinity to a human TIM3 protein in which one or more of the amino acid residues C58, P59, V60, F61, E62, C63, G116, and M118 of SEQ ID NO:286 are changed to another amino acid (e.g., by non-conservative amino acid substitution). In some embodiments, the anti-TIM3 antibody does not significantly bind or binds with only a significantly reduced affinity to a human TIM3 protein in which one or more of the amino acid residues C58, P59, V60, F61, E62, C63, D104, G116, and M118 of SEQ ID NO:286 are changed to another amino acid (e.g., by non-conservative amino acid substitution).

[0504] In some embodiments, the anti-TIM3 antibody binds to an epitope that is substantially the same as 17C3, i.e., an epitope (or region of human TIM3) comprising one or more of the amino acid residues C58, P59, V60, F61, E62, C63, G64, and G116 of SEQ ID NO:286 ( Figure 25 ). In some embodiments, the anti-TIM3 antibody binds to an epitope (or region of human TIM3) comprising one or more of the amino acid residues C58, P59, V60, F61, E62, C63, G64, D104, and G116 of SEQ ID NO:286 ( Figure 25 ). In some embodiments, the anti-TIM3 antibody does not bind significantly or binds only with a significantly reduced affinity to a human TIM3 protein in which one or more of the amino acid residues C58, P59, V60, F61, E62, C63, G64, and G116 of SEQ ID NO:286 are changed to another amino acid (e.g., by non-conservative amino acid substitution). In some embodiments, the anti-TIM3 antibody does not bind significantly or binds only with a significantly reduced affinity to a human TIM3 protein in which one or more of the amino acid residues C58, P59, V60, F61, E62, C63, G64, D104, and G116 of SEQ ID NO:286 are changed to another amino acid (e.g., by non-conservative amino acid substitution).

[0505] In some embodiments, the anti-TIM3 antibody binds to an epitope that is substantially the same as 8B9, i.e., an epitope (or region of human TIM3) comprising one or more of the amino acid residues L48, W78, S80, R81, W83, G86, D87, and R89 of SEQ ID NO:286 ( Figure 25 ). In some embodiments, the anti-TIM3 antibody binds to an epitope (or region of human TIM3) comprising one or more of the amino acid residues L48, W78, S80, R81, W83, L84, G86, D87, and R89 of SEQ ID NO:286 ( Figure 25 ). In some embodiments, the anti-TIM3 antibody binds to an epitope that is substantially the same as 8B9, i.e., an epitope (or region of human TIM3) comprising one or more of the amino acid residues L48, W78, S80, R81, W83, G86, D87, R89, and D104 of SEQ ID NO:286 ( Figure 25 ). In some embodiments, the anti-TIM3 antibody does not bind significantly or binds only with a significantly reduced affinity to such a human TIM3 protein in which one or more of the amino acid residues L48, W78, S80, R81, W83, G86, D87, and R89 of SEQ ID NO:286 ( Figure 25)(e.g., by non-conservative amino acid substitution) a human TIM3 protein that is changed to another amino acid. In some embodiments, the anti-TIM3 antibody does not significantly bind or binds only with a significantly reduced affinity to one or more amino acid residues L48, W78, S80, R81, W83, L84, G86, D87, and R89 of SEQ ID NO:286( Figure 25 )(e.g., by non-conservative amino acid substitution) a human TIM3 protein that is changed to another amino acid. In some embodiments, the anti-TIM3 antibody does not significantly bind or binds only with a significantly reduced affinity to one or more amino acid residues L48, W78, S80, R81, W83, G86, D87, R89, and D104 of SEQ ID NO:286( Figure 25 )(e.g., by non-conservative amino acid substitution) a human TIM3 protein that is changed to another amino acid.

[0506] In some embodiments, the anti-TIM3 antibody competes for binding to human TIM3 (or inhibits binding) with an anti-TIM3 antibody comprising a CDR or variable region as described herein, e.g., those 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 some embodiments, the anti-TIM3 antibody inhibits binding of antibody 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 some 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 the anti-TIM3 antibody to human TIM3 by at least 50%, 60%, 70%, 80%, 90%, or 100%. In some 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 the anti-TIM3 antibody to human TIM3 by at least 50%, 60%, 70%, 80%, 90%, or 100% (e.g., competes in both directions).

[0507] In some embodiments, the anti-TIM3 antibody has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or all of the following characteristics:

[0508] (1) Binds to soluble human TIM3, e.g., with a K D10 nM or less (e.g., 0.01 nM to 10 nM), e.g., as measured by Biacore, e.g., as described in the Examples;

[0509] (2) Bind to soluble cynomolgus TIM3, e.g., with a K D 100 nM or less (e.g., 0.01 nM to 100 nM), e.g., as measured by Biacore, e.g., as described in the Examples;

[0510] (3) Bind to membrane-bound human TIM3, e.g., with an EC 50 Bind at 1 μg / mL or less (e.g., 0.01 μg / mL to 1 μg / mL), e.g., as measured by flow cytometry (e.g., as described in the Examples);

[0511] (4) Bind to membrane-bound human TIM3, e.g., with a K D Bind at 1 nM or less (e.g., 0.01 nM to 10 nM), e.g., as measured by Scatchard analysis, e.g., as described in the Examples;

[0512] (5) Bind to membrane-bound cynomolgus TIM3, e.g., with an EC 50 Bind at 20 μg / mL or less (e.g., 0.01 μg / mL to 20 μg / mL), e.g., as measured by flow cytometry (e.g., as described in the Examples);

[0513] (6) Bind to membrane-bound cynomolgus TIM3, e.g., with a K D 1 nM or less (e.g., 0.01 nM to 10 nM), e.g., as measured by Scatchard analysis, e.g., as described in the Examples;

[0514] (7) Induce or enhance T cell activation (e.g., by blocking or reducing the inhibitory effect of TIM3), as demonstrated by (i) increased IFN-γ production in TIM3-expressing T cells (e.g., Th1 cells or TIL) and / or (ii) enhanced proliferation of TIM3-expressing T cells (e.g., Th1 cells or TIL), e.g., as described in the Examples;

[0515] (8) Stimulate T cell proliferation in a mixed lymphocyte reaction (MLR) assay, e.g., as described in the Examples;

[0516] (9) Inhibit the binding of phosphatidylserine to TIM3, e.g., as measured by a PS-hTIM3 "tandem" blocking assay, e.g., as described in the Examples;

[0517] (10) Does not internalize or down-regulate cell surface TIM3 when bound to TIM3 on cells;

[0518] (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 NO: 296 and 298, respectively); and (d) WTSRYWLNGDFR (SEQ ID NO: 297), e.g., as described in the Examples;

[0519] (12) Has reduced binding to a 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 as in SEQ ID NO: 286 Figure 25 )) are replaced by another amino acid, e.g., as described in the Examples;

[0520] (13) Competitively binds to human TIM3 in one or both directions 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, e.g., as described in the Examples;

[0521] (14) Binds to the human TIM3 region 49 VPVCWGKGACPVFE 62 (SEQ ID NO: 367) and 111 RIQIPGIMNDEKFNLKL 127 (SEQ ID NO: 368), as determined by HDX-MS, e.g., as described in the Examples;

[0522] (15) having a heavy chain variable region and / or a 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; numbered according to SEQ ID NO:286( Figure 25 )); and / or

[0523] (16) (a) having reduced binding to human TIM3 in which 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids C58, P59, F61, E62, C63, R111, D120, and optionally D104 and Q113 (numbered according to SEQ ID NO:286( Figure 25 )) are replaced by another amino acid, relative to binding to wild-type human TIM3; (b) binding to 49 VPVCWGKGACPVFE 62 (SEQ ID NO:367), 111 RIQIPGIMNDEKFNLKL 127 (SEQ ID NO:368) and 119 NDEKFNLKL 127( (SEQ ID NO:373), as determined by HDX-MS, as described in the Examples; and / or (c) competing or cross-blocking the binding of 13A3 or TIM3.18.IgG1.3 to human TIM3, e.g., as described in the Examples;

[0524] (17) binding to hTIM3-IgV with a K D 5x10 -8 M, 2x10 -8 M, 10 -8 M or 5x10 -9 M or less, as determined by the method described in Example 22;

[0525] (18) binding to hTIM3-IgV with a K D 10 -7 M, 2x10 -8 M or 10 -8 M or less, as determined by the method described in Example 22;

[0526] (19) binding to hTIM3-IgV with a K D 5x10 -7M, 2x10 -8 M or 10 -8 M or less that binds cynomolgus TIM3-ECD, as measured by the method described in Example 22; and / or

[0527] (20) in K D 8x10 -8 M, 5x10 -8 M or 10 -8 M or less that binds hTIM3-ECD, as measured by the method described in Example 22.

[0528] Thus, an antibody that exhibits one or more of these functional properties (e.g., biochemical, immunochemical, cytological, physiological, or other biological activities, etc.) as measured by methods known in the art and described herein is understood to exhibit a statistically significant difference in the particular activity as compared to what is seen in the absence of the antibody (e.g., or when a control antibody of irrelevant specificity is present). In some embodiments, the increase in a measured parameter (e.g., T cell proliferation, cytokine production) induced by an anti-TIM3 antibody in a given assay achieves at least a 10% increase in the statistically significant measured parameter, 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 increase; and in some embodiments, the antibodies described herein can increase the measured parameter, e.g., by greater than 92%, 94%, 95%, 97%, 98%, 99%, 100% (i.e., 2-fold), 3-fold, 5-fold, or 10-fold as compared to the same assay conducted in the absence of the antibody. Conversely, the decrease in a measured parameter (e.g., tumor volume, binding of TIM3-L to human TIM3) induced by an anti-TIM3 antibody in a given assay achieves at least a 10% decrease in the statistically significant measured parameter, e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% decrease, and in some embodiments, the antibodies described herein can decrease the measured parameter, e.g., by greater than 92%, 94%, 95%, 97%, 98%, or 99% as compared to the same assay conducted in the absence of the antibody.

[0529] Standard assays for assessing the binding ability of antibodies to TIM3 in 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. The binding kinetics of the antibody (e.g., binding affinity) can also be evaluated by standard assays known in the art, such as by Biacore analysis. Assays for evaluating the effect of the antibody on the functional properties of TIM3 (e.g., ligand binding, T cell proliferation, cytokine production) are described in further detail below and in the Examples.

[0530] In some embodiments, the anti-TIM3 antibody is not a natural antibody or is not a naturally occurring antibody. For example, the anti-TIM3 antibody has post-translational modifications different from those of a naturally occurring antibody, such as having more, fewer, or different types of post-translational modifications.

[0531] In some embodiments, the anti-TIM3 antibody does not have agonist activity, as determined, for example, in the crosslinking of the anti-TIM3 antibody in a CHO-OKT3-CD32:T cell co-culture experiment, where the antibody does not enhance activity above that of the anti-TIM3 alone. In some embodiments, the anti-TIM3 antibody blocks the interaction of TIM3 with its ligand without promoting agonist activity.

[0532] In some embodiments, the anti-TIM3 antibody enhances the production of IL-12 by monocytes or dendritic cells treated with LPS.

[0533] In some embodiments, the anti-TIM3 antibody resurrects tumor-infiltrating CD8 + T cells co-expressing PD-1 and TIM3 by combination therapy, thereby avoiding the exhaustion of CD8 + T cells.

[0534] II. Exemplary anti-TIM3 antibodies

[0535] In particular, the anti-TIM3 antibodies described herein are isolated and structurally characterized antibodies as described herein having the CDR and / or variable region sequences of antibody 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, e.g., monoclonal, recombinant, and / or human antibodies, and antibodies having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or at least 99% identity) to their variable region or CDR sequences. See PCT / US2017 / 041946, which is hereby incorporated by reference in its entirety. The VH amino acid sequences of 13A3, 8B9, 8C4, 17C3, 9F6, 3G4, 17C8, 14H7, and 23B3 are shown in SEQ ID NOs: 34-40, 410, and 411, respectively. The VH amino acid sequences of the mutant forms of 13A3, 8B9, 9F6, and 23B3 are shown in SEQ ID NOs: 112-121, 364, and 412. The VH amino acid sequences of TIM3.20 and TIM3.21 are shown in SEQ ID NO: 449. The VH amino acid sequences of TIM3.22 and TIM3.23 are shown in SEQ ID NO: 456. The VL amino acid sequences of 13A3, 17C3, and 3G4 are shown in SEQ ID NO: 60. The VL amino acid sequences of 8B9, 8C4, and 17C8 are shown in SEQ ID NO: 61. The VL amino acid sequences of 9F6 are shown in SEQ ID NOs: 61, 62, and 63. The VL amino acid sequence of 14H7 is shown in SEQ ID NO: 417. The VL amino acid sequence of 23B3 is shown in SEQ ID NOs: 60 and 418. The VL amino acid sequences of the mutant forms of 13A3, 8B9, 9F6, and 23B3 are those of the corresponding non-mutated antibodies. The VL amino acid sequences of TIM3.20 and TIM3.22 are shown in SEQ ID NO: 450. The VL amino acid sequences of TIM3.21 and TIM3.23 are shown in SEQ ID NO: 63. Figure 16 A summary of SEQ ID NO identities is provided in .

[0536] Accordingly, provided herein are isolated antibodies or antigen-binding portions thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 34-40, 112-121, 364, 410-412, 449, and 456.

[0537] The present invention also provides an isolated antibody or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 60-63, 417, 418 and 450.

[0538] The present invention provides an isolated anti-human TIM3 antibody or antigen-binding portion thereof, comprising:

[0539] (a) heavy and light chain variable region sequences comprising SEQ ID NO: 34 and 60, respectively;

[0540] (b) heavy and light chain variable region sequences comprising SEQ ID NO: 35 and 61, respectively;

[0541] (c) heavy and light chain variable region sequences comprising SEQ ID NO: 36 and 61, respectively;

[0542] (d) heavy and light chain variable region sequences comprising SEQ ID NO: 37 and 60, respectively;

[0543] (e) heavy and light chain variable region sequences comprising SEQ ID NO: 38 and 61, respectively;

[0544] (f) heavy and light chain variable region sequences comprising SEQ ID NO: 38 and 62, respectively;

[0545] (g) heavy and light chain variable region sequences comprising SEQ ID NO: 38 and 63, respectively;

[0546] (h) heavy and light chain variable region sequences comprising SEQ ID NO: 39 and 60, respectively;

[0547] (i) heavy and light chain variable region sequences comprising SEQ ID NO: 40 and 61, respectively;

[0548] (j) heavy and light chain variable region sequences comprising SEQ ID NO: 121 and 63, respectively;

[0549] (k) heavy and light chain variable region sequences comprising SEQ ID NO: 120 and 61, respectively;

[0550] (l) heavy and light chain variable region sequences comprising SEQ ID NO: 112 and 60, respectively;

[0551] (m) heavy and light chain variable region sequences comprising SEQ ID NO: 113 and 60, respectively;

[0552] (n) The heavy chain variable region and light chain variable region sequences respectively comprising SEQ ID NO: 114 and 60;

[0553] (o) The heavy chain variable region and light chain variable region sequences respectively comprising SEQ ID NO: 115 and 60;

[0554] (p) The heavy chain variable region and light chain variable region sequences respectively comprising SEQ ID NO: 116 and 60;

[0555] (q) The heavy chain variable region and light chain variable region sequences respectively comprising SEQ ID NO: 117 and 60;

[0556] (r) The heavy chain variable region and light chain variable region sequences respectively comprising SEQ ID NO: 118 and 60;

[0557] (s) The heavy chain variable region and light chain variable region sequences respectively comprising SEQ ID NO: 119 and 60;

[0558] (t) The heavy chain variable region and light chain variable region sequences respectively comprising SEQ ID NO: 364 and 60;

[0559] (u) The heavy chain variable region and light chain variable region sequences respectively comprising SEQ ID NO: 410 and 417;

[0560] (v) The heavy chain variable region and light chain variable region sequences respectively comprising SEQ ID NO: 411 and 60;

[0561] (w) The heavy chain variable region and light chain variable region sequences respectively comprising SEQ ID NO: 411 and 418;

[0562] (x) The heavy chain variable region and light chain variable region sequences respectively comprising SEQ ID NO: 412 and 60;

[0563] (y) The heavy chain variable region and light chain variable region sequences respectively comprising SEQ ID NO: 449 and 450;

[0564] (z) The heavy chain variable region and light chain variable region sequences respectively comprising SEQ ID NO: 449 and 63;

[0565] (aa) The heavy chain variable region and light chain variable region sequences respectively comprising SEQ ID NO: 456 and 450; or

[0566] (bb) The heavy chain variable region and light chain variable region sequences respectively comprising SEQ ID NO: 456 and 63.

[0567] Anti-TIM3 antibodies may comprise 13A3, 8B9, 8C4, 17C3, 9F6, 3G4, 17C8, 14H7, 23B3 or the heavy chain CDR1, CDR2 and CDR3 and the light chain CDR1, CDR2 and CDR3 of any of TIM3.2 to TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24 or TIM3.25, or combinations thereof. The amino acid sequences of the VH CDR1 of 13A3, 8B9, 8C4 and 17C3 are shown in SEQ ID NO:41-44, respectively. The amino acid sequences of the VH CDR1 of 9F6, 3G4, 17C8, 14H7 and 23B3 are shown in SEQ ID NO 45. The amino acid sequence of the VH CDR1 of the mutant 13A3 antibody (i.e., TIM3.10-TIM3.18) is the same as that of the non-mutant 13A3 antibody, i.e., SEQ ID NO:41. The amino acid sequence of the VH CDR1 of the mutant 8B9 antibody (i.e., TIM3.8) is the same as that of the non-mutant 8B9 antibody, i.e., SEQ ID NO:42. The amino acid sequence of the VH CDR1 of the mutant 9F6 antibody (i.e., TIM3.7) is the same as that of the non-mutant 9F6 antibody, i.e., SEQ ID NO:45. The amino acid sequence of the VH CDR1 of the mutant 23B3 antibody (i.e., TIM3.25) is the same as that of the non-mutant 23B3 antibody, i.e., SEQ ID NO:45.

[0568] The amino acid sequences of the VH CDR2 of 13A3, 8B9, 8C4, 17C3, 9F6, 3G4, 17C8, 14H7 and 23B3 are shown in SEQ ID NO:46-52, 413 and 415, respectively. The amino acid sequences of the VH CDR2 of the mutant 13A3 antibodies TIM3.10, TIM3.17 and TIM3.18 are shown in SEQ ID NO:122. The amino acid sequences of the VH CDR2 of the mutant 13A3 antibodies TIM3.11 and TIM3.12 are shown in SEQ ID NO:123 and 124, respectively. The amino acid sequences of the VH CDR2 of the mutant 13A3 antibodies TIM3.13 and TIM3.16 are the amino acid sequences of the VH CDR2 of the non-mutant 13A3 antibody, i.e., SEQ ID NO:46. The amino acid sequence of the VH CDR2 of the mutant 8B9 antibody (i.e., TIM3.8) is shown in SEQ ID NO:125. The amino acid sequence of the VH CDR2 of the mutant 9F6 antibody (i.e., TIM3.7) is the same as that of the non-mutant 9F6 antibody, i.e., SEQ ID NO:50. The amino acid sequence of the VH CDR2 of the mutant 23B3 antibody (i.e., TIM3.25) is the same as that of the non-mutant 23B3 antibody, i.e., SEQ ID NO:415.

[0569] The amino acid sequences of the VH CDR3s of 13A3, 8B9, 8C4, 17C3, 9F6, 3G4, 17C8, 14H7, and 23B3 are shown in SEQ ID NOs: 53 - 59, 414, and 416, respectively. The amino acid sequences of the VH CDR3s of the mutant 13A3 antibody TIM3.10 to TIM3.12 are the sequences of the non - mutant 13A3 antibody, i.e., SEQ ID NO: 53. The amino acid sequences of the VH CDR3s of the mutant 13A3 antibodies TIM3.13 and TIM3.18 are shown in SEQ ID NO: 126. The amino acid sequences of the VH CDR3s of the mutant 13A3 antibodies TIM3.15 and TIM3.17 are shown in SEQ ID NO: 128. The amino acid sequences of the VH CDR3s of the mutant 13A3 antibodies TIM3.14 and TIM3.16 are shown in SEQ ID NOs: 127 and 129, respectively. The amino acid sequence of the VH CDR3 of the mutant 8B9 antibody (i.e., TIM3.8) is the sequence of the non - mutant 8B9 antibody, i.e., SEQ ID NO: 54. The amino acid sequence of the VH CDR3 of the mutant 9F6 antibody (i.e., TIM3.7) is the same as that of the non - mutant 9F6 antibody, i.e., SEQ ID NO: 57. The amino acid sequence of the VH CDR3 of the mutant 23B3 antibody (i.e., TIM3.25) is the same as that of the non - mutant 23B3 antibody, i.e., SEQ ID NO: 416.

[0570] The amino acid sequences of the VL CDR1s of 13A3, 8B9, 8C4, 17C3, 3G4, 17C8, 14H7, and 23B3 are shown in SEQ ID NO: 64. The amino acid sequences of the VL CDR1s of 9F6 are shown in SEQ ID NOs: 64 and 65. The amino acid sequences of the VL CDR2s of 13A3, 8B9, 8C4, 17C3, 3G4, 17C8, 14H7, and 23B3 are shown in SEQ ID NO: 66. The amino acid sequences of the VL CDR2s of 9F6 are shown in SEQ ID NOs: 66 and 67. The amino acid sequences of the VL CDR3s of 13A3, 17C3, and 3G4 are shown in SEQ ID NO: 68. The amino acid sequences of the VL CDR3s of 8B9, 8C4, 17C8, and 14H7 are shown in SEQ ID NO: 69. The amino acid sequences of the VL CDR3s of 9F6 are shown in SEQ ID NOs: 69, 70, and 71. The amino acid sequence of the VL CDR3 of 23B3 is shown in SEQ ID NOs: 68 and 419. The amino acid sequences of the VL CDRs of the mutant antibodies 13A3, 8B9, 9F6, and 23B3 are those of the corresponding non - mutant antibodies. Figure 16 A table of SEQ ID NOs of the CDRs of the anti - TIM3 antibodies described herein is provided.

[0571] The Kabat system is used to describe the CDR regions (Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242). The Kabat system is the most common numbering system in a scheme known as the EU index or EU numbering system, which is based on the sequential numbering of the first sequenced human IgG1 (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 (enhanced Chothia), or AHo numbering schemes.

[0572] Assuming that each of these antibodies binds human TIM3 and that antigen-binding specificity is provided primarily by the CDR1, 2, and 3 regions, the VH CDR1, 2, and 3 sequences and the VL CDR1, 2, and 3 sequences can be "mixed and matched", e.g., Figure 16 the sequences in

[0573] Isolated anti-human TIM3 antibodies or antigen-binding portions thereof are provided herein, comprising

[0574] (a) The heavy chain variable region CDR1, comprising an amino acid sequence selected from SEQ ID NOs: 41-45 and 469;

[0575] (b) The heavy chain variable region CDR2, comprising an amino acid sequence selected from SEQ ID NOs: 46-52, 122-125, 413, 415 and 470;

[0576] (c) The heavy chain variable region CDR3, comprising an amino acid sequence selected from SEQ ID NOs: 53-59, 126-129, 414, 416 and 471;

[0577] (d) The light chain variable region CDR1, comprising an amino acid sequence selected from SEQ ID NOs: 64-65 and 472;

[0578] (e) The light chain variable region CDR2, comprising an amino acid sequence selected from SEQ ID NOs: 66-67 and 473; and

[0579] (f) The light chain variable region CDR3, comprising an amino acid sequence selected from SEQ ID NOs: 68-71, 419 and 474;

[0580] wherein the antibody specifically binds to human TIM3.

[0581] In some embodiments, the anti-human TIM3 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region CDR1, CDR2 and CDR3 regions comprise:

[0582] (a) SEQ ID NO: 41, 46, 53;

[0583] (b) SEQ ID NO: 42, 47, 54;

[0584] (c) SEQ ID NO: 43, 48, 55;

[0585] (d) SEQ ID NO: 44, 49, 56;

[0586] (e) SEQ ID NO: 45, 50, 57;

[0587] (f) SEQ ID NO: 45, 51, 58;

[0588] (g) SEQ ID NO: 45, 52, 59;

[0589] (h) SEQ ID NO: 41, 122, 53;

[0590] (i) SEQ ID NO: 41, 123, 53;

[0591] (j) SEQ ID NO: 41, 124, 53;

[0592] (k) SEQ ID NO: 41, 46, 126;

[0593] (l) SEQ ID NO: 41, 46, 127;

[0594] (m) SEQ ID NO: 41, 46, 128;

[0595] (n) SEQ ID NO: 41, 46, 129;

[0596] (o) SEQ ID NO: 41, 122, 128;

[0597] (p) SEQ ID NO: 41, 122, 126;

[0598] (q) SEQ ID NO: 45, 413, 414;

[0599] (r) SEQ ID NO: 45, 415, 416; or

[0600] (s) SEQ ID NO: 469, 470 and 471,

[0601] wherein the antibody specifically binds to human TIM3.

[0602] In some embodiments, the anti-human TIM3 antibody comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region CDR1, CDR2 and CDR3 regions comprise:

[0603] (a) SEQ ID NO: 64, 66, 68;

[0604] (b) SEQ ID NO: 64, 66, 69;

[0605] (c) SEQ ID NO: 65, 67, 70;

[0606] (d) SEQ ID NO: 64, 66, 71;

[0607] (e) SEQ ID NO: 64, 66, 419; or

[0608] (f) SEQ ID NO: 472, 473, 474,

[0609] wherein the antibody specifically binds to human TIM3.

[0610] In some embodiments, the anti-TIM3 antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0611] (a1) The heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NO:41, 46, 53, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NO:64, 66, 68, respectively;

[0612] (a2) The heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NO:41, 122, 53, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NO:64, 66, 68, respectively;

[0613] (a3) The heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NO:41, 123, 53, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NO:64, 66, 68, respectively;

[0614] (a4) The heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NO:41, 124, 53, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NO:64, 66, 68, respectively;

[0615] (a5) The heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NO:41, 46, 126, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NO:64, 66, 68, respectively;

[0616] (a6) The heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NO:41, 46, 127, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NO:64, 66, 68, respectively;

[0617] (a7) The heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NO:41, 46, 128, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NO:64, 66, 68, respectively;

[0618] (a8) The heavy chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NO:41, 46, 129, respectively, and the light chain variable region CDR1, CDR2, and CDR3 comprise SEQ ID NO:64, 66, 68, respectively;

[0619] (a9) The CDR1, CDR2 and CDR3 of the heavy chain variable region contain SEQ ID NO:41, 122, 128 respectively, and the CDR1, CDR2 and CDR3 of the light chain variable region contain SEQ ID NO:64, 66, 68 respectively;

[0620] (a10) The CDR1, CDR2 and CDR3 of the heavy chain variable region contain SEQ ID NO:41, 122, 126 respectively, and the CDR1, CDR2 and CDR3 of the light chain variable region contain SEQ ID NO:64, 66, 68 respectively;

[0621] (b1) The CDR1, CDR2 and CDR3 of the heavy chain variable region contain SEQ ID NO:42, 47, 54 respectively, and the CDR1, CDR2 and CDR3 of the light chain variable region contain SEQ ID NO:64, 66, 69 respectively;

[0622] (b2) The CDR1, CDR2 and CDR3 of the heavy chain variable region contain SEQ ID NO:42, 125, 54 respectively, and the CDR1, CDR2 and CDR3 of the light chain variable region contain SEQ ID NO:64, 66, 69 respectively;

[0623] (c) The CDR1, CDR2 and CDR3 of the heavy chain variable region contain SEQ ID NO:43, 48, 55 respectively, and the CDR1, CDR2 and CDR3 of the light chain variable region contain SEQ ID NO:64, 66, 69 respectively;

[0624] (d) The CDR1, CDR2 and CDR3 of the heavy chain variable region contain SEQ ID NO:44, 49, 56 respectively, and the CDR1, CDR2 and CDR3 of the light chain variable region contain SEQ ID NO:64, 66, 68 respectively;

[0625] (e1) The CDR1, CDR2 and CDR3 of the heavy chain variable region contain SEQ ID NO:45, 50, 57 respectively, and the CDR1, CDR2 and CDR3 of the light chain variable region contain SEQ ID NO:64, 66, 69 respectively;

[0626] (e2) The CDR1, CDR2 and CDR3 of the heavy chain variable region contain SEQ ID NO:45, 50, 57 respectively, and the CDR1, CDR2 and CDR3 of the light chain variable region contain SEQ ID NO:64, 66, 71 respectively;

[0627] (e3) The CDR1, CDR2, and CDR3 of the heavy chain variable region respectively contain SEQ ID NO:45, 50, 57, and the CDR1, CDR2, and CDR3 of the light chain variable region respectively contain SEQ ID NO:65, 67, 70;

[0628] (f) The CDR1, CDR2, and CDR3 of the heavy chain variable region respectively contain SEQ ID NO:45, 51, 58, and the CDR1, CDR2, and CDR3 of the light chain variable region respectively contain SEQ ID NO:64, 66, 68;

[0629] (g) The CDR1, CDR2, and CDR3 of the heavy chain variable region respectively contain SEQ ID NO:45, 52, 59, and the CDR1, CDR2, and CDR3 of the light chain variable region respectively contain SEQ ID NO:64, 66, 69;

[0630] (h) The CDR1, CDR2, and CDR3 of the heavy chain variable region respectively contain SEQ ID NO:45, 413, 414, and the CDR1, CDR2, and CDR3 of the light chain variable region respectively contain SEQ ID NO:64, 66, 69;

[0631] (i1) The CDR1, CDR2, and CDR3 of the heavy chain variable region respectively contain SEQ ID NO:45, 415, 416, and the CDR1, CDR2, and CDR3 of the light chain variable region respectively contain SEQ ID NO:64, 66, 68;

[0632] (i2) The CDR1, CDR2, and CDR3 of the heavy chain variable region respectively contain SEQ ID NO:45, 415, 416, and the CDR1, CDR2, and CDR3 of the light chain variable region respectively contain SEQ ID NO:64, 66, 419;

[0633] (j1) The CDR1, CDR2, and CDR3 of the heavy chain variable region respectively contain SEQ ID NO:469, 470, and 471, and the CDR1, CDR2, and CDR3 of the light chain variable region respectively contain SEQ ID NO:472, 473, and 474; or

[0634] (j2) The CDR1, CDR2, and CDR3 of the heavy chain variable region respectively contain SEQ ID NO:469, 470, and 471, and the CDR1, CDR2, and CDR3 of the light chain variable region respectively contain SEQ ID NO:64, 66 and 71,

[0635] wherein the antibody specifically binds to human TIM3.

[0636] The VH domain or one or more CDRs thereof described herein can be linked to a constant domain to form a heavy chain, such as a full-length heavy chain. Similarly, the VL domain or one or more CDRs thereof described herein can be linked to a constant domain to form a light chain, such as a full-length light chain. The full-length heavy chain (except for the C-terminal lysine (K), or except for the C-terminal glycine and lysine (GK), which may be absent) and the full-length light chain combine to form a full-length antibody.

[0637] The VH domain described herein can be fused to the constant domain of a naturally occurring or modified human IgG, such as IgG1, IgG2, IgG3, or IgG4, for example, as further described herein. For example, the VH domain can comprise the amino acid sequence of any VH domain described herein fused to the human IgG, such as the IgG1 constant region, which is, for example, the following wild-type human IgG1 constant domain amino acid sequence:

[0638]

[0639] or an allotypic variant of SEQ ID NO: 291 and having the following amino acid sequence:

[0640] (SEQ ID NO: 277; "IgG1f," the allotype-specific amino acid residues are bold and underlined)

[0641] The VH domain of the anti-TIM3 antibody can comprise the amino acid sequence of any VH domain described herein fused to a constant region without effector function, such as the following human IgG1 constant domain amino acid sequence without effector function:

[0642] (SEQ ID NO: 294; "IgG1.1f," comprising the substitutions L234A, L235E, G237A, A330S, and P331S, which are underlined) underlined)

[0643] or

[0644] (SEQ ID NO: 295; "IgG1.3f," comprising the substitutions L234A, L235E, and G237A, which are underlined)

[0645] For example, an IgG1 allotype variant contains K97R, D239E, and / or L241M (underlined and bolded above) and is numbered according to SEQ ID NOs: 277, 294, and 295. In the full-length heavy chain region, for example, in 8C4 (SEQ ID NO: 3) and according to EU numbering, these amino acid substitutions are numbered K214R, D356E, and L358M. In some embodiments, the constant region of the anti-TIM3 antibody may further contain one or more mutations or substitutions at amino acids L117, A118, G120, A213, and P214 (underlined above) numbered as in SEQ ID NOs: 277, 294, and 295 or at L234, A235, G237, A330, and P331 according to EU numbering. In some embodiments, the constant region of the anti-TIM3 antibody contains one or more mutations or substitutions at L117A, A118E, G120A, A213S, and P214S of SEQ ID NO: 291 or at L234A, L235E, G237A, A330S, and P331S according to EU numbering. The constant region of the anti-TIM3 antibody may also contain one or more mutations or substitutions of L117A, A118E, and G120A of SEQ ID NO: 291 or L234A, L235E, and G237A according to EU numbering.

[0646] Alternatively, the VH domain of the anti-TIM3 antibody may contain the amino acid sequence of any VH domain described herein fused to a human IgG4 constant region, e.g., the human IgG4 constant region is the following human IgG4 amino acid sequence or a variant thereof:

[0647] (SEQ ID NO: 292, containing S228P).

[0648] The VL domain described herein can be fused to the constant domain of a human κ or λ light chain. For example, the VL domain of the anti-TIM3 antibody may contain the amino acid sequence of any VL domain described herein fused to the following human IgG1κ light chain amino acid sequence:

[0649]

[0650] In some embodiments, the heavy chain constant region contains a lysine or another amino acid at the C-terminus, e.g., it contains the following last amino acids 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 and has, for example, a C-terminal sequence of LSPG (SEQ ID NO: 280) or LSP (SEQ ID NO: 281).

[0651] The amino acid sequences of exemplary heavy and light chains 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.

[0652] Provided herein are isolated anti-human TIM3 antibodies or antigen-binding portions thereof, comprising:

[0653] (a1) heavy and light chain sequences comprising SEQ ID NO: 301 (or 302) and 29, respectively;

[0654] (a2) heavy and light chain sequences comprising SEQ ID NO: 1 (or 8) and 29, respectively;

[0655] (a3) heavy and light chain sequences comprising SEQ ID NO: 15 (or 22) and 29, respectively;

[0656] (a4) heavy and light chain sequences comprising SEQ ID NO: 303 (or 304) and 29, respectively;

[0657] (a5) heavy and light chain sequences comprising SEQ ID NO: 72 (or 82) and 29, respectively;

[0658] (a6) heavy and light chain sequences comprising SEQ ID NO: 92 (or 102) and 29, respectively;

[0659] (a7) heavy and light chain sequences comprising SEQ ID NO: 305 (or 306) and 29, respectively;

[0660] (a8) heavy and light chain sequences comprising SEQ ID NO: 73 (or 83) and 29, respectively;

[0661] (a9) heavy and light chain sequences comprising SEQ ID NO: 93 (or 103) and 29, respectively;

[0662] (a10) heavy and light chain sequences comprising SEQ ID NO: 307 (or 308) and 29, respectively;

[0663] (a11) Heavy and light chain sequences containing SEQ ID NO:74 (or 84) and 29 respectively;

[0664] (a12) Heavy and light chain sequences containing SEQ ID NO:94 (or 104) and 29 respectively;

[0665] (a13) Heavy and light chain sequences containing SEQ ID NO:309 (or 310) and 29 respectively;

[0666] (a14) Heavy and light chain sequences containing SEQ ID NO:75 (or 85) and 29 respectively;

[0667] (a15) Heavy and light chain sequences containing SEQ ID NO:95 (or 105) and 29 respectively;

[0668] (a16) Heavy and light chain sequences containing SEQ ID NO:311 (or 312) and 29 respectively;

[0669] (a17) Heavy and light chain sequences containing SEQ ID NO:76 (or 86) and 29 respectively;

[0670] (a18) Heavy and light chain sequences containing SEQ ID NO:96 (or 106) and 29 respectively;

[0671] (a19) Heavy and light chain sequences containing SEQ ID NO:313 (or 314) and 29 respectively;

[0672] (a20) Heavy and light chain sequences containing SEQ ID NO:77 (or 87) and 29 respectively;

[0673] (a21) Heavy and light chain sequences containing SEQ ID NO:97 (or 107) and 29 respectively;

[0674] (a22) Heavy and light chain sequences containing SEQ ID NO:315 (or 316) and 29 respectively;

[0675] (a23) Heavy and light chain sequences containing SEQ ID NO:78 (or 88) and 29 respectively;

[0676] (a24) Heavy and light chain sequences containing SEQ ID NO:98 (or 108) and 29 respectively;

[0677] (a25) Heavy and light chain sequences containing SEQ ID NO:317 (or 318) and 29 respectively;

[0678] (a26) Heavy and light chain sequences containing SEQ ID NO:79 (or 89) and 29, respectively;

[0679] (a27) Heavy and light chain sequences containing SEQ ID NO:99 (or 109) and 29, respectively;

[0680] (a28) Heavy and light chain sequences containing SEQ ID NO:319 (or 320) and 29, respectively;

[0681] (a29) Heavy and light chain sequences containing SEQ ID NO:349 (or 350) and 29, respectively;

[0682] (a30) Heavy and light chain sequences containing SEQ ID NO:351 (or 352) and 29, respectively;

[0683] (a31) Heavy and light chain sequences containing SEQ ID NO:353 (or 354) and 29, respectively;

[0684] (b1) Heavy and light chain sequences containing SEQ ID NO:321 (or 322) and 30, respectively;

[0685] (b2) Heavy and light chain sequences containing SEQ ID NO:2 (or 9) and 30, respectively;

[0686] (b3) Heavy and light chain sequences containing SEQ ID NO:16 (or 23) and 30, respectively;

[0687] (b4) Heavy and light chain sequences containing SEQ ID NO:323 (or 324) and 30, respectively;

[0688] (b5) Heavy and light chain sequences containing SEQ ID NO:80 (or 90) and 30, respectively;

[0689] (b6) Heavy and light chain sequences containing SEQ ID NO:100 (or 110) and 30, respectively;

[0690] (b7) Heavy and light chain sequences containing SEQ ID NO:325 (or 326) and 30, respectively;

[0691] (c1) Heavy and light chain sequences containing SEQ ID NO:327 (or 328) and 30, respectively;

[0692] (c2) Heavy and light chain sequences containing SEQ ID NO:3 (or 10) and 30, respectively;

[0693] (c3) Heavy and light chain sequences containing SEQ ID NO:17 (or 24) and 30 respectively;

[0694] (c4) Heavy and light chain sequences containing SEQ ID NO:329 (or 330) and 30 respectively;

[0695] (d1) Heavy and light chain sequences containing SEQ ID NO:331 (or 332) and 29 respectively;

[0696] (d2) Heavy and light chain sequences containing SEQ ID NO:4 (or 11) and 29 respectively;

[0697] (d3) Heavy and light chain sequences containing SEQ ID NO:18 (or 25) and 29 respectively;

[0698] (d4) Heavy and light chain sequences containing SEQ ID NO:333 (or 334) and 29 respectively;

[0699] (e1.1) Heavy and light chain sequences containing SEQ ID NO:335 (or 336) and 32 respectively;

[0700] (e1.2) Heavy and light chain sequences containing SEQ ID NO:335 (or 336) and 33 respectively;

[0701] (e1.3) Heavy and light chain sequences containing SEQ ID NO:335 (or 336) and 31 respectively;

[0702] (e2) Heavy and light chain sequences containing SEQ ID NO:5 (or 12) and 33 respectively;

[0703] (e3) Heavy and light chain sequences containing SEQ ID NO:19 (or 26) and 33 respectively;

[0704] (e4) Heavy and light chain sequences containing SEQ ID NO:337 (or 338) and 33 respectively;

[0705] (e5) Heavy and light chain sequences containing SEQ ID NO:81 (or 91) and 33 respectively;

[0706] (e6) Heavy and light chain sequences containing SEQ ID NO:101 (or 111) and 33 respectively;

[0707] (e7) Heavy and light chain sequences containing SEQ ID NO:339 (or 340) and 33 respectively;

[0708] (f1) Heavy and light chain sequences containing SEQ ID NO: 341 (or 342) and 29, respectively;

[0709] (f2) Heavy and light chain sequences containing SEQ ID NO: 6 (or 13) and 29, respectively;

[0710] (f3) Heavy and light chain sequences containing SEQ ID NO: 20 (or 27) and 29, respectively;

[0711] (f4) Heavy and light chain sequences containing SEQ ID NO: 343 (or 344) and 29, respectively;

[0712] (g1) Heavy and light chain sequences containing SEQ ID NO: 345 (or 346) and 30, respectively;

[0713] (g2) Heavy and light chain sequences containing SEQ ID NO: 7 (or 14) and 30, respectively;

[0714] (g3) Heavy and light chain sequences containing SEQ ID NO: 21 (or 28) and 30, respectively;

[0715] (g4) Heavy and light chain sequences containing SEQ ID NO: 347 (or 348) and 30, respectively;

[0716] (h1) Heavy and light chain sequences containing SEQ ID NO: 386 (or 387) and 408, respectively;

[0717] (h2) Heavy and light chain sequences containing SEQ ID NO: 388 (or 389) and 408, respectively;

[0718] (h3) Heavy and light chain sequences containing SEQ ID NO: 390 (or 391) and 408, respectively;

[0719] (h4) Heavy and light chain sequences containing SEQ ID NO: 392 (or 393) and 408, respectively;

[0720] (i1.1) Heavy and light chain sequences containing SEQ ID NO: 394 (or 395) and 29, respectively;

[0721] (i1.2) Heavy and light chain sequences containing SEQ ID NO: 394 (or 395) and 409, respectively;

[0722] (i2) Heavy and light chain sequences containing SEQ ID NO: 396 (or 397) and 29, respectively;

[0723] (i3) heavy and light chain sequences containing SEQ ID NO:398 (or 399) and 29 respectively;

[0724] (i4) heavy and light chain sequences containing SEQ ID NO:400 (or 401) and 29 respectively;

[0725] (i5) heavy and light chain sequences containing SEQ ID NO:402 (or 403) and 29 respectively;

[0726] (i6) heavy and light chain sequences containing SEQ ID NO:404 (or 405) and 29 respectively;

[0727] (i7) heavy and light chain sequences containing SEQ ID NO:406 (or 407) and 29 respectively;

[0728] (j1) heavy and light chain sequences containing SEQ ID NO:451 (or 461) and 453 respectively;

[0729] (j2) heavy and light chain sequences containing SEQ ID NO:452 (or 462) and 453 respectively;

[0730] (k1) heavy and light chain sequences containing SEQ ID NO:454 (or 463) and 33 respectively;

[0731] (k2) heavy and light chain sequences containing SEQ ID NO:455 (or 464) and 33 respectively;

[0732] (l1) heavy and light chain sequences containing SEQ ID NO:457 (or 465) and 453 respectively;

[0733] (l2) heavy and light chain sequences containing SEQ ID NO:458 (or 466) and 453 respectively;

[0734] (m1) heavy and light chain sequences containing SEQ ID NO:459 (or 467) and 33 respectively; or

[0735] (m2) heavy and light chain sequences containing SEQ ID NO:460 (or 468) and 33 respectively,

[0736] wherein the antibody specifically binds to human TIM3.

[0737] In some embodiments, the anti-TIM3 antibody comprises a combination of heavy and light chain sequences as described herein, e.g., above, wherein 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 disulfide bonds linking each light chain to each heavy chain.

[0738] Heavy and light chains comprising an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75% or 70% identical to any of the heavy or light chains shown 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 form anti-human TIM3 antibodies with desired characteristics, such as those further described herein. Exemplary variants are those that comprise allotypic variations, e.g., in the constant domain, and / or mutations (e.g., mutations disclosed herein) in the variable or constant region. Heavy and light chains comprising an amino acid sequence that is different by up to 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2 or 1 amino acid (by substitution, addition or deletion) from any of the heavy or light chains (or their variable regions) shown herein can be used to form anti-human TIM3 antibodies with desired characteristics, such as those further described herein.

[0739] In some embodiments, the antibodies described above 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, 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:

[0740] (1) Binds to soluble human TIM3, e.g., with a KD of 10 nM or less (e.g., 0.01 nM to 10 nM), e.g., as determined by Biacore, e.g., as described in the Examples;

[0741] (2) Binds to soluble cynomolgus monkey TIM3, e.g., with a KD of 100 nM or less (e.g., 0.01 nM to 100 nM), e.g., as determined by Biacore, e.g., as described in the Examples;

[0742] (3) Bind to membrane-bound human TIM3, e.g., with an EC50 of 1 μg / mL or less (e.g., 0.01 μg / mL to 1 μg / mL), e.g., as determined by flow cytometry (e.g., as described in the examples);

[0743] (4) Bind to membrane-bound human TIM3, e.g., with a K D d of 1 nM or less (e.g., 0.01 nM to 10 nM), e.g., as determined by Scatchard analysis, e.g., as described in the examples;

[0744] (5) Bind to membrane-bound cynomolgus TIM3, e.g., with an EC50 of 20 μg / mL or less (e.g., 0.01 μg / mL to 20 μg / mL), e.g., as determined by flow cytometry (e.g., as described in the examples);

[0745] (6) Bind to membrane-bound cynomolgus TIM3, e.g., with a K D d of 1 nM or less (e.g., 0.01 nM to 10 nM), e.g., as determined by Scatchard analysis, e.g., as described in the examples;

[0746] (7) Induce or enhance T cell activation (e.g., by blocking or reducing the inhibitory effect of TIM3), as demonstrated by (i) increased IFN-γ production in TIM3-expressing T cells (e.g., Th1 cells or TILs) and / or (ii) enhanced proliferation of TIM3-expressing T cells (e.g., Th1 cells or TILs), e.g., as described in the examples;

[0747] (8) Stimulate T cell proliferation in a mixed lymphocyte reaction (MLR) assay, e.g., as described in the examples;

[0748] (9) Inhibit the binding of phosphatidylserine to TIM3, e.g., as determined by a PS-hTIM3 "tandem" blocking assay, e.g., as described in the examples;

[0749] (10) Do not internalize or downregulate cell surface TIM3 when bound to TIM3 on the cell;

[0750] (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 NO: 296 and 298, respectively); and (d) WTSRYWLNGDFR (SEQ ID NO: 297), e.g., as described in the examples;

[0751] (12) has reduced binding to a 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 as in SEQ ID NO: 286 Figure 25 )) are replaced by another amino acid, e.g., as described in the examples;

[0752] (13) competes 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 for binding to human TIM3 in one or both directions, e.g., as described in the examples;

[0753] (14) binds to the human TIM3 region 49 VPVCWGKGACPVFE 62 (SEQ ID NO: 367) and 111 RIQIPGIMNDEKFNLKL 127 (SEQ ID NO: 368), as determined by HDX-MS, e.g., as described in the examples;

[0754] (15) having a heavy chain variable region and / or a 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; numbered according to SEQ ID NO:286( Figure 25 ));

[0755] (16) (a) having reduced binding to human TIM3 in which 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids C58, P59, F61, E62, C63, R111, D120, and optionally D104 and Q113 (numbered according to SEQ ID NO:286( Figure 25 )) are replaced by another amino acid, as compared to binding to wild-type human TIM3; (b) binding to 49 VPVCWGKGACPVFE 62 (SEQ ID NO:367), 111 RIQIPGIMNDEKFNLKL 127 (SEQ ID NO:368) and 119 NDEKFNLKL 127( (SEQ ID NO:373), as determined by HDX-MS, as described in the Examples; and / or (c) competing with or cross-blocking the binding of 13A3 or TIM3.18.IgG1.3 to human TIM3, e.g., as described in the Examples

[0756] (17) binding to hTIM3-IgV with a K D 5x10 -8 M, 2x10 -8 M, 10 -8 M or 5x10 -9 M or less, as determined by the method described in Example 22;

[0757] (18) binding to hTIM3-IgV with a K D 10 -7 M, 2x10 -8 M or 10 -8 M or less, as determined by the method described in Example 22;

[0758] (19) binding to hTIM3-IgV with a K D 5x10 -7 M, 2x10-8 M or 10 -8 M or less binds to cynomolgus TIM3-ECD, as determined by the method described in Example 22; and / or

[0759] (20) in K D 8x10 -8 M, 5x10 -8 M or 10 -8 M or less binds to hTIM3-ECD, as determined by the method described in Example 22.

[0760] Such antibodies include, for example, human antibodies, humanized antibodies or chimeric antibodies.

[0761] In some embodiments, the anti-TIM3 antibodies described herein bind to a conformational epitope.

[0762] In some embodiments, the anti-TIM3 antibodies described herein bind to amino acid residues within the following regions of the mature human TIM3 extracellular domain (SEQ ID NO: 290):

[0763] SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLN GDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMND (SEQ ID NO: 299), corresponding to amino acid residues 1-99 of the mature human TIM3 extracellular domain (SEQ ID NO: 290) or amino acids 22 to 120 of human TIM3 having SEQ ID NO: 286.

[0764] In some embodiments, the anti-TIM3 antibodies described herein bind to amino acid residues within the following region of the mature human TIM3 extracellular domain (SEQ ID NO: 290): CPVFECG (SEQ ID NO: 296), corresponding to amino acid residues 37-43 of the mature human TIM3 extracellular domain (SEQ ID NO: 290).

[0765] In some embodiments, the anti-TIM3 antibodies described herein bind to amino acid residues within the following region of the mature human TIM3 extracellular domain (SEQ ID NO: 290): WTSRYWLNGDFR (SEQ ID NO: 297), corresponding to amino acid residues 57-83 of the mature human TIM3 extracellular domain (SEQ ID NO: 290).

[0766] In some embodiments, the anti-TIM3 antibodies described herein bind to amino acid residues within the following regions of the mature human TIM3 extracellular domain (SEQ ID NO: 290): RIQIPGIMND (SEQ ID NO: 298), corresponding to amino acid residues 90 - 99 of the mature human TIM3 extracellular domain (SEQ ID NO: 290).

[0767] In some embodiments, the binding of the anti-TIM3 antibodies to wild-type and mutant human TIM3 has the same pattern as one or more of antibodies 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3 and TIM3.2 to TIM3.18. In some embodiments, the anti-TIM3 antibodies bind 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).

[0768] In some embodiments, the anti-TIM3 antibody binds (1) 49 VPVCWGKGACPVFE 62 (SEQ ID NO: 367) and

[0769] 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), as determined by HDX-MS, e.g., as described in the Examples. In some 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 between amino acid residues E62 and R111, and the region C-terminal to amino acid residue L127, as determined by HDX-MS, e.g., as described in the Examples.

[0770] In some embodiments, relative to binding wild-type human TIM3, the anti-TIM3 antibody has 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 as in SEQ ID NO:286 Figure 25 )) are replaced by another amino acid and the antibody binds to (1) 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), as determined by HDX-MS, e.g., as described in the Examples.

[0771] In some embodiments, the binding of the anti-TIM3 antibody to wild-type and mutant human TIM3 has a pattern similar to TIM3.18.IgG1.3 or 13A3, i.e., the antibody:

[0772] (i) binds to (1) 49 VPVCWGKGACPVFE 62(SEQ ID NO:367), 111 RIQIPGIMNDEKFNLKL 127 (SEQ ID NO:368), and 119 NDEKFNLKL 127 (SEQ ID NO:373), and, for example, but not significantly bind to (a) a peptide having a sequence located N-terminal to amino acid residue 49; (b) a peptide having a sequence located between amino acid residues 62 and 111 (e.g., 78 WTSRYWLNGDFRKGDVSL 95 (SEQ ID NO:371)); and (c) a peptide having a sequence located C-terminal to amino acid residue 127, as determined by HDX-MS (e.g., as described in the Examples);

[0773] (ii) cannot bind to such human TIM3 or binds to such human TIM3 significantly less, said human TIM3 having one or more of the following amino acid mutations, which are determined, for example, using yeast surface display methods (e.g., as described in the Examples): C58, P59, F61, E62, C63, R111, D120, and optionally D104 and Q113 (numbered according to SEQ ID NO:286 ( Figure 25 )); and / or

[0774] (iii) having a heavy chain variable region and / or a 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; numbered according to SEQ ID NO:286 ( Figure 25 ));

[0775] In some embodiments, the anti-TIM3 antibody comprises a heavy chain and a light chain, wherein the heavy chain is selected from SEQ ID NO: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 SEQ ID NO:29 - 33 and 453.

[0776] As further discussed herein, the heavy chain constant region of the anti-TIM3 antibodies described herein can be any isotype, e.g., IgG1, IgG2, IgG3, and IgG4, or combinations and / or modifications thereof. The anti-TIM3 antibodies can have effector functions or can have reduced or no effector functions. In some embodiments, the anti-TIM3 antibody comprises a modified heavy chain constant region that provides enhanced properties to the antibody.

[0777] In some embodiments, the anti-TIM3 antibody comprises a heavy chain and a light chain, wherein the heavy chain is selected from 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 SEQ ID NOs: 29-33 and 453.

[0778] III. Antibodies with Specific Germline Sequences

[0779] In some embodiments, the anti-TIM3 antibody comprises a heavy chain variable region from a specific germline heavy chain immunoglobulin gene and / or a light chain variable region from a specific germline light chain immunoglobulin gene.

[0780] As described herein, human antibodies specific for TIM3 have been prepared that comprise a heavy chain variable region that is a product of or derived from a 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 are isolated monoclonal antibodies or antigen-binding portions thereof that comprise a heavy chain variable region that is a product of or derived from a human VH germline gene selected from 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, VHJH4b, VH JH5b, VH JH6b, and any combination thereof.

[0781] Human antibodies specific for TIM3 have been prepared that comprise a light chain variable region that is a product of or derived from a human germline VKA27 gene, VK JK5 gene, VK JK4 gene, VK JK3, VK L18 gene, and / or VK JK1 gene. Accordingly, provided herein are isolated monoclonal antibodies or antigen-binding portions thereof that comprise a light chain variable region that is a product of or derived from a human VK germline gene selected from VK A27, VK JK5, VK JK4, VK JK3, VK L18, VK JK1, and any combination thereof.

[0782] The anti-TIM3 antibodies described herein include those antibodies that comprise a heavy chain variable region and also comprise a light chain variable region, wherein the heavy chain variable region is a product of or derived from one of the human germline VH genes listed above, and the light chain variable region is a product of or derived from one of the human germline VK genes listed above, as shown in the figures.

[0783] As used herein, a human antibody comprises a heavy chain variable region and a light chain variable region, and if the variable regions of the antibody are obtained from a system using human germline immunoglobulin genes, the heavy chain variable region and the light chain variable region are "products" of or "derived from" specific germline sequences. Such systems include immunizing transgenic mice carrying human immunoglobulin genes with an antigen of interest or screening a library of human immunoglobulin genes displayed on phage with an antigen of interest. A human antibody that is a "product" of or "derived from" a human germline immunoglobulin sequence can be identified by comparing the amino acid sequence of the human antibody with the amino acid sequences of human germline immunoglobulins and selecting the human germline immunoglobulin sequence that is most closely related (i.e., has the greatest % identity) in sequence to the sequence of the human antibody. A human antibody that is a "product" of or "derived from" a specific human germline immunoglobulin sequence can contain amino acids that are different from the germline sequence being compared, due to, for example, naturally occurring somatic mutations or intentionally introduced site-directed mutations. However, the selected human antibody is typically at least 90% identical in amino acid sequence to the amino acid sequence encoded by the human germline immunoglobulin gene and contains amino acid residues that identify the human antibody as human when compared to the amino acid sequences of germline immunoglobulins of other species (e.g., murine germline sequences). In some cases, the human antibody can be at least 95% or even 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 specific human germline sequence differs from the amino acid sequence encoded by the human germline immunoglobulin gene by no more than 10 amino acids. In certain cases, the human antibody can be shown to have no more than 5, or even no more than 4, 3, 2 or 1 amino acid difference compared to the amino acid sequence encoded by the germline immunoglobulin gene.

[0784] IV. Homologous Antibodies

[0785] Antibodies having heavy chain variable regions and light chain variable regions that comprise amino acid sequences homologous to the amino acid sequences of the anti-TIM3 antibodies described herein are encompassed herein, and wherein the antibodies retain the desired functional properties of the anti-TIM3 antibodies described herein.

[0786] For example, an isolated anti-TIM3 antibody or an antigen-binding portion thereof can comprise heavy chain variable regions and light chain variable regions, wherein:

[0787] (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 SEQ ID NOs: 34 - 40, 112 - 121, 364, 410 - 412, 449 and 456, or comprises 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 changes (i.e., amino acid substitutions, additions or deletions) relative to an amino acid sequence selected from 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;

[0788] (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 SEQ ID NOs: 60 - 63, 417 and 418, or comprises 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 changes (i.e., amino acid substitutions, additions or deletions) relative to an amino acid sequence selected from 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;

[0789] (c) The antibody specifically binds to human TIM3, and

[0790] (d) The antibody exhibits 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19 or all of the following functional characteristics:

[0791] (1) Binds to soluble human TIM3, e.g., with a K D 10 nM or less (e.g., 0.01 nM to 10 nM), e.g., as measured by Biacore, e.g., as described in the Examples;

[0792] (2) Binds to soluble cynomolgus monkey TIM3, e.g., with a K D 100 nM or less (e.g., 0.01 nM to 100 nM), e.g., as measured by Biacore, e.g., as described in the Examples;

[0793] (3) Binds to membrane - bound human TIM3, e.g., with an EC 501 ug / mL or less (e.g., 0.01 ug / mL to 1 ug / mL), e.g., as determined by flow cytometry (e.g., as described in the Examples);

[0794] (4) Bind to membrane-bound human TIM3, e.g., with a K D 1 nM or less (e.g., 0.01 nM to 10 nM), e.g., as determined by Scatchard analysis, e.g., as described in the Examples;

[0795] (5) Bind to membrane-bound cynomolgus TIM3, e.g., with an EC50 of 20 ug / mL or less (e.g., 0.01 ug / mL to 20 ug / mL), e.g., as determined by flow cytometry (e.g., as described in the Examples);

[0796] (6) Bind to membrane-bound cynomolgus TIM3, e.g., with a K D 1 nM or less (e.g., 0.01 nM to 10 nM), e.g., as determined by Scatchard analysis, e.g., as described in the Examples;

[0797] (7) Induce or enhance T cell activation (e.g., by blocking or reducing the inhibitory effect of TIM3), as demonstrated by (i) increased IFN-γ production in TIM3-expressing T cells (e.g., Th1 cells or TILs) and / or (ii) enhanced proliferation of TIM3-expressing T cells (e.g., Th1 cells or TILs), e.g., as described in the Examples;

[0798] (8) Stimulate T cell proliferation in a mixed lymphocyte reaction (MLR) assay, e.g., as described in the Examples;

[0799] (9) Inhibit the binding of phosphatidylserine to TIM3, e.g., as determined by a PS-hTIM3 "tandem" blocking assay, e.g., as described in the Examples;

[0800] (10) Do not internalize or downregulate cell surface TIM3 when bound to TIM3 on the cell;

[0801] (11) Bind to one of the following regions of the extracellular domain of human TIM3 (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), e.g., as described in the Examples;

[0802] (12) Having reduced binding to a 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 as in SEQ ID NO:286 Figure 25 )) are replaced by another amino acid, e.g., as described in the Examples;

[0803] (13) Competing 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 for binding to human TIM3 in one or both directions, e.g., as described in the Examples;

[0804] (14) Binding to the human TIM3 region 49 VPVCWGKGACPVFE 62 (SEQ ID NO:367) and 111 RIQIPGIMNDEKFNLKL 127 (SEQ ID NO:368), as determined by HDX-MS, e.g., as described in the Examples;

[0805] (15) Having a heavy chain variable region and / or a 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; numbered according to SEQ ID NO:286 Figure 25 ));

[0806] (16) (a) Having reduced binding to human TIM3 as compared to binding to wild-type human TIM3, wherein one, two, three, four, five, six, seven, eight, or nine amino acids C58, P59, F61, E62, C63, R111, D120, and optionally D104 and Q113 (numbered according to SEQ ID NO: 286 Figure 25 )) are replaced by another amino acid; (b) Binding to 49 VPVCWGKGACPVFE 62 (SEQ ID NO: 367), 111 RIQIPGIMNDEKFNLKL 127 (SEQ ID NO: 368) and 119 NDEKFNLKL 127( SEQ ID NO: 373), as determined by HDX-MS, as described in the examples; and / or (c) Competing or cross-blocking the binding of 13A3 or TIM3.18.IgG1.3 to human TIM3, e.g., as described in the examples;

[0807] (17) Binding to hTIM3-IgV with a K D 5x10 -8 M, 2x10 -8 M, 10 -8 M or 5x10 -9 M or less, as determined by the method described in Example 22;

[0808] (18) Binding to hTIM3-IgV with a K D 10 -7 M, 2x10 -8 M or 10 -8 M or less, as determined by the method described in Example 22;

[0809] (19) Binding to cynomolgus monkey TIM3-ECD with a K D 5x10 -7 M, 2x10 -8 M or 10 -8 M or less, as determined by the method described in Example 22; and / or

[0810] (20) Binding to hTIM3-ECD with a K D 8x10 -8 M, 5x10 -8 M or 10 -8 M or less, as determined by the method described in Example 22.

[0811] In some 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, or all of the functional characteristics listed in (1) to (20) above. The antibody can be, for example, a human antibody, a humanized antibody or a chimeric antibody.

[0812] The isolated anti-TIM3 antibody or antigen-binding portion thereof can comprise a heavy chain and a light chain, wherein:

[0813] (a) The heavy 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 SEQ ID NO: 1-28, 72-111, and 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 IgG1f and IgG1.3f), 451, 452, 461, 462 (TIM3.20HC IgG1f and IgG1.3f), 454, 455, 463, 464 (TIM3.21 HC IgG1f and IgG1.3f), 457, 458, 465, 466 (TIM3.22HC IgG1f and IgG1.3f), and 459, 460, 467, 468 (TIM3.23 HC IgG1f and IgG1.3f); or comprises 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 variations (i.e., amino acid substitutions, additions or deletions) relative to an amino acid sequence selected from SEQ ID NO: 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 (TIM3.23HC IgG1f and IgG1.3f), provided that, in some embodiments, if the sequence is a sequence of a heavy chain with no effector function, mutations that render the heavy chain with no effector function are not modified for the IgG1.1 constant region (e.g., R214, A234, E235, A237, S330 and S331 are not modified), and R214, A234 and E235 are not modified for the IgG1.3 constant region, wherein optionally, the heavy chain variable region comprises the CDR sequence of one of the anti-TIM3 antibodies described herein;

[0814] (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 SEQ ID NO: 29 - 33, 408, 409 and 453; or comprises 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 variations (i.e., amino acid substitutions, additions or deletions) relative to an amino acid sequence selected from SEQ ID NO: 29 - 33, 408, 409 and 453, wherein optionally, the variable region of the light chain comprises the CDR sequences of one of the anti - TIM3 antibodies described herein;

[0815] (c) The antibody specifically binds to human TIM3, and

[0816] (d) The antibody exhibits 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or all of the following functional characteristics:

[0817] (1) Binds to soluble human TIM3, e.g., with a K D 10 nM or less (e.g., 0.01 nM to 10 nM), e.g., as determined by Biacore, e.g., as described in the Examples;

[0818] (2) Binds to soluble cynomolgus TIM3, e.g., with a K D 100 nM or less (e.g., 0.01 nM to 100 nM), e.g., as determined by Biacore, e.g., as described in the Examples;

[0819] (3) Binds to membrane - bound human TIM3, e.g., with an EC 50 1 μg / mL or less (e.g., 0.01 μg / mL to 1 μg / mL), e.g., as determined by flow cytometry (e.g., as described in the Examples);

[0820] (4) Binds to membrane - bound human TIM3, e.g., with a K D 1 nM or less (e.g., 0.01 nM to 10 nM), e.g., as determined by Scatchard analysis, e.g., as described in the Examples;

[0821] (5) Binds to membrane - bound cynomolgus TIM3, e.g., with an EC50 of 20 μg / mL or less (e.g., 0.01 μg / mL to 20 μg / mL), e.g., as determined by flow cytometry (e.g., as described in the Examples);

[0822] (6)Bind to membrane-bound cynomolgus TIM3, for example, with a K D of 1 nM or less (e.g., 0.01 nM to 10 nM), for example, as determined by Scatchard analysis, for example, as described in the examples;

[0823] (7)Induce or enhance T cell activation (e.g., by blocking or reducing the inhibitory effect of TIM3), as demonstrated by (i) increased IFN-γ production in TIM3-expressing T cells (e.g., Th1 cells or TILs) and / or (ii) enhanced proliferation of TIM3-expressing T cells (e.g., Th1 cells or TILs), for example, as described in the examples;

[0824] (8)Stimulate T cell proliferation in a mixed lymphocyte reaction (MLR) assay, for example, as described in the examples;

[0825] (9)Inhibit the binding of phosphatidylserine to TIM3, for example, as determined by a PS-hTIM3 "tandem" blocking assay, for example, as described in the examples;

[0826] (10)Do not internalize or downregulate cell surface TIM3 when bound to TIM3 on the cell;

[0827] (11)Bind to one of the following regions of the extracellular domain of human TIM3 (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), for example, as described in the examples;

[0828] (12)Have reduced binding to a 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 as in SEQ ID NO: 286 Figure 25 )) are replaced by another amino acid, for example, as described in the examples;

[0829] (13) Antibodies comprising 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 compete for binding to human TIM3 in one or both directions, e.g., as described in the Examples;

[0830] (14) Binds to the human TIM3 region 49 VPVCWGKGACPVFE 62 (SEQ ID NO:367) and 111 RIQIPGIMNDEKFNLKL 127 (SEQ ID NO:368), as determined by HDX-MS, e.g., as described in the Examples;

[0831] (15) Having a heavy chain variable region and / or a 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; numbered according to SEQ ID NO:286( Figure 25 ));

[0832] (16) (a) Having reduced binding to human TIM3 in which 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids C58, P59, F61, E62, C63, R111, D120, and optionally D104 and Q113 (numbered according to SEQ ID NO:286( Figure 25 )) are replaced by another amino acid, relative to binding to wild-type human TIM3; (b) Binds to 49 VPVCWGKGACPVFE 62 (SEQ ID NO:367), 111 RIQIPGIMNDEKFNLKL 127 (SEQ ID NO:368) and 119 NDEKFNLKL 127((SEQ ID NO:373), as determined by HDX-MS, as described in the Examples; and / or (c) competes with or cross-blocks 13A3 or TIM3.18.IgG1.3 binding to human TIM3, e.g., as described in the Examples;

[0833] (17) with a K D 5x10 -8 M, 2x10 -8 M, 10 -8 M or 5x10 -9 M binds hTIM3-IgV, as determined by the method described in Example 22;

[0834] (18) with a K D 10 -7 M, 2x10 -8 M or 10 -8 M binds hTIM3-IgV, as determined by the method described in Example 22;

[0835] (19) with a K D 5x10 -7 M, 2x10 -8 M or 10 -8 M binds cynomolgus monkey TIM3-ECD, as determined by the method described in Example 22; and / or

[0836] (20) with a K D 8x10 -8 M, 5x10 -8 M or 10 -8 M binds hTIM3-ECD, as determined by the method described in Example 22.

[0837] Also provided are anti-TIM3 antibodies that comprise CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 that are different from those of 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3 or the corresponding CDRs of any of TIM3.2 to TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24, and TIM3.25, said differences being changes of 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, or 1-5 amino acids (i.e., amino acid substitutions, additions, or deletions). In some embodiments, relative to the corresponding sequences 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, the anti-TIM3 antibody comprises 1-5 amino acid changes in each CDR of 1, 2, 3, 4, 5, or 6 CDRs. In some embodiments, relative 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, the anti-TIM3 antibody comprises a total of 1-5 amino acid changes in all CDRs.

[0838] In some embodiments, the anti-TIM3 antibody comprises VH and VL CDRs that consist of those of 13A3, wherein one or more amino acids in one or more CDRs are those of one of the other anti-TIM3 antibodies disclosed herein.

[0839] For example, in some embodiments, the 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.

[0840] In some embodiments, the anti-TIM3 antibody comprises a VH CDR2 having one or more amino acid modifications relative to SIYYSGFTYYNPSLKS (SEQ ID NO:46), and may comprise, for example, the following degenerate sequences: X1IX2X3X4GX5X6X7X8YX9X10X 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; X10 is any amino acid, e.g., P, S, Q or D; X11 is any amino acid, e.g., S or K; X12 is any amino acid, e.g., L, F or V; X13 is any amino acid, e.g., K or Q; and X14 is any amino acid, e.g., S or G.

[0841] In some embodiments, the anti-TIM3 antibody comprises a VH CDR3 having one or more amino acid modifications relative to GGPYGDYAHWFDP (SEQ ID NO:53), and may comprise, for example, the following degenerate sequences:

[0842] 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 none; X2 is any amino acid, e.g., F, G, R or none; X3 is any amino acid, e.g., Y, M, I or none; X4 is any amino acid, e.g., G, S, V or none; X5 is any amino acid, e.g., G, T, R, S or none; X6 is any amino acid, e.g., G, S or none; X7 is any amino acid, e.g., M, N, W or none; X8 is any amino acid, e.g., Y, S, E, N or none; X9 is any amino acid, e.g., Y or none; X10 is any amino acid, e.g., F, P or Y; X11 is any amino acid, e.g., Y or F; X12 is any amino acid, e.g., G or none; X13 is any amino acid, e.g., D or none; X14 is any amino acid, e.g., Y or none; X15 is any amino acid, e.g., A or none; X16 is any amino acid, e.g., H or none; X17 is any amino acid, e.g., W or none; X18 is any amino acid, e.g., F, M or none; X19 is any amino acid, e.g., D or E; and X20 is any amino acid, e.g., P, I, V, Y or L.

[0843] In some 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.

[0844] In some 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.

[0845] In some embodiments, the anti-TIM3 antibody comprises a VL CDR3 having one or more amino acid modifications relative to QQYGSSPIT (SEQ ID NO:68) and may comprise, for example, the following degenerate sequences: 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; X6 is any amino acid, e.g., T or none.

[0846] Antibodies having VH and VL regions homologous to those of 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, e.g., SEQ ID NO:34-40, 112-121, 364 and 410-412, and SEQ ID NO:60-63, 417 and 418 respectively, or heavy and light chains homologous to those of SEQ ID NO:1-28, 72-111, 349-352, 388-391, 396-399 or 402-405, and SEQ ID NO:29-33, 408 or 409 respectively, or sequences having homology to the CDRs can be obtained by mutating (e.g., site-directed mutagenesis or PCR-mediated mutagenesis) nucleic acid molecules encoding SEQ ID NO:167-173 and / or SEQ ID NO:193-196 or SEQ ID NO:134-161, 430-437 and / or SEQ ID NO:162-166 and 442-444, and then testing the function retained by the encoded altered antibodies (i.e., the functions shown in (1) to (16) above) using the functional assays described herein.

[0847] V. Antibodies with conservative modifications

[0848] An anti-TIM3 antibody can comprise a heavy chain variable region containing CDR1, CDR2 and CDR3 sequences and a light chain variable region containing CDR1, CDR2 and CDR3 sequences, wherein one or more of these CDR sequences comprise specific amino acid sequences based on the anti-TIM3 antibodies 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 conservative modifications thereof, and wherein the antibody retains the functional properties of the anti-TIM3 antibodies described herein. Accordingly, an isolated anti-TIM3 antibody or antigen-binding portion thereof can comprise a heavy chain variable region containing CDR1, CDR2 and CDR3 sequences and a light chain variable region containing CDR1, CDR2 and CDR3 sequences, wherein:

[0849] (a) The CDR3 sequence of the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 53-59, 126-129, 414, 416 and 471 and conservative modified amino acid sequences thereof, said conservative modifications being, for example, 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;

[0850] (b) The CDR3 sequence of the light chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 68-71, 419 and 474 and conservative modified amino acid sequences thereof, said conservative modifications being, for example, 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;

[0851] (c) The antibody specifically binds to human TIM3, and

[0852] (d) The antibody exhibits 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or all of the following characteristics:

[0853] (1) Binds to soluble human TIM3, for example, with a K D of 10 nM or less (e.g., 0.01 nM to 10 nM), for example, as determined by Biacore, for example, as described in the examples;

[0854] (2) Binds to soluble cynomolgus TIM3, for example, with a K D of 100 nM or less (e.g., 0.01 nM to 100 nM), for example, as determined by Biacore, for example, as described in the examples;

[0855] (3) Binds to membrane-bound human TIM3, for example, with an EC 50 of 1 μg / mL or less (e.g., 0.01 μg / mL to 1 μg / mL), for example, as determined by flow cytometry (e.g., as described in the examples);

[0856] (4) Binds to membrane-bound human TIM3, for example, with a K D of 1 nM or less (e.g., 0.01 nM to 10 nM), for example, as determined by Scatchard analysis, for example, as described in the examples;

[0857] (5) Binds to membrane-bound cynomolgus TIM3, for example, with an EC 5020 ug / mL or less (e.g., 0.01 ug / mL to 20 ug / mL), e.g., as determined by flow cytometry (e.g., as described in the Examples);

[0858] (6) Bind to membrane-bound cynomolgus TIM3, e.g., with a K D 1 nM or less (e.g., 0.01 nM to 10 nM), e.g., as determined by Scatchard analysis, e.g., as described in the Examples;

[0859] (7) Induce or enhance T cell activation (e.g., by blocking or reducing the inhibitory effect of TIM3), as demonstrated by (i) increased IFN-γ production and / or (ii) enhanced proliferation in T cells expressing TIM3 (e.g., Th1 cells or TILs), e.g., as described in the Examples;

[0860] (8) Stimulate T cell proliferation in a mixed lymphocyte reaction (MLR) assay, e.g., as described in the Examples;

[0861] (9) Inhibit the binding of phosphatidylserine to TIM3, e.g., as determined by a PS-hTIM3 "tandem" blocking assay, e.g., as described in the Examples;

[0862] (10) Do not internalize or downregulate cell surface TIM3 when bound to TIM3 on the cell;

[0863] (11) Bind to one of the following regions of the extracellular domain of human TIM3 (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), e.g., as described in the Examples;

[0864] (12) Have reduced binding to a 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 as in SEQ ID NO: 286 Figure 25 )) are replaced by another amino acid, e.g., as described in the Examples;

[0865] (13) Antibodies comprising VH and VL domains of any one 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 compete for binding to human TIM3 in one or both directions, e.g., as described in the Examples;

[0866] (14) Binds to the human TIM3 region 49 VPVCWGKGACPVFE 62 (SEQ ID NO:367) and 111 RIQIPGIMNDEKFNLKL 127 (SEQ ID NO:368), as determined by HDX-MS, e.g., as described in the Examples;

[0867] (15) Having a heavy chain variable region and / or a 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; numbered according to SEQ ID NO:286( Figure 25 ));

[0868] (16) (a) Having reduced binding to human TIM3 in which 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids C58, P59, F61, E62, C63, R111, D120, and optionally D104 and Q113 (numbered according to SEQ ID NO:286( Figure 25 )) are replaced by another amino acid, relative to binding to wild-type human TIM3; (b) Binds to 49 VPVCWGKGACPVFE 62 (SEQ ID NO:367), 111 RIQIPGIMNDEKFNLKL 127 (SEQ ID NO:368) and 119 NDEKFNLKL 127((SEQ ID NO:373), as determined by HDX-MS, as described in the Examples; and / or (c) competes with or cross-blocks 13A3 or TIM3.18.IgG1.3 binding to human TIM3, e.g., as described in the Examples;

[0869] (17) With a K D 5x10 -8 M, 2x10 -8 M, 10 -8 M or 5x10 -9 M or less binds to hTIM3-IgV, as determined by the method described in Example 22;

[0870] (18) With a K D 10 -7 M, 2x10 -8 M or 10 -8 M or less binds to hTIM3-IgV, as determined by the method described in Example 22;

[0871] (19) With a K D 5x10 -7 M, 2x10 -8 M or 10 -8 M or less binds to cynomolgus TIM3-ECD, as determined by the method described in Example 22; and / or

[0872] (20) With a K D 8x10 -8 M, 5x10 -8 M or 10 -8 M or less binds to hTIM3-ECD, as determined by the method described in Example 22.

[0873] In some embodiments, the heavy chain variable region CDR2 sequence comprises an amino acid sequence selected from SEQ ID NO: 46-52, 122-125, 413, 415, and 470 and conservatively modified amino acid sequences thereof, said conservative modifications being, e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, or 1-5 conservative amino acid substitutions; and the light chain variable region CDR2 sequence comprises an amino acid sequence selected from SEQ ID NO: 66-67 and 473 and conservatively modified amino acid sequences thereof, said conservative modifications being, e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, or 1-5 conservative amino acid substitutions.

[0874] In some embodiments, the heavy chain variable region CDR1 sequence comprises an amino acid sequence selected from SEQ ID NOs: 41-45 and 469 and conservatively modified amino acid sequences thereof, such conservative modifications being, for example, 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, or 1-5 conservative amino acid substitutions; and the light chain variable region CDR1 sequence comprises an amino acid sequence selected from SEQ ID NOs: 64-65 and 472 and conservatively modified amino acid sequences thereof, such conservative modifications being, for example, 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, or 1-5 conservative amino acid substitutions.

[0875] In some 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, or all of the functional characteristics listed in (1) to (20) above. Such antibodies can be, for example, human antibodies, humanized antibodies or chimeric antibodies.

[0876] Conservative amino acid substitutions can be made in parts of the antibody other than or in addition to the CDRs. For example, conservative amino acid modifications can be made in the framework region or the Fc region. The variable region or the heavy or light chain can contain 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-50 conservative amino acid substitutions relative to the anti-TIM3 antibody sequences provided herein. In some embodiments, the anti-TIM3 antibody comprises a combination of conservative and non-conservative amino acid modifications.

[0877] VI. Antibodies that Bind to the Same Epitope or Compete for Binding

[0878] Antibodies that compete with one or more of the specific anti-TIM3 antibodies described herein (e.g., antibody 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) for binding to human TIM3 are also provided. Such competing antibodies can be identified in a standard TIM3 binding assay based on the ability of such competing antibodies to competitively inhibit the binding of one or more 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 human TIM3. In a standard TIM3 binding assay, 0.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24, and TIM3.25. 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 unlabeled first antibody are added, the plate is washed, a labeled second antibody is added, and the amount of label is measured. If an increase in the concentration of the unlabeled (first) antibody (also referred to as the "blocking antibody") inhibits the binding of the labeled (second) antibody, it can be said that the first antibody inhibits the second antibody from binding to the target on the plate, or it can be said that the first antibody competes with the second antibody for binding to the target. Additionally or alternatively, Biacore analysis can be used to evaluate the ability of the antibody to compete. The ability of the test antibody to inhibit the binding of the anti-TIM3 antibodies described herein to TIM3 demonstrates that the test antibody can compete with the said antibodies for binding to human TIM3.

[0879] Accordingly, provided herein are anti-TIM3 antibodies that inhibit the 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 inhibit their binding to human 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%, e.g., as determined by ELISA or FACS (e.g., using the assay methods described in the following paragraphs).

[0880] An exemplary competition experiment to determine, for example, whether a first antibody blocks the binding of a second antibody (i.e., "competes with it") can be carried out 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 seeded in a 96-well plate at a density of 10 5 cells per sample well. The plate is placed on ice and then unconjugated first antibody at a concentration range of 0 to 50 μg / mL (starting with a three-fold titration from the highest concentration of 50 μg / mL) is added. Unrelated IgG can be used as an isotype control for the first antibody and is added at the same concentration (starting with a three-fold titration from the highest concentration of 50 μg / mL). Samples pre-incubated with 50 μg / mL unlabeled second antibody can be included as a positive control for complete block (100% inhibition), and samples without antibody in the first incubation can be used as a negative control (no competition; 0% inhibition). After incubation for 30 minutes, without washing, labeled, e.g., biotinylated, second antibody is added at a concentration of 2 μg / mL per well. The samples are incubated on ice for an additional 30 minutes. Unbound antibody is removed by washing the cells with FACS buffer. The labeled second antibody bound to the cells is detected with a reagent that detects the label, such as PE-conjugated streptavidin (Invitrogen, catalog number S21388) for detecting biotin. Samples are acquired on a FACS Calibur FlowCytometer (BD, San Jose) and analyzed using software (Tree Star, Inc., Ashland, OR). Results can be expressed as % inhibition (i.e., subtracting the amount of label at each concentration divided by the amount of label obtained without the blocking antibody from 100%). Typically, then the same experiment is carried out conversely, i.e., the first antibody is the second antibody described and the second antibody is the first antibody described.

[0881] In some embodiments, an antibody blocks the binding of another antibody to a target (e.g., human TIM3 or a portion thereof) at least partially (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) or completely (100%), and regardless of whether inhibition occurs when one or the other antibody is the first antibody. When antibodies compete with each other in both ways (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), then the first antibody and the second antibody "mutually block" the binding to the target.

[0882] In some embodiments, the anti-TIM3 antibody binds to the same epitope as the anti-TIM3 antibodies 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), e.g., as determined by a given epitope mapping technique. Techniques for determining that an antibody binds to the "same epitope on TIM3" as the anti-TIM3 antibodies described herein include, for example, epitope mapping methods such as X-ray analysis of crystals of the antigen:antibody complex, which provides atomic resolution of the epitope. Other methods monitor the binding of the antibody to antigen fragments or mutant variants of the antigen, and a loss of binding due to modification of an amino acid residue within the antigen sequence is generally considered an indication of an epitope component (see Figure 25 ). Additionally, computer combinatorial methods for epitope mapping can also be used. The method can also depend on the ability of the antibody of interest to affinity isolate (in native three-dimensional form or denatured form) specific short peptides from a combinatorial phage display peptide library. The peptides are then regarded as guides to the epitope definition for the antibody used to screen the peptide library. Computer algorithms have also been developed for epitope mapping, which have been shown to map conformational discontinuous epitopes.

[0883] Antibodies that compete for binding or bind to the same epitope as the anti-TIM3 antibodies described herein can be identified by using methods known in the art. For example, mice can be immunized with the human TIM3 described herein, hybridomas can be generated, and the resulting monoclonal antibodies can be screened for their ability to compete for binding to human TIM3 with the antibodies described herein. Mice can also be immunized with smaller TIM3 fragments that contain the epitope to which the antibody binds. The epitope or the region containing the epitope can be mapped, 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 and thus have properties similar to the anti-TIM3 antibodies described herein. Using phage display, the heavy chain of the anti-TIM3 antibody is first paired with a (human) light chain repertoire to select TIM3-binding antibodies, and then the new light chain is paired with a (human) heavy chain repertoire to select (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 mutagenizing the cDNAs encoding the heavy and light chains of the antibody.

[0884] Alanine scanning mutagenesis as described by Cunningham and Wells (1989) Science 244:1081-1085 or some other form of point mutagenesis of amino acid residues in TIM3 can also be used to obtain TIM3 antibody binding characteristics.

[0885] The binding characteristics of an antibody can also be determined by evaluating the binding of a specific antibody to a peptide containing a fragment of TIM3, such as a non-denatured or denatured fragment. A series of overlapping peptides containing the TIM3 (e.g., human TIM3) sequence can be synthesized and screened for binding, e.g., in a direct ELISA, a competitive ELISA (where the ability of the peptide to prevent the antibody from binding to TIM3 that has been bound to the wells of a microtiter plate is evaluated), or screened for binding on a chip.

[0886] The binding characteristics of anti-TIM3 antibodies can also be obtained by MS-based protein footprinting, 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, the methods of which are hereby specifically incorporated by reference. FPOP can be performed, for example, as described in Hambley and Gross (2005) J. American Soc. Mass Spectrometry 16:2057, the methods of which are hereby specifically incorporated by reference.

[0887] The binding characteristics of anti-TIM3 antibodies can also be obtained by structural methods, such as X-ray crystallography (e.g., WO2005 / 044853), molecular modeling, and nuclear magnetic resonance (NMR) spectroscopy, including NMR determination of the H-D exchange rates of labile amide hydrogens in TIM3 when TIM3 is free and when bound to the antibody of interest in a complex (Zinn-Justin et al. (1992) Biochemistry 31, 11335-11347; Zinn-Justin et al. (1993) Biochemistry 32, 6884-6891).

[0888] Regarding X-ray crystallography, crystallization can be accomplished using any known method in the art (e.g., Giege et al. (1994) Acta Crystallogr. D50:339-350; McPherson (1990) Eur. J. Biochem. 189:1-23), 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. A protein preparation at a concentration of at least about 1 mg / mL or about 10 mg / mL to about 20 mg / mL is desired. Crystallization can be best achieved in a precipitant solution containing polyethylene glycol 1000-20000 (PEG; average molecular weight ranging from about 1000 to about 20000 Da), about 5000 to about 7000 Da or about 6000 Da at a concentration of about 10% to about 30% (w / v). It is also desirable to include a protein stabilizer such as glycerol at a concentration of about 0.5% to about 20%. Suitable salts in the precipitant solution, such as sodium chloride, lithium chloride, or sodium citrate, at a concentration ranging from about 1 mM to about 1000 mM, may also be desired. The precipitant is buffered to a pH of about 3.0 to about 5.0. The specific buffers useful in the precipitant solution can vary and are well known in the art (Scopes, Protein Purification: Principles and Practice, Third Edition, (1994) Springer-Verlag, New York). Examples of useful buffers include, but are not limited to, HEPES, Tris, MES, and acetate. Crystals can be grown over a wide range of temperatures, including 2°C, 4°C, 8°C, and 26°C.

[0889] Well-known X-ray diffraction techniques can be used to study antibody:antigen crystals, and refinement can be carried out 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, eds. H.W. Wyckoff et al., Academic Press; U.S. Patent Application Publication No. 2004 / 0014194), and BUSTER (Bricogne (1993) Acta Cryst. D49:37-60; Bricogne (1997) Meth. Enzymol. 276A:361-423, Carter &

[0890] Compiled by Sweet; Roversi et al. (2000) Acta Cryst. D56: 1313-1323), the disclosures of which are incorporated herein by reference in their entirety.

[0891] An anti-TIM3 antibody can bind to the same epitope as any anti-TIM3 antibody having the amino acid sequence described herein, as determined by epitope mapping techniques such as those described herein.

[0892] This document includes antibodies that bind to human TIM3 and optionally to cynomolgus TIM3, which are antibodies having binding characteristics similar to those of the anti-TIM3 antibodies described herein and are determined by one of the methods used in the examples.

[0893] In some embodiments, the anti-TIM3 antibodies described herein bind to the extracellular portion of human TIM3 (e.g., in the Ig-like domain or IgV domain in the extracellular region), i.e., the epitope in amino acids 22 to 130 of SEQ ID NO: 286 ( Figure 25 ), such as a conformational epitope. In some embodiments, the anti-TIM3 antibody binds to an epitope located in amino acids 22 to 120 of the extracellular domain of human TIM3 (SEQ ID NO: 286) or in amino acids 1-99 of mature human TIM3 (SEQ ID NO: 290) (see Examples). In some embodiments, the anti-TIM3 antibody binds to or binds to an epitope in the region consisting of amino acids 58-64 of human TIM3 having SEQ ID NO: 286, which corresponds to amino acid residues 37-43 of mature human TIM3 (CPVFECG, SEQ ID NO: 296; see Figure 25 ). In some embodiments, the anti-TIM3 antibody binds to or binds to an epitope in the region consisting of amino acids 111-120 of human TIM3 having SEQ ID NO: 286, which corresponds to amino acid residues 90-99 of mature human TIM3 (RIQIPGIMND, SEQ ID NO: 298; see Figure 25 ). In some embodiments, the anti-TIM3 antibody binds to or binds to an epitope in the region consisting of amino acids 58-64 (CPVFECG, SEQ ID NO: 296) of human TIM3 having SEQ ID NO: 286 and in the region consisting of amino acids 111-120 (RIQIPGIMND, SEQ ID NO: 298; see Figure 25Epitopes in the region composed of. In some embodiments, the anti-TIM3 antibody binds to or binds an epitope in the region composed of amino acids 78 - 89 (WTSRYWLNGDFR, SEQ ID NO:297; see Figure 25 ) of human TIM3 having SEQ ID NO:286, which corresponds to amino acid residues 57 - 83 of mature human TIM3.

[0894] In some embodiments, the anti-TIM3 antibody binds to an epitope substantially the same as 13A3. In some embodiments, the anti-TIM3 antibody binds an epitope (or region of human TIM3) comprising one or more of the amino acid residues C58, P59, F61, E62, C63, R111, and D120 of SEQ ID NO:286 ( Figure 25 ). In some embodiments, the anti-TIM3 antibody binds an epitope (or region of human TIM3) comprising one or more of the amino acid residues C58, P59, F61, E62, C63, D104, R111, Q113, and D120 of SEQ ID NO:286 ( Figure 25 ). In some embodiments, the anti-TIM3 antibody does not significantly bind or binds only with a significantly reduced affinity to a human TIM3 protein in which one or more of the amino acid residues C58, P59, F61, E62, C63, R111, and D120 of SEQ ID NO:286 are changed to another amino acid (e.g., by non-conservative amino acid substitution). In some embodiments, the anti-TIM3 antibody does not significantly bind or binds only with a significantly reduced affinity to a human TIM3 protein in which one or more of the amino acid residues C58, P59, F61, E62, C63, D104, R111, Q113, and D120 of SEQ ID NO:286 are changed to another amino acid (e.g., by non-conservative amino acid substitution).

[0895] In some embodiments, the anti-TIM3 antibody binds to an epitope substantially the same as 3G4. In some embodiments, the anti-TIM3 antibody binds an epitope (or region of human TIM3) comprising one or more of the amino acid residues C58, P59, V60, F61, E62, C63, G116, and M118 of SEQ ID NO:286 ( Figure 25 ). In some embodiments, the anti-TIM3 antibody binds an epitope (or region of human TIM3) comprising one or more of the amino acid residues C58, P59, V60, F61, E62, C63, D104, G116, and M118 of SEQ ID NO:286 ( Figure 25Epitopes (or regions of human TIM3). In some embodiments, the anti-TIM3 antibody does not significantly bind or binds only with a significantly reduced affinity to a human TIM3 protein in which one or more amino acid residues C58, P59, V60, F61, E62, C63, G116, and M118 of SEQ ID NO:286 are changed to another amino acid (e.g., by non-conservative amino acid substitution). In some embodiments, the anti-TIM3 antibody does not significantly bind or binds only with a significantly reduced affinity to a human TIM3 protein in which one or more amino acid residues C58, P59, V60, F61, E62, C63, D104, G116, and M118 of SEQ ID NO:286 are changed to another amino acid (e.g., by non-conservative amino acid substitution).

[0896] In some embodiments, the anti-TIM3 antibody binds to an epitope substantially the same as 17C3. In some embodiments, the anti-TIM3 antibody binds to an epitope (or region of human TIM3) comprising one or more amino acid residues C58, P59, V60, F61, E62, C63, G64, and G116 of SEQ ID NO:286( Figure 25 )Epitopes (or regions of human TIM3). In some embodiments, the anti-TIM3 antibody binds to an epitope (or region of human TIM3) comprising one or more amino acid residues C58, P59, V60, F61, E62, C63, G64, D104, and G116 of SEQ ID NO:286 Figure 25 )Epitopes (or regions of human TIM3). In some embodiments, the anti-TIM3 antibody does not significantly bind or binds only with a significantly reduced affinity to a human TIM3 protein in which one or more amino acid residues C58, P59, V60, F61, E62, C63, G64, and G116 of SEQ ID NO:286 are changed to another amino acid (e.g., by non-conservative amino acid substitution). In some embodiments, the anti-TIM3 antibody does not significantly bind or binds only with a significantly reduced affinity to a human TIM3 protein in which one or more amino acid residues C58, P59, V60, F61, E62, C63, G64, D104, and G116 of SEQ ID NO:286 are changed to another amino acid (e.g., by non-conservative amino acid substitution).

[0897] In some embodiments, the anti-TIM3 antibody binds to an epitope substantially the same as 8B9. In some embodiments, the anti-TIM3 antibody binds to an epitope (or region of human TIM3) comprising one or more amino acid residues L48, W78, S80, R81, W83, G86, D87, and R89 of SEQ ID NO:286 Figure 25) epitope (or region of human TIM3). In some embodiments, the anti-TIM3 antibody binds to one or more amino acid residues L48, W78, S80, R81, W83, L84, G86, D87, and R89 of SEQ ID NO:286( Figure 25 ) epitope (or region of human TIM3). In some embodiments, the anti-TIM3 antibody binds to an epitope substantially the same as that of 8B9. In some embodiments, the anti-TIM3 antibody binds to one or more amino acid residues L48, W78, S80, R81, W83, G86, D87, R89, and D104 of SEQ ID NO:286( Figure 25 ) epitope (or region of human TIM3). In some embodiments, the anti-TIM3 antibody does not bind significantly or binds only with a significantly reduced affinity to a human TIM3 protein in which one or more amino acid residues L48, W78, S80, R81, W83, G86, D87, and R89 of SEQ ID NO:286 are changed to another amino acid (e.g., by non-conservative amino acid substitution). In some embodiments, the anti-TIM3 antibody does not bind significantly or binds only with a significantly reduced affinity to a human TIM3 protein in which one or more amino acid residues L48, W78, S80, R81, W83, L84, G86, D87, and R89 of SEQ ID NO:286 are changed to another amino acid (e.g., by non-conservative amino acid substitution). In some embodiments, the anti-TIM3 antibody does not bind significantly or binds only with a significantly reduced affinity to a human TIM3 protein in which one or more amino acid residues L48, W78, S80, R81, W83, G86, D87, R89, and D104( Figure 25 )(e.g., by non-conservative amino acid substitution) are changed to another amino acid of the human TIM3 protein.

[0898] In some embodiments, the anti-TIM3 antibody competes (or inhibits binding) for binding to human TIM3 with an anti-TIM3 antibody comprising a CDR or variable region as described herein, wherein the anti-TIM3 antibody comprising a CDR or variable region as described herein is, for example, antibody 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 some embodiments, the anti-TIM3 antibody inhibits binding of antibody 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 some 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 the anti-TIM3 antibody to human TIM3 by at least 50%, 60%, 70%, 80%, 90%, or 100%. In some 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 the anti-TIM3 antibody to human TIM3 by at least 50%, 60%, 70%, 80%, 90%, or 100% (e.g., competes in both directions).

[0899] In some embodiments, the anti-TIM3 antibody competes with an anti-TIM3 antibody comprising a CDR or variable region of anti-TIM3 antibodies 13A3, 3G4, 17C3, 17C8, 9F6, 14H7, and 23B3 (and variants thereof) for binding to human TIM3. In some embodiments, the anti-TIM3 antibody does not compete with an anti-TIM3 antibody comprising a CDR or variable region of anti-TIM3 antibodies 8B9 and 8C4 (and variants thereof) for binding to human TIM3.

[0900] VII. Engineered and Modified Antibodies

[0901] Also provided are engineered and modified antibodies, which can be engineered using an antibody having one or more VH and / or VL sequences disclosed herein as a starting material to modify the antibody, and the modified antibody can have altered properties compared to the starting antibody. Antibodies can be engineered by modifying one or more residues in one or both variable regions (i.e., VH and / or VL), such as in one or more CDR regions and / or one or more framework regions. Additionally or alternatively, antibodies can be engineered by modifying residues in the constant region, for example, to alter the effector function of the antibody.

[0902] One type of variable region engineering that can be implemented is CDR grafting. Antibodies interact with target antigens mainly through amino acid residues located in six heavy chain complementarity determining regions and light chain complementarity determining regions (CDRs). Thus, the amino acid sequences in the CDRs differ more between individual antibodies than the sequences outside the CDRs. Since the CDR sequences are responsible for most antibody-antigen interactions, it is possible to construct an expression vector to express a recombinant antibody that mimics the properties of a particular naturally occurring antibody, wherein the expression vector comprises the CDR sequences from a particular naturally occurring antibody, and the CDR sequences are grafted onto the framework region sequences from a different antibody with different properties (see, for example, 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. U.S.A. 86:10029-10033; U.S. Patent No. 5,225,539 to Winter, and U.S. Patent No. 5,530,101 to Queen et al.;

[0903] 5,585,089; 5,693,762 and 6,180,370).

[0904] Accordingly, some embodiments described herein relate to isolated monoclonal antibodies or antigen-binding portions thereof, comprising a heavy-chain variable region and a light-chain variable region, the heavy-chain variable region comprising CDR1, CDR2, and CDR3 sequences comprising amino acid sequences selected from 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 the light-chain variable region comprising CDR1, CDR2, and CDR3 sequences comprising amino acid sequences selected from SEQ ID NOs: 64-65 and 472; SEQ ID NOs: 66-67 and 473; and SEQ ID NOs: 68-71, 419, and 474, respectively. Accordingly, such antibodies comprise the VH CDR sequences 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, but may comprise framework region sequences different from those of these antibodies.

[0905] Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, the germline DNA sequences of the human heavy-chain variable region and light-chain variable region genes can be found in the "VBase" human germline sequence database (available from the Internet at www.mrc-cpe.cam.ac.uk / vbase), and also in Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, I.M. et al. (1992) "The Repertoire of Human Germline V H Sequences Reveals about Fifty Groups of V H Segments with Different Hypervariable Loops" / . Mol. Biol. 227:776-798; and Cox, J.P.L. et al. (1994) "A Directory of Human Germ-line V HSegments Reveals a Strong Bias in their Usage" can be found in "Eur. J. Immunol. 24:827-836", the content of each of which is hereby incorporated by reference in its entirety for all purposes.

[0906] In some embodiments, the framework region sequences for the anti-TIM3 antibodies described herein are those that are structurally similar to the framework region sequences used for the anti-TIM3 antibodies described herein. The VH CDR1, 2, and 3 sequences and the VL CDR1, 2, and 3 sequences can be grafted onto framework regions that have the same sequences as those found in the germline immunoglobulin genes from which the framework region sequences are derived, or the CDR sequences can be grafted onto framework regions that contain one or more mutations compared to the germline sequences. For example, it has been found that in some cases it is beneficial to mutate residues within the framework region to maintain or enhance the antigen-binding ability of the antibody (see, e.g., U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762; and 6,180,370 to Queen et al.).

[0907] The engineered anti-TIM3 antibodies described herein include antibodies in which framework region residues within the VH and / or VL have been modified, e.g., to improve antibody properties, such as the mutation of amino acid 107 in 9F6. Generally, such framework region modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework region residues to the corresponding germline sequences. More specifically, an antibody that has undergone somatic mutation can contain framework region residues that are different from the germline sequences from which the antibody is derived. Such residues can be identified by comparing the antibody framework region sequence to the germline sequences from which the antibody is derived. To revert the framework region sequence to its germline configuration, the somatic mutations can be "backmutated" to the germline sequences by, for example, site-directed mutagenesis or PCR-mediated mutagenesis. The present invention also contemplates such "backmutated" antibodies. Another type of framework region modification involves mutating one or more residues within the framework region or even one or more residues within one or more CDR regions to remove T cell epitopes and thereby reduce the potential immunogenicity of the antibody. This method is also referred to as "deimmunization" and is described in more detail in U.S. Patent Publication No. 20030153043 to Carr et al.

[0908] Another type of variable region modification is to mutate amino acid residues within the VH and / or VL CDR1, CDR2, and / or CDR3 regions to improve one or more binding properties (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutations, and the effects on antibody binding or other desired functions can be evaluated in in vitro or in vivo assays as described herein and provided in the Examples. In some embodiments, conservative modifications (as described above) are introduced. The mutations can be amino acid substitutions, additions, or deletions. Moreover, generally no more than 1, 2, 3, 4, or 5 residues are altered in the CDR regions.

[0909] Accordingly, the present invention also provides an isolated anti-TIM3 monoclonal antibody or an antigen-binding portion thereof, comprising a heavy chain variable region containing:

[0910] (a) a VH CDR1 region that contains an amino acid sequence selected from SEQ ID NOs: 41-45 and 469, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions as compared to SEQ ID NOs: 41-45 and 469;

[0911] (b) a VH CDR2 region that contains an amino acid sequence selected from SEQ ID NOs: 46-52, 122-125, 413, 415, and 470, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions as compared to SEQ ID NOs: 46-52, 122-125, 413, 415, and 470;

[0912] (c) a VH CDR3 region that contains an amino acid sequence selected from SEQ ID NOs: 53-59, 126-129, 414, 416, and 471, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions as compared to SEQ ID NOs: 53-59, 126-129, 414, 416, and 471;

[0913] (d) a VL CDR1 region that contains an amino acid sequence selected from 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 as compared to SEQ ID NOs: 64-65 and 472;

[0914] (e) a VL CDR2 region that contains an amino acid sequence selected from SEQ ID NOs: 66-67 and 473, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions as compared to SEQ ID NOs: 66-67 and 473; and

[0915] (f) The VL CDR3 region, which contains an amino acid sequence selected from SEQ ID NOs: 68 - 71, 419, and 474, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions as compared to SEQ ID NOs: 68 - 71, 419, and 474.

[0916] Methionine residues in the CDRs of antibodies can be oxidized, leading to potential chemical degradation and thus reducing the efficacy of the antibody. Accordingly, the present invention also provides anti - TIM3 antibodies having one or more methionine residues replaced by amino acid residues that do not undergo oxidative degradation in the heavy - chain CDR and / or light - chain CDR. In some embodiments, the methionine residues in the CDRs of antibody 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 replaced with amino acid residues that do not undergo oxidative degradation.

[0917] Similarly, deamidation sites can be removed from anti - TIM3 antibodies, particularly in the CDRs.

[0918] The anti - TIM3 variable regions described herein can be linked (e.g., covalently linked or fused) to an Fc, such as IgG1, IgG2, IgG3, or IgG4 Fc, which can be any allotype or isoallotype, e.g., for IgG1: G1m, G1ml(a), G1m2(x), G1m3(f), G1m17(z); for IgG2: G2m, G2m23(n); for IgG3: G3m, G3m21(g1), G3m28(g5), G3ml l(b0), G3m5(b1), G3ml3(b3), G3ml4(b4), G3ml0(b5), G3ml5(s), G3ml6(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).

[0919] In some embodiments, the anti - TIM3 variable regions described herein are linked to an effectorless or substantially effectorless Fc, such as IgG1.

[0920] Generally, the variable regions described herein can be linked to an Fc comprising one or more modifications that typically alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cytotoxicity. In addition, the antibodies described herein can be chemically modified (e.g., one or more chemical moieties can be linked to the antibody) or modified to alter their glycosylation to change one or more functional properties of the antibody. Each of these embodiments will be described in further detail below. Residue numbering in the Fc region is according to the EU index of Kabat.

[0921] The Fc region comprises domains derived from immunoglobulin constant regions, including fragments, analogs, variants, mutants, or derivatives of the constant regions. Suitable immunoglobulins include IgG1, IgG2, IgG3, IgG4, and other classes such as IgA, IgD, IgE, and IgM. 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 the immunoglobulin and can individually or in combination include the CH1 domain, hinge, CH2 domain, CH3 domain, or CH4 domain.

[0922] Ig molecules interact with multiple classes of cellular receptors. For example, IgG molecules interact with three classes of Fcγ receptors (FcγR) specific for IgG class antibodies, namely FcγRI, FcγRII, and FcγRIII. It has been reported that the important sequences for IgG binding to FcγR receptors are located in the CH2 and CH3 domains. The serum half-life of an antibody is affected by the ability of the antibody to bind to an Fc receptor (FcR).

[0923] In some 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 parental Fc sequence (e.g., an unmodified Fc polypeptide that is subsequently modified to produce the variant) to provide desired structural features and / or biological activities.

[0924] Generally, variants of the constant region or portions thereof (e.g., CH1, CL, hinge, CH2, or CH3 domains) can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations and / or at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutation, or 1 - 10 or 1 - 5 mutations, or contain 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 necessary biological activity.

[0925] For example, one can make modifications in the Fc region to generate Fc variants that (a) increase or decrease antibody-dependent cell-mediated cytotoxicity (ADCC) relative to the parental Fc, (b) increase or decrease complement-mediated cytotoxicity (CDC), (c) increase or decrease the affinity for C1q, and / or (d) increase or decrease the affinity for Fc receptors. Such Fc region variants typically contain at least one amino acid modification in the Fc region. Combinatorial amino acid modifications are considered particularly desirable. For example, the variant Fc region can include two, three, four, five, etc. substitutions therein (e.g., at specific Fc region positions identified herein).

[0926] The variant Fc region can also contain sequence alterations where the 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 with the removal of cysteine residues, the single-chain Fc domain can still form a dimeric Fc domain that binds non-covalently together. In some embodiments, the Fc region can be modified to be more compatible with the selected host cell. For example, the PA sequence near the N-terminus of the typical native Fc region can be removed, which can be recognized by digestive enzymes such as proline iminopeptidase in E. coli. In some embodiments, one or more glycosylation sites in the Fc domain can be removed. Residues that are typically glycosylated (e.g., asparagine) can confer a cytolytic response. Such residues can be deleted or replaced with non-glycosylated residues (e.g., alanine). In some embodiments, sites that interact 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 some embodiments, sites that affect binding to Fc receptors can be removed, preferably sites other than the salvage receptor binding site. In some embodiments, the Fc region can be modified to remove the ADCC site. ADCC sites are known in the art. For the ADCC site in IgG1, see, for example, Molec. Immunol. 29(5):633-9 (1992). Specific examples of variant Fc domains are disclosed in, for example, WO 97 / 34631 and WO 96 / 32478.

[0927] In some embodiments, the hinge region of the Fc is modified such 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 No. 5,677,425 to Bodmer et al. Altering the number of cysteine residues in the hinge region of the Fc, e.g., 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 the mutant 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 such that the antibody has impaired Staphylococcus protein A (SpA) binding relative to the native Fc-hinge domain SpA binding. This method is further described in detail in U.S. Patent 6,165,745 to Ward et al.

[0928] 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 different amino acid residues such that the antibody has an altered affinity for an effector ligand while retaining the antigen-binding ability of the parental antibody. The effector ligand with altered affinity can be, for example, an Fc receptor or the C1 component of complement. This method is described in more detail in two U.S. Patent Nos. 5,624,821 and 5,648,260 to Winter et al.

[0929] In another example, one or more amino acids selected from amino acid residues 329, 331, and 322 can be replaced with different amino acid residues to cause the antibody to have altered C1q binding and / or reduced or eliminated complement-dependent cytotoxicity (CDC). This method is described in more detail in U.S. Patent No. 6,194,551 to Idusogie et al.

[0930] In another example, one or more amino acid residues at amino acid positions 231 and 239 are altered, thereby altering the ability of the antibody to fix complement. This method is further described in PCT Publication WO 94 / 29351 to Bodmer et al.

[0931] In yet another example, the Fc region can be modified by modifying one or more amino acids at the following positions to reduce antibody-dependent cell cytotoxicity (ADCC) and / or reduce the affinity for Fcγ receptors: 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. Exemplary substitutions include 236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D and 332E. Exemplary variants include 239D / 332E, 236A / 332E, 236A / 239D / 332E, 268F / 324T, 267E / 268F, 267E / 324T, and 267E / 268F7324T. Other modifications for enhancing FcγR and complement interactions include, but are not limited to, substituting 298A, 333A, 334A, 326A, 2471, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 305I, and 396L. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691.

[0932] Fc modifications that increase binding to Fcγ receptors include amino acid modifications at any one or more of 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, 324, 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 in the Fc region, wherein the numbering of residues in the Fc region is the numbering in the EU index (WO00 / 42072).

[0933] Other Fc modifications that can be made to 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), wherein the 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), wherein the numbering is according to the EU index. The Fc variant can comprise 236R / 328R. Other modifications for reducing FcγR and complement interactions include substituting 297A, 234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 331S, 220S, 226S, 229S, 238S, 233P, and 234V, and removing the glycosylation at position 297 by mutagenesis or enzymatic methods or by methods of 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.

[0934] Optionally, the Fc region may contain non-naturally occurring amino acid residues at additional and / or alternative positions known to those of skill in the art (see, e.g., U.S. Patent Nos. 5,624,821; 6,277,375; 6,737,056; 6,194,551; 7,317,091; 8,101,720; PCT Patent Publications WO 00 / 42072; WO 01 / 58957; WO 02 / 06919; WO 04 / 016750; WO 04 / 029207; WO 04 / 035752; WO 04 / 074455; WO 04 / 099249; WO 04 / 063351; WO05 / 070963; WO 05 / 040217, WO 05 / 092925 and WO 06 / 020114).

[0935] Fc variants that enhance the 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 some embodiments, the Fc variant provides a selectively enhanced affinity for FcγRIIb relative to one or more activating receptors. Modifications for altering the binding to FcγRIIb include one or more modifications at positions selected from positions 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327, 328, 330, 331, and 332 according to the EU index. Exemplary substitutions for enhancing 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. Exemplary substitutions include 235Y, 236D, 239D, 266M, 267E, 268D, 268E, 328F, 328W, and 328Y. Other Fc variants for enhancing binding to FcγRIIb include 235Y / 267E, 236D / 267E, 239D / 268D, 239D / 267E, 267E / 268D, 267E / 268E, and 267E / 328F.

[0936] The affinity and binding characteristics of the Fc region to its ligands can be determined by a variety of in vitro assays known in the art (biochemical or immunological based assays), including but not limited to equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)) or kinetic methods (e.g., BIACORE analysis), as well as 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 labels on one or more of the components being examined 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 kinetics can be found in Paul, W.E., Fundamental Immunology, 4th Edition, Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions.

[0937] In some embodiments, the antibody is modified to increase its biological half-life. A variety of methods are possible. For example, this can be accomplished by increasing the binding affinity of the Fc region for FcRn. For example, one or more of the following residues can be mutated: 252, 254, 256, 433, 435, 436, as described in U.S. Patent No. 6,277,375. Specific exemplary substitutions include one or more of the following: T252L, T254S, and / or T256F. Alternatively, to increase the biological half-life, the antibody can be altered in the CH1 or CL region, as described in U.S. Patent Nos. 5,869,046 and 6,121,022 to Presta et al., to incorporate a salvage receptor binding epitope derived from two loops of the CH2 domain of the Fc region of IgG. Other exemplary variants that increase binding to FcRn and / or improve pharmacokinetic properties include substitutions at positions 259, 308, 428, and 434, including, for example, 259I, 308F, 428L, 428M, 434S, 434I, 434F, 434Y, and 434X1. Other variants that increase Fc-FcRn binding 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, 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, 433R, 433S, 433I, 433P, 433Q, 434H, 434F, 434Y, 252Y / 254T / 256E, 433K / 434F / 436H, 308T / 309P / 311S (Dall Acqua et al., Journal of Immunology, 2002, 169:5171-5180, Dall'Acqua et al., 2006, Journal of Biological Chemistry 281:23514-23524). Other modifications for modulating FcRn binding are described in Yeung et al., 2010, J Immunol, 182:7663-7671.

[0938] In some embodiments, hybrid IgG isotypes with specific biological characteristics can be used. For example, an IgG1 / IgG3 hybrid variant can be constructed by replacing the IgG1 positions in the CH2 and / or CH3 regions with amino acids from IgG3 that are different at the positions of the two isotypes. Thus, a hybrid variant IgG antibody containing one or more substitutions (e.g., 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 422I, 435R, and 436F) can be constructed. In some embodiments described herein, an IgG1 / IgG2 hybrid variant can be constructed by replacing the IgG2 positions in the CH2 and / or CH3 regions with amino acids from IgG1 that are different at the positions of the two isotypes. Thus, a hybrid variant IgG antibody containing one or more substitutions, e.g., one or more of the following amino acid substitutions: 233E, 234L, 235L, -236G (referring to the insertion of glycine at position 236), and 327A, can be constructed.

[0939] In addition, the binding sites on human IgG1 to FcγRI, FcγRII, FcγRIII, and FcRn have been mapped, and variants with improved binding have been described (see Shields, R.L. 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. In addition, the following combination mutations have been shown to improve FcγRIII binding: T256A / S298A, S298A / E333A, S298A / K224A, and S298A / E333A / K334A, which have 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, which show the greatest increase in affinity for FcγRIIIa, a decrease in FcγRIIb binding, and strong cytotoxic activity in cynomolgus monkeys (Lazar et al., 2006). Introduction of the triple mutations into antibodies such as alemtuzumab (specific for CD52), trastuzumab (specific for HER2 / neu), rituximab (specific for CD20), and cetuximab (specific for EGFR) converts them to greatly enhanced in vitro ADCC activity, and the S239D / I332E varia...

Claims

1. An isolated antibody that binds to human T cell immunoglobulin and mucin domain-containing protein 3 (TIM3), comprising 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 according to claim 1, wherein the heavy chain CDR1 comprises SEQ ID NO:

45.

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

415.

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

64.

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

66.

6. The antibody according to 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. An isolated antibody that binds to human TIM3, comprising heavy chain CDR1, CDR2, and CDR3, and 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.

8. The antibody according to any one of claims 1 to 7, wherein: (a) the heavy chain CDR1, CDR2, and CDR3 comprise SEQ ID NO: 45, 413, 414, respectively, and the light chain CDR1, CDR2, and CDR3 comprise SEQ ID NO: 64, 66, 69, respectively; (b) the heavy chain CDR1, CDR2, and CDR3 comprise SEQ ID NO: 45, 415, 416, respectively, and the light chain CDR1, CDR2, and CDR3 comprise SEQ ID NO: 64, 66, 68, respectively; or (c) the heavy chain CDR1, CDR2, and CDR3 comprise SEQ ID NO: 45, 415, 416, respectively, and the light chain CDR1, CDR2, and CDR3 comprise SEQ ID NO: 64, 66, 419, respectively.

9. The antibody according to claim 8, wherein the heavy chain CDR1, CDR2, and CDR3 comprise SEQ ID NO: 45, 413, 414, respectively, and the light chain CDR1, CDR2, and CDR3 comprise SEQ ID NO: 64, 66, 69, respectively.

10. The antibody according to claim 8, wherein the heavy chain CDR1, CDR2, and CDR3 comprise SEQ ID NO: 45, 415, and 416, respectively, and the light chain CDR1, CDR2, and CDR3 comprise SEQ ID NO: 64, 66, and 68, respectively.

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