Anti-TIM3 antibodies and uses thereof

Abstract

Provided herein are antibodies or antigen binding portions thereof that bind to T cell immunoglobulins and mucin domain-containing protein 3 (TIM3) proteins. 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 that produce antibodies or antigen binding portions thereof, polynucleotides encoding the heavy chain region and / or light chain region of the antibodies or antigen binding portions thereof, and vectors comprising polynucleotides encoding the heavy chain region and / or light chain region of the antibodies or antigen binding portions thereof.

Description

[0001] References to sequence lists submitted electronically via EFS-WEB

[0002] The contents of the sequence list in the ASCII text file (name: 3338_098PC01_SequenceListing.txt; size: 912,981 bytes; creation date: January 11, 2019) submitted electronically with this application are incorporated herein by reference in their entirety. Background of the Invention

[0004] T-cell immunoglobulin and mucin-domain containing protein 3 (TIM3), also known as hepatitis A virus cell receptor 2 (HAVCR2), is a type I transmembrane protein that functions as a key regulator of the immune response. TIM3 is initially detected on activated IFN-γ-producing T cells (e.g., type 1 helper CD4+). + T cells and cytotoxic CD8 + It has been identified on T cells and shown to induce T cell death or consumption upon binding to galactagogue 9. Recent studies have also shown that TIM3 expression is important in regulating the activity of many innate immune cells, such as macrophages, monocytes, dendritic cells, mast cells, and natural killer cells. See Han G et al., Front Immunol. 4:449 (2013).

[0005] Like many inhibitory receptors (such as PD-1 and CTLA-4), TIM3 expression is associated with a variety of chronic diseases, including cancer. TIM3 has been detected in patients with advanced melanoma, non-small cell lung cancer, or follicular B-cell non-Hodgkin lymphoma. + T cells. And TIM3 + The presence of regulatory T cells has been described as an effective indicator of lung cancer progression. See Anderson AC, Cancer Immunol Res. 2:393-8 (2014).

[0006] Several potential ligands for TIM3 have been identified: galactagogue-9, HMGB1, brain signaling protein-4A, CEACAM-1, ILT-4, and phosphatidylserine (PtdSer or PS). PS is an important cell membrane component and is normally located in the inner lobules of the cell membrane. However, with apoptosis, PS is redistributed and exposed to 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).

[0007] 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 exhibit more dysfunction compared to T cells that express only PD-1 or TIM3. See Fourcade J et al., J Exp Med. 207:2175-2186 (2010).

[0008] Therefore, there is a great need for drugs that target TIM3 and methods for using such drugs in designing new cancer immunotherapies and improving traditional cancer immunotherapies. Invention Overview

[0010] This document provides isolated antibodies, such as monoclonal antibodies or their antigen-binding moieties, particularly human (e.g., monoclonal) antibodies, that specifically bind to human T-cell immunoglobulins and protein 3 (TIM3) containing a mucin domain (“anti-TIM3 antibody”), and comprise heavy chains CDR1, CDR2, and CDR3 and light chains 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.

[0011] In some embodiments, the anti-TIM3 antibody or its antigen-binding moiety comprises heavy chains CDR1, CDR2, and CDR3, and light chains CDR1, CDR2, and CDR3, wherein:

[0012] (a) Heavy chain CDR1 contains SEQ ID NO:45;

[0013] (b) Heavy chain CDR2 contains SEQ ID NO:413 or SEQ ID NO:415;

[0014] (c) Heavy chain CDR3 contains SEQ ID NO:414 or SEQ ID NO:416;

[0015] (d) Light chain CDR1 contains SEQ ID NO:64;

[0016] (e) The light chain CDR2 contains SEQ ID NO:66; and

[0017] (f) The light chain CDR3 contains SEQ ID NO:69, SEQ ID NO:68 or SEQ ID NO:419.

[0018] In some implementations, (a) the heavy chains CDR1, CDR2, and CDR3 contain SEQ ID NO:45, 413, and 414, respectively, and the light chains CDR1, CDR2, and CDR3 contain SEQ ID NO:64, 66, and 69, respectively; (b) the heavy chains CDR1, CDR2, and CDR3 contain SEQ ID NO:45, 415, and 416, respectively, and the light chains CDR1, CDR2, and CDR3 contain SEQ ID NO:64, 66, and 68, respectively; or (c) the heavy chains CDR1, CDR2, and CDR3 contain SEQ ID NO:45, 415, and 416, respectively, and the light chains CDR1, CDR2, and CDR3 contain SEQ ID NO:64, 66, and 419, respectively.

[0019] In some embodiments, the heavy chains CDR1, CDR2, and CDR3 comprise SEQ ID NO:45, 413, and 414, respectively, and the light chains CDR1, CDR2, and CDR3 comprise SEQ ID NO:64, 66, and 69, respectively. In some embodiments, the heavy chains CDR1, CDR2, and CDR3 comprise SEQ ID NO:45, 415, and 416, respectively, and the light chains CDR1, CDR2, and CDR3 comprise SEQ ID NO:64, 66, and 68, respectively. In some embodiments, the heavy chains CDR1, CDR2, and CDR3 comprise SEQ ID NO:45, 415, and 416, respectively, and the light chains CDR1, CDR2, and CDR3 comprise SEQ ID NO:64, 66, and 419, respectively.

[0020] In some embodiments, the anti-TIM3 antibody or its antigen-binding portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH contains 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 shown in SEQ ID NO:410.

[0021] In some embodiments, the anti-TIM3 antibody or its antigen-binding portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VL contains 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 shown in SEQ ID NO:411.

[0022] In some embodiments, the anti-TIM3 antibody or its antigen-binding portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VL contains 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 shown in SEQ ID NO:412.

[0023] In some embodiments, the anti-TIM3 antibody or its antigen-binding moiety cross-competes with a reference antibody for binding to TIM3, comprising:

[0024] (a) Containing the heavy chain variable region (VH) of SEQ ID NO:410 and the light chain variable region (VL) of SEQ ID NO:417;

[0025] (b) A heavy chain variable region (VH) containing SEQ ID NO:411 and a light chain variable region (VL) containing SEQ ID NO:60;

[0026] (c) Containing the heavy chain variable region (VH) of SEQ ID NO:411 and the light chain variable region (VL) of SEQ ID NO:418; or

[0027] (d) A heavy chain variable region (VH) containing SEQ ID NO:412 and a light chain variable region (VL) containing SEQ ID NO:60.

[0028] In some embodiments, the anti-TIM3 antibody or its antigen-binding portion comprises:

[0029] (a) Heavy chain CDR1, CDR2 and CDR3 containing the heavy chain variable region (VH) of SEQ ID NO:410 and light chain CDR1, CDR2 and CDR3 containing the light chain variable region (VL) of SEQ ID NO:417;

[0030] (b) Heavy chain CDR1, CDR2 and CDR3 containing the heavy chain variable region (VH) of SEQ ID NO:411 and light chain CDR1, CDR2 and CDR3 containing the light chain variable region (VL) of SEQ ID NO:60;

[0031] (c) Heavy chain CDR1, CDR2, and CDR3 containing the heavy chain variable region (VH) of SEQ ID NO:411 and light chain CDR1, CDR2, and CDR3 containing the light chain variable region (VL) of SEQ ID NO:418; or

[0032] (d) Heavy chain CDR1, CDR2 and CDR3 containing the heavy chain variable region (VH) of SEQ ID NO:412 and light chain CDR1, CDR2 and CDR3 containing the light chain variable region (VL) of SEQ ID NO:60.

[0033] In some embodiments, the anti-TIM3 antibody or its antigen-binding moiety binds to the same epitope as a reference antibody, as determined by HDX, and the anti-TIM3 antibody or its antigen-binding moiety comprises:

[0034] (a) Containing the heavy chain variable region (VH) of SEQ ID NO:410 and the light chain variable region (VL) of SEQ ID NO:417;

[0035] (b) A heavy chain variable region (VH) containing SEQ ID NO:411 and a light chain variable region (VL) containing SEQ ID NO:60;

[0036] (c) Containing the heavy chain variable region (VH) of SEQ ID NO:411 and the light chain variable region (VL) of SEQ ID NO:418; or

[0037] (d) The heavy chain variable region (VH) containing SEQ ID NO:412 and the light chain variable region (VL) containing SEQ ID NO:60.

[0038] In some embodiments, the anti-TIM3 antibody or its antigen-binding portion comprises:

[0039] (a) Heavy chain CDR1, CDR2 and CDR3 containing the heavy chain variable region (VH) of SEQ ID NO:410 and light chain CDR1, CDR2 and CDR3 containing the light chain variable region (VL) of SEQ ID NO:417;

[0040] (b) Heavy chain CDR1, CDR2 and CDR3 containing the heavy chain variable region (VH) of SEQ ID NO:411 and light chain CDR1, CDR2 and CDR3 containing the light chain variable region (VL) of SEQ ID NO:60;

[0041] (c) Heavy chain CDR1, CDR2, and CDR3 containing the heavy chain variable region (VH) of SEQ ID NO:411 and light chain CDR1, CDR2, and CDR3 containing the light chain variable region (VL) of SEQ ID NO:418; or

[0042] (d) Heavy chain CDR1, CDR2 and CDR3 containing the heavy chain variable region (VH) of SEQ ID NO:412 and light chain CDR1, CDR2 and CDR3 containing the light chain variable region (VL) of SEQ ID NO:60.

[0043] In some embodiments, the anti-TIM3 antibody or its antigen-binding portion comprises VH and 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.

[0044] In some embodiments, the anti-TIM3 antibody disclosed herein comprises heavy chains CDR1, CDR2, and CDR3, and light chains CDR1, CDR2, and CDR3, wherein: (a) heavy chain CDR1 comprises SEQ ID NO:469; (b) heavy chain CDR2 comprises SEQ ID NO:470; and (c) heavy chain CDR3 comprises SEQ ID NO:471. In some embodiments, (a) light chain CDR1 comprises SEQ ID NO:472; (b) light chain CDR2 comprises SEQ ID NO:473; and (c) light chain CDR3 comprises SEQ ID NO:474.

[0045] 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.

[0046] 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.

[0047] In some embodiments, the anti-TIM3 antibody or its antigen-binding portion comprises

[0048] (a1) Contains heavy chain and light chain sequences of SEQ ID NO:386 (or 387) and 408, respectively;

[0049] (a2) Contains heavy chain and light chain sequences of SEQ ID NO:388 (or 389) and 408, respectively;

[0050] (a3) Contains heavy chain and light chain sequences of SEQ ID NO:390 (or 391) and 408, respectively;

[0051] (a4) Contains heavy chain and light chain sequences of SEQ ID NO:392 (or 393) and 408, respectively;

[0052] (b1) Contains heavy chain and light chain sequences of SEQ ID NO:394 (or 395) and 29, respectively;

[0053] (b2) Contains heavy chain and light chain sequences of SEQ ID NO:394 (or 395) and 409, respectively;

[0054] (b3) Contains heavy chain and light chain sequences of SEQ ID NO:396 (or 397) and 29, respectively;

[0055] (b4) Contains heavy chain and light chain sequences of SEQ ID NO:398 (or 399) and 29, respectively;

[0056] (b5) Contains heavy chain and light chain sequences of SEQ ID NO:400 (or 401) and 29, respectively;

[0057] (b6) Contains heavy chain and light chain sequences of SEQ ID NO:402 (or 403) and 29, respectively;

[0058] (b7) Contains heavy chain and light chain sequences of SEQ ID NO:404 (or 405) and 29, respectively;

[0059] (b8) Contains heavy chain and light chain sequences of SEQ ID NO:406 (or 407) and 29, respectively;

[0060] (c1) Contains heavy chain and light chain sequences of SEQ ID NO:451 (or 461) and 453, respectively;

[0061] (c2) Contains heavy chain and light chain sequences of SEQ ID NO:452 (or 462) and 453, respectively;

[0062] (d1) Contains heavy and light chain sequences of SEQ ID NO:457 (or 465) and 453, respectively; or

[0063] (d2) Contains heavy chain and light chain sequences of SEQ ID NO:458 (or 466) and 453, respectively.

[0064] In some embodiments, the anti-TIM3 antibody or its antigen-binding portion has one or more of the following characteristics:

[0065] (1) Binding to membrane-bound human TIM3, for example, EC 50 1 μg / mL or less (e.g., 0.01 μg / mL to 0.05 μg / mL), for example, as determined by flow cytometry;

[0066] (2) Binding to membrane-bound cyno TIM3, for example, as determined by flow cytometry;

[0067] (3) K D 5x10 -8 M, 2x10 -8 M, 10 -8 M or 5x10 -9 M or smaller binds to hTIM3-IgV, as determined by the method described in Example 22;

[0068] (4) K D 10 -7 M, 2x10 -8 M or 10 -8 M or smaller binds to hTIM3-IgV, as determined by the method described in Example 22;

[0069] (5) K D 5x10 -7 M, 2x10 -8 M or 10 -8M or smaller bound to cynomolgus monkey TIM3-ECD, as determined by the method described in Example 22;

[0070] (6) K D 8x10 -8 M, 5x10 -8 M or 10 -8 M or smaller binds to hTIM3-ECD, as determined by the method described in Example 22;

[0071] (7) (e.g., by blocking or reducing the inhibitory effect of TIM3) induce or enhance T cell activation, as demonstrated by enhanced proliferation of T cells expressing TIM3 (e.g., Th1 cells or TILs); and / or

[0072] (8) Antibodies containing any of the following VH and VL molecules: 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 compete for binding to human TIM3 in one or both directions.

[0073] In some embodiments, the anti-TIM3 antibody comprises: (i) VHCDR1, CDR2, and CDR3 of SEQ ID NO:45, 415, and 416, respectively, and VL CDR1, CDR2, and CDR3 of SEQ ID NO:64, 66, and 68, respectively; or (ii) VH of SEQ ID NO:412 and VL of SEQ ID NO:60; wherein the antibody is in KD 5x10 -9 M or smaller binding to human TIM3-IgV; with KD 10 -7 M or smaller combined with cyno TIM3-IgV; and / or with KD 5x10 -6 M or smaller binds to cynoTIM3-ECD.

[0074] In some embodiments, the anti-TIM3 antibody comprises: (i) VHCDR1, CDR2, and CDR3 of SEQ ID NO:45, 413, and 414, respectively, and VL CDR1, CDR2, and CDR3 of SEQ ID NO:64, 66, and 69, respectively; or (ii) VH of SEQ ID NO:410 and VL of SEQ ID NO:417; wherein the antibody is in KD 5x10 -8M or smaller bound to human TIM3-IgV; with KD5x10 -9 M or smaller combined with cyno TIM3-IgV; and / or with KD 5x10 -9 M or smaller binds to cynoTIM3-ECD.

[0075] In some embodiments, the heavy chain of the anti-TIM3 antibody disclosed herein contains a C-terminal lysine residue. In other embodiments, the heavy chain of the anti-TIM3 antibody does not contain a C-terminal lysine residue.

[0076] This document provides a bispecific molecule comprising an anti-TIM3 antibody or its antigen-binding moiety linked to a molecule having a second binding specificity.

[0077] This article provides nucleic acids encoding VH and / or VL of an anti-TIM3 antibody or its antigen-binding moiety, an expression vector containing the nucleic acid, and cells transformed with the expression vector.

[0078] This article provides immunoconjugates comprising the anti-TIM3 antibody or its antigen-binding moiety described herein linked to a reagent.

[0079] This document provides compositions comprising the anti-TIM3 antibody described herein or its antigen-binding moiety, a bispecific molecule or immunoconjugate, and a carrier. A kit comprising the anti-TIM3 antibody described herein or its antigen-binding moiety, a bispecific molecule or immunoconjugate, and instructions for use are also provided.

[0080] This article provides a method for preparing an anti-TIM3 antibody or its antigen-binding moiety thereof, the method comprising expressing the anti-TIM3 antibody or its antigen-binding moiety thereof in cells, and isolating the antibody or its antigen-binding moiety thereof from the cells.

[0081] This article provides a method for stimulating an antigen-specific T cell response, which includes contacting T cells with an anti-TIM3 antibody or its antigen-binding moiety, a bispecific molecule or an immunoconjugate as described herein, thereby stimulating an antigen-specific T cell response (e.g., by inhibiting the negative effects of TIM3 on cells such as T cells).

[0082] This article provides a method for activating or co-stimulating T cells (e.g., effector T cells, such as Th1 cells) by contacting cells (e.g., effector T cells) with an anti-TIM3 antibody or its antigen-binding moiety, a bispecific molecule or immunoconjugate as described herein, and CD3, wherein (e.g., by inhibiting the negative effects of TIM3 on cells (e.g., T cells)) the effector T cells are activated or co-stimulated.

[0083] This article provides a method for increasing IFN-γ production and / or proliferation of T cells, such as Th1 cells or TILs, which includes contacting T cells with an effective amount of the anti-TIM3 antibody or antigen-binding moiety, bispecific molecule or immunoconjugate described herein.

[0084] This article provides a method for increasing IFN-γ production by T cells in subjects, which includes administering to subjects an effective amount of the anti-TIM3 antibody described herein or its antigen-binding moiety, bispecific molecule or immunoconjugate to increase IFN-γ production from T cells.

[0085] This article provides a method for stimulating TIL activity in a subject, the method comprising administering to the subject a therapeutically effective amount of the anti-TIM3 antibody described herein or its antigen-binding portion thereof to induce TIL proliferation or secretion of cytokines, such as IFN-γ.

[0086] This article 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), comprising administering to the subject an effective amount of the anti-TIM3 antibody described herein or its antigen-binding moiety, bispecific molecule, or immunoconjugate thereof. For example, the anti-TIM3 antibody described herein can increase IL-12 secretion by antigen-presenting cells in contact with the TIM3 antibody.

[0087] This article provides a method for stimulating an immune response in a subject, the method comprising administering to the subject an anti-TIM3 antibody or its antigen-binding moiety, a bispecific molecule, or an immune conjugate as described herein, thereby stimulating an immune response in the subject. In some embodiments, the subject has a tumor, and an immune response against the tumor is stimulated.

[0088] This article provides a method for inhibiting tumor growth or reducing tumor size in subjects, which includes administering the anti-TIM3 antibody described herein or its antigen-binding moiety, bispecific molecule or immune conjugate to the subject to inhibit tumor growth in the subject.

[0089] This document provides methods for treating cancer, for example, through immunotherapy, comprising administering a therapeutically effective amount of the anti-TIM3 antibody described herein or its antigen-binding moiety, bispecific molecule, or immunoconjugate to a subject in need 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-related cancers, and any combination thereof. In some embodiments, the cancer is metastatic, refractory, or recurrent. In some embodiments, the cancer is a cold tumor.

[0090] In some embodiments, the methods described herein further include administering one or more additional therapeutic agents, such as anti-PD-1 antibodies, anti-LAG-3 antibodies, anti-CTLA-4 antibodies, anti-GITR antibodies, and / or anti-PD-L1 antibodies, in combination with anti-TIM3 antibodies.

[0091] This article provides a method for detecting the presence of TIM3 protein in a sample, which includes contacting the sample with an anti-TIM3 antibody or its antigen-binding portion under conditions that allow the formation of a complex between an antibody or its antigen-binding portion and TIM3, and detecting the formation of the complex.

[0092] Implementation Plan

[0093] Implementation Scheme 1. An isolated antibody (e.g., a human antibody) or its antigen-binding moiety, which binds to human T-cell immunoglobulin and protein 3 (TIM3) containing a mucin domain and exhibits the following properties:

[0094] (a) Binds to soluble human TIM3;

[0095] (b) Human TIM3 bound to the membrane;

[0096] (c) Inducing or enhancing T cell activation; and optionally:

[0097] (d) Binding to soluble cynomolgus monkey TIM3; and

[0098] (e) Binding membrane cynomolgus monkey TIM3.

[0099] Implementation Scheme 2. An antibody or its antigen-binding portion according to Implementation Scheme 1, wherein the antibody stimulates an antitumor immune response.

[0100] Implementation Scheme 3. An antibody or its antigen-binding portion according to Implementation Scheme 1 or 2, wherein the antibody stimulates an antigen-specific T cell response.

[0101] Implementation Scheme 4. An antibody or its antigen-binding portion according to any of the foregoing embodiments, wherein the antibody increases IFN-γ production in T cells expressing TIM3.

[0102] Implementation Scheme 5. An antibody or its antigen-binding portion according to any of the foregoing embodiments, wherein the antibody increases T cell proliferation.

[0103] Implementation Scheme 6. An antibody or its antigen-binding portion according to any of the foregoing embodiments, wherein the antibody does not bind to an Fc receptor or wherein the antibody lacks effector function.

[0104] Implementation Scheme 7. An antibody or its antigen-binding portion according to any of the foregoing embodiments, wherein the antibody is K D 10 nM or less of bound soluble human TIM3, as determined by Biacore.

[0105] Implementation Scheme 8. An antibody or its antigen-binding portion according to any of the foregoing embodiments, wherein the antibody is K D 100 nM or less binds to soluble cynomolgus monkey TIM3, as determined by Biacore.

[0106] Implementation Scheme 9. An antibody or antigen-binding fragment thereof according to any of the foregoing embodiments, wherein the antibody is an antagonistic antibody that inhibits signal transduction of negative cells (e.g., T cells) via TIM3.

[0107] Implementation Scheme 10. An antibody or its antigen-binding portion according to any of the foregoing embodiments, wherein the antibody is EC 50 Human TIM3 bound to the membrane at concentrations of 0.1 or 1 μg / mL or less, as determined by flow cytometry.

[0108] Implementation Scheme 11. An antibody or its antigen-binding portion according to any of the foregoing embodiments, wherein the antibody is K D Human TIM3 with a binding membrane of 1 nM or less, as determined by Scatchard analysis.

[0109] Implementation Scheme 12. An antibody or its antigen-binding portion according to any of the foregoing embodiments, wherein the antibody is EC 50 1 μg / mL or less of membrane-bound cynomolgus monkey TIM3, as determined by flow cytometry; or said antibody in K D Cyno TIM3 bound to a membrane at a concentration of 1 nM or less, as determined by Scatchard analysis.

[0110] Implementation Scheme 13. An antibody or antigen-binding portion thereof according to any of the foregoing embodiments, wherein the antibody or antigen-binding portion thereof comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3, wherein the heavy chain CDR3 comprises an amino acid sequence selected from 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.

[0111] Implementation Scheme 14. The antibody or its antigen-binding portion according to Implementation Scheme 13, wherein the heavy chain CDR1 comprises X1, X2, X3, X4, Y, X5 and X6, wherein X1 is S or absent, X2 is R or absent, X3 is S, R or D, X4 is Y or H, X5 is W or M, and X6 is G, N, S or H.

[0112] Implementation Scheme 15. An antibody or its antigen-binding portion according to Implementation Scheme 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.

[0113] Implementation Scheme 16. An antibody or its antigen-binding portion according to Implementation Scheme 13 or 14, wherein the heavy chain CDR1 comprises R, X1, Y, W and X2, and wherein X1 is H or Y and X2 are N or S.

[0114] Implementation Scheme 17. An antibody or its antigen-binding portion according to any one of Implementation Schemes 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 none, 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.

[0115] Implementation Scheme 18. An antibody or its antigen-binding portion according to any one of Implementation Schemes 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.

[0116] Implementation Scheme 19. An antibody or its antigen-binding portion according to any one of Implementation Schemes 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.

[0117] Implementation Scheme 20. An antibody or its antigen-binding portion according to any one of Implementation Schemes 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.

[0118] Implementation Scheme 21. An antibody or its antigen-binding portion according to any one of Implementation Schemes 13 to 20, wherein the light chain CDR1 comprises SEQ ID NO:64 or SEQ ID NO:65.

[0119] Implementation Scheme 22. An antibody or its antigen-binding portion according to any one of Implementation Schemes 13 to 21, wherein the light chain CDR2 comprises SEQ ID NO:66 or SEQ ID NO:67.

[0120] Implementation Scheme 23. An antibody or its antigen-binding portion according to any one of Implementation Schemes 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.

[0121] Implementation Scheme 24. An antibody or its antigen-binding portion according to any of the foregoing embodiments, wherein the antibody or its antigen-binding portion comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3, wherein

[0122] (a) 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;

[0123] (b) 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;

[0124] (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;

[0125] (d) Light chain CDR1 contains SEQ ID NO:64 or SEQ ID NO:65;

[0126] (e) The light chain CDR2 contains SEQ ID NO:66 or SEQ ID NO:67; and

[0127] (f) The light chain CDR3 includes SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70 or SEQ ID NO:71.

[0128] Implementation Scheme 25. An isolated antibody or its antigen-binding moiety, which binds to human TIM3, comprising:

[0129] (a1) The heavy chain variable regions CDR1, CDR2 and CDR3 respectively containing SEQ ID NO:41, 46 and 53, and the light chain variable regions CDR1, CDR2 and CDR3 respectively containing SEQ ID NO:64, 66 and 68;

[0130] (a2) The heavy chain variable regions CDR1, CDR2 and CDR3 respectively containing SEQ ID NO:41, 122 and 53, and the light chain variable regions CDR1, CDR2 and CDR3 respectively containing SEQ ID NO:64, 66 and 68;

[0131] (a3) The heavy chain variable regions CDR1, CDR2 and CDR3 respectively containing SEQ ID NO:41, 123 and 53, and the light chain variable regions CDR1, CDR2 and CDR3 respectively containing SEQ ID NO:64, 66 and 68;

[0132] (a4) The heavy chain variable regions CDR1, CDR2 and CDR3 respectively containing SEQ ID NO:41, 124 and 53, and the light chain variable regions CDR1, CDR2 and CDR3 respectively containing SEQ ID NO:64, 66 and 68;

[0133] (a5) The heavy chain variable regions CDR1, CDR2 and CDR3 respectively containing SEQ ID NO:41, 46 and 126, and the light chain variable regions CDR1, CDR2 and CDR3 respectively containing SEQ ID NO:64, 66 and 68;

[0134] (a6) The heavy chain variable regions CDR1, CDR2 and CDR3 respectively containing SEQ ID NO:41, 46 and 127, and the light chain variable regions CDR1, CDR2 and CDR3 respectively containing SEQ ID NO:64, 66 and 68;

[0135] (a7) The heavy chain variable regions CDR1, CDR2 and CDR3 respectively containing SEQ ID NO:41, 46 and 128, and the light chain variable regions CDR1, CDR2 and CDR3 respectively containing SEQ ID NO:64, 66 and 68;

[0136] (a8) contains heavy chain variable regions CDR1, CDR2 and CDR3 of SEQ ID NO:41, 46 and 129 respectively, and light chain variable regions CDR1, CDR2 and CDR3 of SEQ ID NO:64, 66 and 68 respectively;

[0137] (a9) contains heavy chain variable regions CDR1, CDR2 and CDR3 of SEQ ID NO:41, 122 and 128 respectively, and light chain variable regions CDR1, CDR2 and CDR3 of SEQ ID NO:64, 66 and 68 respectively;

[0138] (a10) contains heavy chain variable regions CDR1, CDR2 and CDR3 of SEQ ID NO:41, 122 and 126 respectively, and light chain variable regions CDR1, CDR2 and CDR3 of SEQ ID NO:64, 66 and 68 respectively;

[0139] (b1) The heavy chain variable regions CDR1, CDR2 and CDR3 respectively containing SEQ ID NO:42, 47 and 54, and the light chain variable regions CDR1, CDR2 and CDR3 respectively containing SEQ ID NO:64, 66 and 69;

[0140] (b2) The heavy chain variable regions CDR1, CDR2 and CDR3 respectively containing SEQ ID NO:42, 125 and 54, and the light chain variable regions CDR1, CDR2 and CDR3 respectively containing SEQ ID NO:64, 66 and 69;

[0141] (c) The heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO:43, 48 and 55 respectively, and / or the light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO:64, 66 and 69 respectively;

[0142] (d) Containing heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:44, 49 and 56 respectively, and / or containing light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:64, 66 and 68 respectively;

[0143] (e) The heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO:45, 50 and 57 respectively, and / or the light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO:64, 66 and 69 respectively;

[0144] (f) The heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO:45, 50 and 57 respectively, and / or the light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO:64, 66 and 71 respectively;

[0145] (g1) Contains heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:45, 50 and 57 respectively, and / or contains light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:65, 67 and 70 respectively;

[0146] (g2) Contains heavy chain variable regions CDR1, CDR2 and CDR3 of SEQ ID NO:45, 50 and 57 respectively, and light chain variable regions CDR1, CDR2 and CDR3 of SEQ ID NO:64, 66 and 71 respectively;

[0147] (g3) contains heavy chain variable regions CDR1, CDR2 and CDR3 of SEQ ID NO:45, 50 and 57 respectively, and light chain variable regions CDR1, CDR2 and CDR3 of SEQ ID NO:64, 66 and 69 respectively;

[0148] (h) Contains heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:45, 51 and 58 respectively, and / or contains light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:64, 66 and 68 respectively;

[0149] (i) The heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO:45, 52 and 59 respectively, and / or the light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO:64, 66 and 69 respectively.

[0150] Implementation Scheme 26. An antibody or its antigen-binding portion according to Implementation Scheme 25, wherein the antibody comprises heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO:41, 46 and 53 respectively, and / or light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO:64, 66 and 68 respectively.

[0151] Implementation Scheme 27. An antibody or its antigen-binding portion according to Implementation Scheme 25, wherein the antibody comprises heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO:42, 47 and 54, respectively, and / or light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO:64, 66 and 69, respectively.

[0152] Implementation Scheme 28. An antibody or its antigen-binding portion according to Implementation Scheme 25, wherein the antibody comprises heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO:43, 48 and 55, respectively, and / or light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO:64, 66 and 69, respectively.

[0153] Implementation Scheme 29. An antibody or its antigen-binding portion according to Implementation Scheme 25, wherein the antibody comprises heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO:44, 49 and 56, respectively, and / or light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO:64, 66 and 68, respectively.

[0154] Implementation Scheme 30. An antibody or its antigen-binding portion according to Implementation Scheme 25, wherein the antibody comprises heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO:45, 50 and 57 respectively, and / or light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO:64, 66 and 69 respectively.

[0155] Implementation Scheme 31. An antibody or its antigen-binding portion according to Implementation Scheme 25, wherein the antibody comprises heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO:45, 50 and 57 respectively, and / or light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO:64, 66 and 71 respectively.

[0156] Implementation Scheme 32. An antibody or its antigen-binding portion according to Implementation Scheme 25, wherein the antibody comprises heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO:45, 50 and 57 respectively, and / or light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO:65, 67 and 70 respectively.

[0157] Implementation Scheme 33. An antibody or its antigen-binding portion according to Implementation Scheme 25, wherein the antibody comprises heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO:45, 51 and 58 respectively, and / or light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO:64, 66 and 68 respectively.

[0158] Implementation Scheme 34. An antibody or its antigen-binding portion according to Implementation Scheme 25, wherein the antibody comprises heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO:45, 52 and 59 respectively, and / or light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO:64, 66 and 69 respectively.

[0159] Implementation Scheme 35. An isolated antibody or its antigen-binding moiety, which 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 NO: 34, 35, 36, 37, 38, 39, 40, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, and 364.

[0160] Implementation Scheme 36. An isolated antibody or its antigen-binding moiety, which 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 NO: 60, 61, 62, and 63.

[0161] Implementation Scheme 37. An isolated antibody or its antigen-binding moiety, which binds to human TIM3 and cross-competes with a reference antibody for binding to human TIM3, said reference antibody comprising VH and VL, wherein said VH and said VL are selected from:

[0162] (a) VH containing the amino acid sequence shown in SEQ ID NO:34 and VL containing the amino acid sequence shown in SEQ ID NO:60;

[0163] (b) VH containing the amino acid sequence shown in SEQ ID NO:35 and VL containing the amino acid sequence shown in SEQ ID NO:61;

[0164] (c) VH containing the amino acid sequence shown in SEQ ID NO:36 and VL containing the amino acid sequence shown in SEQ ID NO:61;

[0165] (d) VH containing the amino acid sequence shown in SEQ ID NO:37 and VL containing the amino acid sequence shown in SEQ ID NO:60;

[0166] (e) VH containing the amino acid sequence shown in SEQ ID NO:38 and VL containing the amino acid sequence shown in SEQ ID NO:61;

[0167] (f) VH containing the amino acid sequence shown in SEQ ID NO:38 and VL containing the amino acid sequence shown in SEQ ID NO:62;

[0168] (g) VH containing the amino acid sequence shown in SEQ ID NO:38 and VL containing the amino acid sequence shown in SEQ ID NO:63;

[0169] (h) VH containing the amino acid sequence shown in SEQ ID NO:39 and VL containing the amino acid sequence shown in SEQ ID NO:60;

[0170] (i) VH containing the amino acid sequence shown in SEQ ID NO:40 and VL containing the amino acid sequence shown in SEQ ID NO:61.

[0171] (j) VH containing the amino acid sequence shown in SEQ ID NO:121 and VL containing the amino acid sequence shown in SEQ ID NO:63, respectively;

[0172] (k) VH containing the amino acid sequence shown in SEQ ID NO:120 and VL containing the amino acid sequence shown in SEQ ID NO:61, respectively;

[0173] (l) VH containing the amino acid sequence shown in SEQ ID NO:112 and VL containing the amino acid sequence shown in SEQ ID NO:60, respectively;

[0174] (m) respectively contain VH containing the amino acid sequence shown in SEQ ID NO:113 and VL containing the amino acid sequence shown in SEQ ID NO:60;

[0175] (n) VH, which contains the amino acid sequence shown in SEQ ID NO:114, and VL, which contains the amino acid sequence shown in SEQ ID NO:60, respectively;

[0176] (o) VH, which contains the amino acid sequence shown in SEQ ID NO:115, and VL, which contains the amino acid sequence shown in SEQ ID NO:60, respectively;

[0177] (p) VH, which contains the amino acid sequence shown in SEQ ID NO:116, and VL, which contains the amino acid sequence shown in SEQ ID NO:60, respectively;

[0178] (q) VH containing the amino acid sequence shown in SEQ ID NO:117 and VL containing the amino acid sequence shown in SEQ ID NO:60, respectively;

[0179] (r) VH, which contains the amino acid sequence shown in SEQ ID NO:118, and VL, which contains the amino acid sequence shown in SEQ ID NO:60, respectively;

[0180] (s) VH, which contains the amino acid sequence shown in SEQ ID NO:119, and VL, which contains the amino acid sequence shown in SEQ ID NO:60, respectively; and

[0181] (t) VH, which contains the amino acid sequence shown in SEQ ID NO:364, and VL, which contains the amino acid sequence shown in SEQ ID NO:60, respectively.

[0182] Implementation Scheme 38. An antibody or its antigen-binding portion according to Implementation Scheme 37, which binds to TIM3 at the same epitope as a reference antibody, as determined, for example, by one or more methods provided herein.

[0183] Implementation Scheme 39. The antibody or its antigen-binding portion according to Implementation Scheme 37 or 38 comprises VH and VL selected from:

[0184] (a) VH containing the amino acid sequence shown in SEQ ID NO:34 and VL containing the amino acid sequence shown in SEQ ID NO:60;

[0185] (b) VH containing the amino acid sequence shown in SEQ ID NO:35 and VL containing the amino acid sequence shown in SEQ ID NO:61;

[0186] (c) VH containing the amino acid sequence shown in SEQ ID NO:36 and VL containing the amino acid sequence shown in SEQ ID NO:61;

[0187] (d) VH containing the amino acid sequence shown in SEQ ID NO:37 and VL containing the amino acid sequence shown in SEQ ID NO:60;

[0188] (e) VH containing the amino acid sequence shown in SEQ ID NO:38 and VL containing the amino acid sequence shown in SEQ ID NO:61;

[0189] (f) VH containing the amino acid sequence shown in SEQ ID NO:38 and VL containing the amino acid sequence shown in SEQ ID NO:62;

[0190] (g) VH containing the amino acid sequence shown in SEQ ID NO:38 and VL containing the amino acid sequence shown in SEQ ID NO:63;

[0191] (h) VH containing the amino acid sequence shown in SEQ ID NO:39 and VL containing the amino acid sequence shown in SEQ ID NO:60;

[0192] (i) VH containing the amino acid sequence shown in SEQ ID NO:40 and VL containing the amino acid sequence shown in SEQ ID NO:61;

[0193] (j) VH containing the amino acid sequence shown in SEQ ID NO:121 and VL containing the amino acid sequence shown in SEQ ID NO:63, respectively;

[0194] (k) VH containing the amino acid sequence shown in SEQ ID NO:120 and VL containing the amino acid sequence shown in SEQ ID NO:61, respectively;

[0195] (l) VH containing the amino acid sequence shown in SEQ ID NO:112 and VL containing the amino acid sequence shown in SEQ ID NO:60, respectively;

[0196] (m) respectively contain VH containing the amino acid sequence shown in SEQ ID NO:113 and VL containing the amino acid sequence shown in SEQ ID NO:60;

[0197] (n) VH, which contains the amino acid sequence shown in SEQ ID NO:114, and VL, which contains the amino acid sequence shown in SEQ ID NO:60, respectively;

[0198] (o) VH, which contains the amino acid sequence shown in SEQ ID NO:115, and VL, which contains the amino acid sequence shown in SEQ ID NO:60, respectively;

[0199] (p) VH, which contains the amino acid sequence shown in SEQ ID NO:116, and VL, which contains the amino acid sequence shown in SEQ ID NO:60, respectively;

[0200] (q) VH and VL60, respectively, contain the amino acid sequence shown in SEQ ID NO:117;

[0201] (r) VH, which contains the amino acid sequence shown in SEQ ID NO:118, and VL, which contains the amino acid sequence shown in SEQ ID NO:60, respectively;

[0202] (s) VH, which contains the amino acid sequence shown in SEQ ID NO:119, and VL, which contains the amino acid sequence shown in SEQ ID NO:60, respectively; and

[0203] (t) VH, which contains the amino acid sequence shown in SEQ ID NO:364, and VL, which contains the amino acid sequence shown in SEQ ID NO:60, respectively.

[0204] Implementation Scheme 40. The antibody or its antigen-binding portion according to Implementation Scheme 37 or 38, comprising a VH containing 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 containing the amino acid sequence shown in SEQ ID NO:60.

[0205] Implementation Scheme 41. The antibody or its antigen-binding portion according to Implementation Scheme 37 or 38, comprising a VH containing the amino acid sequence shown in SEQ ID NO:35 or SEQ ID NO:120 and a VL containing the amino acid sequence shown in SEQ ID NO:61.

[0206] Implementation Scheme 42. The antibody or its antigen-binding portion according to Implementation Scheme 37 or 38, comprising a VH containing the amino acid sequence shown in SEQ ID NO:36 and a VL containing the amino acid sequence shown in SEQ ID NO:61.

[0207] Implementation Scheme 43. The antibody or its antigen-binding portion according to Implementation Scheme 37 or 38, comprising 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.

[0208] Implementation Scheme 44. The antibody or its antigen-binding portion according to Implementation Scheme 37 or 38, comprising 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.

[0209] Implementation Scheme 45. The antibody or its antigen-binding portion according to Implementation Scheme 37 or 38, comprising 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.

[0210] Implementation Scheme 46. The antibody or its antigen-binding portion according to Implementation Scheme 37 or 38, comprising 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.

[0211] Implementation Scheme 47. An antibody or its antigen-binding portion according to any of the foregoing embodiments, wherein the antibody is selected from IgG1, IgG2, IgG3, IgG4 or variants thereof.

[0212] Implementation Scheme 48. The antibody or its antigen-binding portion according to Implementation Scheme 47, wherein the antibody is an IgG1 antibody.

[0213] Implementation Scheme 49. An antibody or its antigen-binding portion according to Implementation Scheme 47, wherein said antibody comprises IgG1 Fc without effector function.

[0214] Implementation Scheme 50. An antibody or antigen-binding portion thereof according to Implementation Scheme 49, wherein the antibody or antigen-binding portion thereof comprises IgG1 Fc with no effector function containing the following mutations L234A, L235E, G237A and optionally A330S and P331S.

[0215] Implementation Scheme 51. The antibody or its antigen-binding portion according to any of the preceding embodiments comprises a heavy chain constant region containing an amino acid sequence selected from SEQ ID NO:263-266.

[0216] Implementation Scheme 52. An antibody or antigen-binding portion thereof according to any one of Implementation Schemes 1-51, wherein the antibody or antigen-binding portion thereof is a human antibody or a humanized antibody.

[0217] Implementation Scheme 53. The antibody according to any one of Implementation Schemes 1-52, wherein the antibody comprises:

[0218] (a1) Contains heavy chain and light chain sequences of SEQ ID NO:301 (or 302) and 29, respectively;

[0219] (a2) Contains heavy chain and light chain sequences of SEQ ID NO:1 (or 8) and 29, respectively;

[0220] (a3) Contains heavy chain and light chain sequences of SEQ ID NO:15 (or 22) and 29, respectively;

[0221] (a4) Contains heavy chain and light chain sequences of SEQ ID NO:303 (or 304) and 29, respectively;

[0222] (a5) contains heavy chain and light chain sequences of SEQ ID NO:72 (or 82) and 29, respectively;

[0223] (a6) contains heavy chain and light chain sequences of SEQ ID NO:92 (or 102) and 29, respectively;

[0224] (a7) Contains heavy chain and light chain sequences of SEQ ID NO:305 (or 306) and 29, respectively;

[0225] (a8) contains the heavy chain and light chain sequences of SEQ ID NO:73 (or 83) and 29, respectively;

[0226] (a9) contains heavy chain and light chain sequences of SEQ ID NO:93 (or 103) and 29, respectively;

[0227] (a10) contains the heavy chain and light chain sequences of SEQ ID NO:307 (or 308) and 29, respectively;

[0228] (a11) contains the heavy chain and light chain sequences of SEQ ID NO:74 (or 84) and 29, respectively;

[0229] (a12) contains the heavy chain and light chain sequences of SEQ ID NO:94 (or 104) and 29, respectively;

[0230] (a13) contains heavy chain and light chain sequences of SEQ ID NO:309 (or 310) and 29, respectively;

[0231] (a14) contains the heavy chain and light chain sequences of SEQ ID NO:75 (or 85) and 29, respectively;

[0232] (a15) contains the heavy chain and light chain sequences of SEQ ID NO:95 (or 105) and 29, respectively;

[0233] (a16) contains the heavy chain and light chain sequences of SEQ ID NO:311 (or 312) and 29, respectively;

[0234] (a17) contains the heavy chain and light chain sequences of SEQ ID NO:76 (or 86) and 29, respectively;

[0235] (a18) contains the heavy chain and light chain sequences of SEQ ID NO:96 (or 106) and 29, respectively;

[0236] (a19) contains the heavy chain and light chain sequences of SEQ ID NO:313 (or 314) and 29, respectively;

[0237] (a20) contains the heavy chain and light chain sequences of SEQ ID NO:77 (or 87) and 29, respectively;

[0238] (a21) contains heavy chain and light chain sequences of SEQ ID NO:97 (or 107) and 29, respectively;

[0239] (a22) contains the heavy chain and light chain sequences of SEQ ID NO:315 (or 316) and 29, respectively;

[0240] (a23) contains the heavy chain and light chain sequences of SEQ ID NO:78 (or 88) and 29, respectively;

[0241] (a24) contains the heavy chain and light chain sequences of SEQ ID NO:98 (or 108) and 29, respectively;

[0242] (a25) contains the heavy chain and light chain sequences of SEQ ID NO:317 (or 318) and 29, respectively;

[0243] (a26) contains the heavy chain and light chain sequences of SEQ ID NO:79 (or 89) and 29, respectively;

[0244] (a27) contains heavy chain and light chain sequences of SEQ ID NO:99 (or 109) and 29, respectively;

[0245] (a28) contains the heavy chain and light chain sequences of SEQ ID NO:319 (or 320) and 29, respectively;

[0246] (a29) contains the heavy chain and light chain sequences of SEQ ID NO:349 (or 350) and 29, respectively;

[0247] (a30) contains the heavy chain and light chain sequences of SEQ ID NO:351 (or 352) and 29, respectively;

[0248] (a31) contains heavy chain and light chain sequences of SEQ ID NO:353 (or 354) and 29, respectively;

[0249] (b1) Contains heavy chain and light chain sequences of SEQ ID NO:321 (or 322) and 30, respectively;

[0250] (b2) Contains heavy chain and light chain sequences of SEQ ID NO:2 (or 9) and 30, respectively;

[0251] (b3) Contains heavy chain and light chain sequences of SEQ ID NO:16 (or 23) and 30, respectively;

[0252] (b4) Contains heavy chain and light chain sequences of SEQ ID NO:323 (or 324) and 30, respectively;

[0253] (b5) Contains heavy chain and light chain sequences of SEQ ID NO:80 (or 90) and 30, respectively;

[0254] (b6) Contains heavy chain and light chain sequences of SEQ ID NO: 100 (or 110) and 30, respectively;

[0255] (b7) Contains heavy chain and light chain sequences of SEQ ID NO:325 (or 326) and 30, respectively;

[0256] (c1) Contains heavy chain and light chain sequences of SEQ ID NO:327 (or 328) and 30, respectively;

[0257] (c2) Contains heavy chain and light chain sequences of SEQ ID NO:3 (or 10) and 30 respectively;

[0258] (c3) Contains heavy chain and light chain sequences of SEQ ID NO:17 (or 24) and 30, respectively;

[0259] (c4) Contains heavy chain and light chain sequences of SEQ ID NO:329 (or 330) and 30, respectively;

[0260] (d1) contains heavy chain and light chain sequences of SEQ ID NO:331 (or 332) and 29, respectively;

[0261] (d2) Contains heavy chain and light chain sequences of SEQ ID NO:4 (or 11) and 29, respectively;

[0262] (d3) contains the heavy chain and light chain sequences of SEQ ID NO:18 (or 25) and 29, respectively;

[0263] (d4) Contains heavy chain and light chain sequences of SEQ ID NO:333 (or 334) and 29, respectively;

[0264] (e1.1) Contains heavy chain and light chain sequences of SEQ ID NO:335 (or 336) and 32, respectively;

[0265] (e1.2) Contains heavy chain and light chain sequences of SEQ ID NO:335 (or 336) and 33, respectively;

[0266] (e1.3) contains the heavy chain and light chain sequences of SEQ ID NO:335 (or 336) and 31, respectively;

[0267] (e2) Contains heavy chain and light chain sequences of SEQ ID NO:5 (or 12) and 33, respectively;

[0268] (e3) contains heavy chain and light chain sequences of SEQ ID NO:19 (or 26) and 33, respectively;

[0269] (e4) Contains heavy chain and light chain sequences of SEQ ID NO:337 (or 338) and 33, respectively;

[0270] (e5) contains heavy chain and light chain sequences of SEQ ID NO:81 (or 91) and 33, respectively;

[0271] (e6) contains heavy chain and light chain sequences of SEQ ID NO: 101 (or 111) and 33, respectively;

[0272] (e7) Contains heavy chain and light chain sequences of SEQ ID NO:339 (or 340) and 33, respectively;

[0273] (f1) contains heavy chain and light chain sequences of SEQ ID NO:341 (or 342) and 29, respectively;

[0274] (f2) Contains heavy chain and light chain sequences of SEQ ID NO:6 (or 13) and 29, respectively;

[0275] (f3) contains heavy chain and light chain sequences of SEQ ID NO:20 (or 27) and 29, respectively;

[0276] (f4) contains heavy chain and light chain sequences of SEQ ID NO:343 (or 344) and 29, respectively;

[0277] (g1) contains heavy chain and light chain sequences of SEQ ID NO:345 (or 346) and 29, respectively;

[0278] (g2) contains heavy chain and light chain sequences of SEQ ID NO:7 (or 43) and 30, respectively;

[0279] (g3) contains the heavy chain and light chain sequences of SEQ ID NO:21 (or 28) and 30, respectively; or

[0280] (g4) contains the heavy chain and light chain sequences of SEQ ID NO:347 (or 348) and 30, respectively;

[0281] The antibody specifically binds to human TIM3.

[0282] Implementation Scheme 54. An antibody or antigen-binding portion thereof according to any one of Implementation Schemes 1-53, wherein the antibody or antigen-binding portion thereof has one or more of the following characteristics:

[0283] (1) Binding to soluble human TIM3, for example, binding at KD 10 nM or less (e.g., 0.01 nM to 10 nM), for example, as determined by Biacore;

[0284] (2) Binding to soluble cynomolgus monkey TIM3, for example, binding at KD 100 nM or less (e.g., 0.01 nM to 100 nM), for example, as determined by Biacore;

[0285] (3) Binding to membrane-bound human TIM3, for example, at EC50 1 ug / mL or less (e.g. 0.01 ug / mL to 1 ug / mL), for example, as determined by flow cytometry;

[0286] (4) Binding to membrane-bound human TIM3, for example, binding at KD 1 nM or less (e.g., 0.01 nM to 10 nM), for example, as determined by Scatchard analysis;

[0287] (5) Binding to membrane-bound cynomolgus monkey TIM3, for example, at EC50 20 ug / mL or less (e.g. 0.01 ug / mL to 20 ug / mL), for example, as determined by flow cytometry;

[0288] (6) Binding to membrane-bound cynomolgus monkey TIM3, for example, at KD 1 nM or less (e.g., 0.01 nM to 10 nM), for example, as determined by Scatchard analysis;

[0289] (7) (e.g., by blocking or reducing the inhibitory effect of TIM3) induce or enhance T cell activation, as demonstrated by (i) increased IFN-γ production in T cells expressing TIM3 (e.g., Th1 cells or TILs) and / or (ii) enhanced proliferation of T cells expressing TIM3 (e.g., Th1 cells or TILs).

[0290] (8) Stimulate T cell proliferation in a mixed lymphocyte response (MLR) assay;

[0291] (9) Inhibit the binding of phosphatidylserine to TIM3, for example, as determined by the PS-hTIM3 "in-tandem" blocking assay;

[0292] (10) When bound to TIM3 on the cell, it is not internalized or downregulated on the cell surface;

[0293] (11) Binding 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);

[0294] (12) Compared to binding with wild-type human TIM3, it has reduced binding with one or more of the following 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 (as indicated in SEQ ID NO: 286). Figure 30 The binding of human TIM3 substituted with another amino acid;

[0295] (13) Antibodies containing any of the VH and VL domains 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 compete for binding to human TIM3 in one or both directions;

[0296] (14) Binding to human TIM3 region 49VPVCWGKGACPVFE62 (SEQ ID NO:367) and 111RIQIPGIMNDEKFNLKL127 (SEQ ID NO:368), as determined by HDX-MS;

[0297] (15) Having a heavy chain variable region and / or a light chain variable region interacting 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; according to SEQ ID NO:286). Figure 30 )); and / or

[0298] (16)(a) Compared to binding to wild-type human TIM3, it has reduced binding to 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids therein: C58, P59, F61, E62, C63, R111, D120, and optionally D104 and Q113 (according to SEQ ID NO:286). Figure 30 (a) human TIM3 substituted with another amino acid; (b) binding 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) competitively or cross-blocking 13A3 or TIM3.18.IgG1.3 binding to human TIM3.

[0299] Implementation Scheme 55. A bispecific molecule comprising an antibody as described in any of the preceding embodiments, linked to a molecule having a second binding specificity.

[0300] Implementation Scheme 56. Nucleic acid, which encodes the heavy chain variable region and / or light chain variable region of the antibody or its antigen-binding moiety as described in any one of Implementation Schemes 1-54.

[0301] Implementation scheme 57. Expression vector, which contains the nucleic acid molecule of implementation scheme 56.

[0302] Implementation scheme 58. Cells, which are transformed by the expression vector of implementation scheme 57.

[0303] Implementation Scheme 59. An immunoconjugate comprising an antibody, according to any one of Implementation Schemes 1-54, linked to a reagent.

[0304] Implementation Scheme 60. A composition comprising an antibody or antigen-binding moiety thereof, a bispecific molecule or immunoconjugate, and a carrier, as described in any one of Implementation Schemes 1-55 and 59.

[0305] Implementation Scheme 61. A kit comprising an antibody or its antigen-binding moiety or bispecific molecule or immunoconjugate as described in any one of Implementation Schemes 1-55 and 59, and instructions for use.

[0306] Implementation Scheme 62. A method for preparing an anti-TIM3 antibody or an antigen-binding portion thereof, comprising expressing the anti-TIM3 antibody or an antigen-binding portion thereof in cells according to Implementation Scheme 58, and isolating the antibody or the antigen-binding portion thereof from the cells.

[0307] Implementation Scheme 63. A method for stimulating an antigen-specific T cell response, the method comprising contacting T cells with an antibody or its antigen-binding portion, a bispecific molecule or an immunoconjugate as described in any one of Implementation Schemes 1-55 and 59, thereby stimulating an antigen-specific T cell response.

[0308] Implementation Scheme 64. A method for activating or co-stimulating effector T cells, comprising contacting effector T cells with any one of Implementation Schemes 1-55 and 59, an anti-TIM3 antibody or its antigen-binding portion, a bispecific molecule or immunoconjugate, and CD3, wherein the effector T cells are activated or co-stimulated.

[0309] Implementation Scheme 65. A method for increasing IFN-γ production in T cells, comprising contacting the T cells with an effective amount of an antibody or its antigen-binding portion, a bispecific molecule or an immunoconjugate as described in any one of Implementation Schemes 1-55 and 59.

[0310] Implementation Scheme 66. A method for increasing T cell proliferation, comprising contacting the cells with an effective amount of an antibody or its antigen-binding portion, a bispecific molecule or an immunoconjugate as described in any one of Implementation Schemes 1-55 and 59.

[0311] Implementation Scheme 67. A method for increasing IFN-γ production by T cells in a subject, comprising administering an effective amount of an antibody or its antigen-binding portion, bispecific molecule or immunoconjugate as described in any one of Implementation Schemes 1-55 and 59 to increase IFN-γ production from T cells.

[0312] Implementation Scheme 68. A method for stimulating TIL activity in a subject, the method comprising administering to the subject a therapeutically effective amount of the anti-TIM3 antibody as described in any one of Implementation Schemes 1-54.

[0313] Implementation Scheme 69. A method for stimulating an immune response in a subject, the method comprising administering to the subject an antibody or antigen-binding moiety thereof, a bispecific molecule or an immune conjugate as described in any one of Implementation Schemes 1-55 and 59, thereby stimulating an immune response in the subject.

[0314] Implementation Scheme 70. The method according to Implementation Scheme 69, wherein the subject has a tumor and an immune response against the tumor is stimulated.

[0315] Implementation Scheme 71. A method for inhibiting tumor growth or reducing tumor size in a subject, comprising administering to the subject an antibody or its antigen-binding moiety, bispecific molecule or immunoconjugate described in any one of Implementation Schemes 1-55 and 59, thereby inhibiting tumor growth in the subject.

[0316] Implementation Scheme 72. A method for treating cancer, comprising administering a therapeutically effective amount of any one of Implementation Schemes 1-55 and 59, or an antigen-binding portion thereof, a bispecific molecule, or an immunoconjugate thereof, to a subject in need to treat cancer.

[0317] Implementation Scheme 73. The method of Implementation Scheme 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, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system tumors, lymphoma, leukemia, myeloma, sarcoma, and virus-related cancers.

[0318] Implementation scheme 74. The method of implementation scheme 72 or 73, wherein the cancer is a metastatic cancer, a refractory cancer, or a recurrent cancer.

[0319] Implementation Scheme 75. The method according to any one of Implementation Schemes 67-74 further includes the application of one or more additional therapeutic agents.

[0320] Implementation Scheme 76. The method according to Implementation Scheme 75, wherein the additional treatment is an anti-PD-1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, an anti-GITR antibody, or an anti-PD-L1 antibody.

[0321] Implementation Scheme 77. A method for detecting the presence of T-cell immunoglobulins and protein 3 containing a mucin domain (TIM3) in a sample, the method comprising contacting the sample with the antibody or antigen-binding portion thereof as described in any one of Implementation Schemes 1-54, under conditions that allow the formation of a complex between an antibody or its antigen-binding portion and TIM3, and detecting the formation of the complex. Brief description of the attached diagram

[0323] Figure 1A The nucleotide sequence (SEQ ID NO: 167) and amino acid sequence (SEQ ID NO: 34) of the mature heavy chain variable (VH) region of anti-TIM3 monoclonal antibody 13A3 are shown. CDR1 (SEQ ID NO: 41), CDR2 (SEQ ID NO: 46), and CDR3 (SEQ ID NO: 53) are plotted, and the V, D, and J germline derivatives are indicated.

[0324] Figure 1B The nucleotide sequence (SEQ ID NO: 193) and amino acid sequence (SEQ ID NO: 60) of the mature light chain variable (VL) region of the anti-TIM3 monoclonal antibody 13A3 are shown. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66), and CDR3 (SEQ ID NO: 68) are plotted, and the V and J germline derivatives are indicated.

[0325] Figure 1C The images show the nucleotide sequence (SEQ ID NO: 167) and amino acid sequence (SEQ ID NO: 34) of the heavy chain VH region of anti-TIM3 monoclonal antibody 13A3, which has a signal sequence (nucleotide sequence and amino acid sequence, respectively SEQ ID NO: 274 and 269), 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 13A3, which has a signal sequence (nucleotide sequence and amino acid sequence, respectively SEQ ID NO: 273 and 268).

[0326] Figure 2A The nucleotide sequence (SEQ ID NO: 168) and amino acid sequence (SEQ ID NO: 35) of the mature heavy chain variable (VH) region of the anti-TIM3 monoclonal antibody 8B9 are shown. CDR1 (SEQ ID NO: 42), CDR2 (SEQ ID NO: 47), and CDR3 (SEQ ID NO: 54) are plotted, and the V, D, and J lineage derivatives are indicated.

[0327] Figure 2B The nucleotide sequence (SEQ ID NO: 194) and amino acid sequence (SEQ ID NO: 61) of the mature light chain variable (VL) region of the anti-TIM3 monoclonal antibody 8B9 are shown. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66), and CDR3 (SEQ ID NO: 69) are plotted, and the V and J germline derivatives are indicated.

[0328] Figure 2C The images show the nucleotide sequence (SEQ ID NO: 168) and amino acid sequence (SEQ ID NO: 35) of the heavy chain VH region of the anti-TIM3 monoclonal antibody 8B9, which has a signal sequence (nucleotide sequence and amino acid sequence, respectively SEQ ID NO: 274 and 269), and the nucleotide sequence (SEQ ID NO: 194) and amino acid sequence (SEQ ID NO: 61) of the light chain VL region of the anti-TIM3 monoclonal antibody 8B9, which has a signal sequence (nucleotide sequence and amino acid sequence, respectively SEQ ID NO: 273 and 268).

[0329] Figure 3A The nucleotide sequence (SEQ ID NO: 169) and amino acid sequence (SEQ ID NO: 36) of the mature heavy chain variable (VH) region of the anti-TIM3 monoclonal antibody 8C4 are shown. CDR1 (SEQ ID NO: 43), CDR2 (SEQ ID NO: 48), and CDR3 (SEQ ID NO: 55) are plotted, and the V, D, and J germline derivatives are indicated.

[0330] Figure 3B The nucleotide sequence (SEQ ID NO: 194) and amino acid sequence (SEQ ID NO: 61) of the mature light chain variable (VL) region of the anti-TIM3 monoclonal antibody 8C4 are shown. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66), and CDR3 (SEQ ID NO: 69) are plotted, and the V and J germline derivatives are indicated.

[0331] Figure 3C The images show the nucleotide sequence (SEQ ID NO: 169) and amino acid sequence (SEQ ID NO: 36) of the heavy chain VH region of the anti-TIM3 monoclonal antibody 8C4, which has a signal sequence (nucleotide sequence and amino acid sequence, respectively SEQ ID NO: 274 and 269), and the nucleotide sequence (SEQ ID NO: 194) and amino acid sequence (SEQ ID NO: 61) of the light chain VL region of the anti-TIM3 monoclonal antibody 8C4, which has a signal sequence (nucleotide sequence and amino acid sequence, respectively SEQ ID NO: 273 and 268).

[0332] Figure 4AThe nucleotide sequence (SEQ ID NO: 170) and amino acid sequence (SEQ ID NO: 37) of the mature heavy chain variable (VH) region of the anti-TIM3 monoclonal antibody 17C3 are shown. CDR1 (SEQ ID NO: 44), CDR2 (SEQ ID NO: 49), and CDR3 (SEQ ID NO: 56) are plotted, and the V, D, and J lineage derivatives are indicated.

[0333] Figure 4B The nucleotide sequence (SEQ ID NO: 193) and amino acid sequence (SEQ ID NO: 60) of the mature light chain variable (VL) region of the anti-TIM3 monoclonal antibody 17C3 are shown. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66), and CDR3 (SEQ ID NO: 68) are plotted, and the V and J germline derivatives are indicated.

[0334] Figure 4C The images show the nucleotide sequence (SEQ ID NO: 170) and amino acid sequence (SEQ ID NO: 37) of the heavy chain VH region of the anti-TIM3 monoclonal antibody 17C3, which has a signal sequence (nucleotide sequence and amino acid sequence, respectively SEQ ID NO: 272 and 267), and the nucleotide sequence (SEQ ID NO: 193) and amino acid sequence (SEQ ID NO: 60) of the light chain VL region of the anti-TIM3 monoclonal antibody 17C3, which has a signal sequence (nucleotide sequence and amino acid sequence, respectively SEQ ID NO: 273 and 268).

[0335] Figure 5A The nucleotide sequence (SEQ ID NO: 171) and amino acid sequence (SEQ ID NO: 38) of the mature heavy chain variable (VH) region of anti-TIM3 monoclonal antibody 9F6 are shown. CDR1 (SEQ ID NO: 45), CDR2 (SEQ ID NO: 50), and CDR3 (SEQ ID NO: 57) are plotted, and the V, D, and J lineage derivatives are indicated.

[0336] Figure 5B The nucleotide sequence (SEQ ID NO:195) and amino acid sequence (SEQ ID NO:62) of the mature light chain variable (VL) region of VK1 of the anti-TIM3 monoclonal antibody 9F6 are shown. CDR1 (SEQ ID NO:65), CDR2 (SEQ ID NO:67), and CDR3 (SEQ ID NO:70) are plotted, and the V and J germline derivatives are indicated.

[0337] Figure 5CThe 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 are shown. CDR1 (SEQ ID NO:64), CDR2 (SEQ ID NO:66), and CDR3 (SEQ ID NO:71) are plotted, and the V and J germline derivatives are indicated.

[0338] Figure 5D The nucleotide sequence (SEQ ID NO:194) and amino acid sequence (SEQ ID NO:61) of the mature light chain variable (VL) region of VK3 of anti-TIM3 monoclonal antibody 9F6 are shown. CDR1 (SEQ ID NO:64), CDR2 (SEQ ID NO:66), and CDR3 (SEQ ID NO:69) are plotted, and the V and J germline derivatives are indicated.

[0339] Figure 5E The images show 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, which has a signal sequence (nucleotide and amino acid sequences, respectively SEQ ID NO: 275 and 270), and the nucleotide sequences (SEQ ID NO: 195, 196 and 194) and amino acid sequences (SEQ ID NO: 62, 63 and 61, respectively) of the light chain VL regions of the anti-TIM3 monoclonal antibody 9F6, which have a signal sequence (nucleotide and amino acid sequences, respectively SEQ ID NO: 276 and 271).

[0340] Figure 6A The nucleotide sequence (SEQ ID NO: 172) and amino acid sequence (SEQ ID NO: 39) of the mature heavy chain variable (VH) region of the anti-TIM3 monoclonal antibody 3G4 are shown. CDR1 (SEQ ID NO: 45), CDR2 (SEQ ID NO: 51), and CDR3 (SEQ ID NO: 58) are plotted, and the V, D, and J germline derivatives are indicated.

[0341] Figure 6B The nucleotide sequence (SEQ ID NO: 193) and amino acid sequence (SEQ ID NO: 60) of the mature light chain variable (VL) region of the anti-TIM3 monoclonal antibody 3G4 are shown. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66), and CDR3 (SEQ ID NO: 68) are plotted, and the V and J germline derivatives are indicated.

[0342] Figure 6C The images show the nucleotide sequence (SEQ ID NO: 172) and amino acid sequence (SEQ ID NO: 39) of the heavy chain VH region of the anti-TIM3 monoclonal antibody 3G4, which has a signal sequence (nucleotide sequence and amino acid sequence, respectively SEQ ID NO: 275 and 270), and the nucleotide sequence (SEQ ID NO: 193) and amino acid sequence (SEQ ID NO: 60) of the light chain VL region of the anti-TIM3 monoclonal antibody 3G4, which has a signal sequence (nucleotide sequence and amino acid sequence, respectively SEQ ID NO: 273 and 268).

[0343] Figure 7A The nucleotide sequence (SEQ ID NO: 173) and amino acid sequence (SEQ ID NO: 40) of the variable (VH) region of the mature heavy chain of the anti-TIM3 monoclonal antibody 17C8 are shown. CDR1 (SEQ ID NO: 45), CDR2 (SEQ ID NO: 52), and CDR3 (SEQ ID NO: 59) are plotted, and the V, D, and J lineage derivatives are indicated.

[0344] Figure 7B The nucleotide sequence (SEQ ID NO: 194) and amino acid sequence (SEQ ID NO: 61) of the mature light chain variable (VL) region of the anti-TIM3 monoclonal antibody 17C8 are shown. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66), and CDR3 (SEQ ID NO: 69) are plotted, and the V and J germline derivatives are indicated.

[0345] Figure 7C The images show the nucleotide sequence (SEQ ID NO: 173) and amino acid sequence (SEQ ID NO: 40) of the heavy chain VH region of the anti-TIM3 monoclonal antibody 17C8, which has a signal sequence (nucleotide sequence and amino acid sequence, respectively SEQ ID NO: 275 and 270), and the nucleotide sequence (SEQ ID NO: 194) and amino acid sequence (SEQ ID NO: 61) of the light chain VL region of the anti-TIM3 monoclonal antibody 17C8, which has a signal sequence (nucleotide sequence and amino acid sequence, respectively SEQ ID NO: 273 and 268).

[0346] Figure 8AThe nucleotide sequence (SEQ ID NO:420) and amino acid sequence (SEQ ID NO:410) of the mature heavy chain variable (VH) region of anti-TIM3 monoclonal antibody 14H7 are shown. CDR1 (SEQ ID NO:45), CDR2 (SEQ ID NO:413), and CDR3 (SEQ ID NO:414) are plotted, and the V, D, and J lineage derivatives are indicated.

[0347] Figure 8B The nucleotide sequence (SEQ ID NO:427) and amino acid sequence (SEQ ID NO:417) of the mature light chain variable (VL) region of anti-TIM3 monoclonal antibody 14H7 are shown. CDR1 (SEQ ID NO:64), CDR2 (SEQ ID NO:66), and CDR3 (SEQ ID NO:69) are plotted, and the V and J germline derivatives are indicated.

[0348] Figure 8C The images show the nucleotide sequence (SEQ ID NO:420) and amino acid sequence (SEQ ID NO:410) of the heavy chain VH region of the anti-TIM3 monoclonal antibody 14H7, which has a signal sequence (nucleotide sequence and amino acid sequence, respectively SEQ ID NO:362 and 361), and the nucleotide sequence (SEQ ID NO:427) and amino acid sequence (SEQ ID NO:417) of the light chain VL region of the anti-TIM3 monoclonal antibody 14H7, which has a signal sequence (nucleotide sequence and amino acid sequence, respectively SEQ ID NO:363 and 361).

[0349] Figure 9A The nucleotide sequence (SEQ ID NO:421) and amino acid sequence (SEQ ID NO:411) of the mature heavy chain variable (VH) region of anti-TIM3 monoclonal antibody 23B3 are shown. CDR1 (SEQ ID NO:45), CDR2 (SEQ ID NO:415), and CDR3 (SEQ ID NO:416) are plotted, and the V, D, and J lineage derivatives are indicated.

[0350] Figure 9B The nucleotide sequence (SEQ ID NO:193) and amino acid sequence (SEQ ID NO:60) of the mature light chain variable (VL) region of VK1 of the anti-TIM3 monoclonal antibody 23B3 are shown. CDR1 (SEQ ID NO:64), CDR2 (SEQ ID NO:66), and CDR3 (SEQ ID NO:68) are plotted, and the V and J germline derivatives are indicated.

[0351] Figure 9CThe nucleotide sequence (SEQ ID NO:428) and amino acid sequence (SEQ ID NO:418) of the mature light chain variable (VL) region of VK2 of the anti-TIM3 monoclonal antibody 23B3 are shown. CDR1 (SEQ ID NO:64), CDR2 (SEQ ID NO:66), and CDR3 (SEQ ID NO:419) are plotted, and the V and J germline derivatives are indicated.

[0352] Figure 9D The images show 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, which has a signal sequence (nucleotide sequence and amino acid sequence, respectively SEQ ID NO: 362 and 361). They also show the nucleotide sequences (SEQ ID NO: 193 and 428) and amino acid sequences (SEQ ID NO: 60 and 418) of the light chain VL regions of the anti-TIM3 monoclonal antibody 23B3, which have a signal sequence (nucleotide sequence and amino acid sequence, respectively SEQ ID NO: 363 and 361).

[0353] Figure 10A The sequence alignments of the heavy chain variable (VH) regions of monoclonal antibodies 13A3, 8B9, 8C4, 17C3, 9F6, 3G4, 17C8, 23B3, and 14H7 are shown. Complementarity-determining regions (CDRs) are indicated in the boxes.

[0354] Figure 10B The VH region of the antibody, each CDR, and its mutant SEQ ID NO are listed.

[0355] Figure 11A The sequence alignments of the light chain variable (VL) regions of monoclonal antibodies 13A3, 8B9, 8C4, 17C3, 9F6_VK1, 9F6_VK2, 9F6_VK3, 3G4, 17C8, 23B3_VK1, 23B3_VK2, and 14H7 are shown. Complementarity-determining regions (CDRs) are indicated in the boxes.

[0356] Figure 11B The VL region of the antibody and the SEQ ID NO for each CDR are listed.

[0357] Figure 12The sequence alignments of the mature full-length heavy chains (HC) of the monoclonal antibody TIM3.5 (13A3) and its example variants are shown: 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.

[0358] Figure 13 The sequence alignment of the mature full-length HC of monoclonal antibody 9F6 and its example variant TIM3.7 (A108T) is shown. The VH region of each heavy chain is underlined.

[0359] Figure 14 The sequence alignment of the mature full-length HC of monoclonal antibody 8B9 and its example variant TIM3.8 (S61P) is shown. The VH region of each heavy chain is underlined.

[0360] Figure 15 The sequence alignment of the mature full-length HC of monoclonal antibody 23B3 and its example variant TIM3.25 (G6E / D79Y) is shown. The VH region of each heavy chain is underlined.

[0361] Figure 16 The SEQ ID NOs for the full-length heavy and light chains, variable regions, and CDRs of hybridoma-derived antibodies (13A3, 8B9, 8C4, 17C3, 9F6, 3G4, 17C8, 14H7, and 23B3) and recombinant (TIM3.2-TIM3.18, TIM3.24, and TIM3.25) anti-human TIM3 antibodies are listed. The isotypes of the heavy and light chains are also indicated. "Hn" refers to the hybridoma name. Figure 16 The heavy and light chains referred to herein may be derived from their elements, such as the variable and constant regions disclosed herein. If the SEQ ID NO does not appear in a given column on the second or third page of this table, the SEQ ID NO is provided in a column on the preceding page or on a page preceding that page.

[0362] Figure 17A and 17B Several anti-TIM3 antibodies were shown in relation to human TIM3-transfected CHO cells. Figure 17A ) and activated human T cells ( Figure 17B The combination curve and EC 50 Value. In Figure 17A The following anti-TIM3 antibodies were shown: 13A3, 17C3, 17C8, 3G4, 8B9, and 9F6. Figure 17B The following anti-TIM3 antibodies were shown: 13A3, 17C3, 17C8, 3G4, 8B9, 8C4, and 9F6. Figure 17A and 17B In these two figures, the isotype control antibodies human IgG1 (hG1), IgG2 (hG2), and IgG4 (hG4) were used as negative controls.

[0363] Figure 18 The binding curves of anti-TIM3 antibodies 14H7 and 23B3 to anti-TIM3 antibody 13A3 and EC2 binding curves of anti-TIM3 antibodies to human TIM3-positive cells are shown. 50 Comparison of values. Isotype control antibodies IgG1, IgG1.1 (hG1.1), IgG2, and IgG4 were used as negative controls.

[0364] Figure 19A , 19B 19C showed the interaction between anti-TIM3 antibody and cynomolgus monkey TIM3-transfected CHO cell lines ( Figure 19A ) and activated cynomolgus monkey T cells ( Figure 19B and 19C The combined analysis of ) . In Figure 19A The study provides binding curves and EC50 values ​​for anti-TIM3 antibodies 13A3, 17C3, 17C8, 3G4, 8B9, and 9F6. Figure 19B The binding curve and EC50 value of the anti-TIM3 antibody 13A3 are provided. Figure 19C In this study, the binding of 100 μg of the relevant antibody to activated cynomolgus monkey CD8+ T cells was tested. Binding data were presented as mean fluorescence index (MFI). Figures 19A-19C In this study, relevant isotype control antibodies (hIgG1, hIgG2, and hIgG4) or antibody-free treatment (no Ab) were used as negative controls.

[0365] Figure 20 Flow cytometry images show the binding of anti-TIM3 antibodies 14H7 (left) and 23B3 (right) to CHO cells transfected with human TIM3 ("1") or cynomolgus monkey TIM3 ("2"). CHO cells transfected with an isotype control antibody were used as a negative control ("3").

[0366] Figure 21 The anti-TIM3 activity promoting IFN-γ production by tumor-infiltrating leukocytes (TILs) in renal cell carcinoma (RCC) is shown (at multiple antibody concentrations shown in the boxes). As illustrated, the eight columns for each antibody represent different antibody concentrations. Data for anti-TIM3 antibodies 13A3, 3G4, 17C3, 17C8, 8B9, and 9F6 are shown. An isotype control antibody was used as a control. Dashed horizontal lines indicate background threshold levels.

[0367] Figure 22A and 22B This demonstrates anti-TIM3 activity that promotes IFN-γ production by lung cancer TILs (at multiple antibody concentrations shown in the boxes). Figure 22A IFN-γ ELISA; Figure 22B (Intracellular IFN-γ staining). In Figure 22A In the diagram, each column for each antibody (13A3 and 3G4) represents the different antibody concentrations shown. Figure 22B The image above shows CD4. + T cells, the image below shows CD8 + T cells. TIM3 levels were measured using 8B9 (x-axis). Figure 22B In this study, 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.

[0368] Figure 23 This study demonstrated the efficacy of anti-TIM3 antibodies (i.e., antibodies 13A3 and 3G4) in promoting IFN-γ secretion by TILs isolated from various patient tissues (i.e., kidney, pancreas, lung, and thyroid) and cultured in vitro with either anti-TIM3 antibody 13A3 or 3G4. IFN-γ secretion was measured in the presence of CHO-OKT3 cells. The data showed a fold increase in IFN-γ levels produced by TILs relative to the absence of anti-TIM3 antibodies.

[0369] Figure 24A and 24B Data on cross-blockade of anti-TIM3 antibody against activated human T cells are presented. Figure 24A In the study, anti-TIM3 antibodies 17C3 (dark gray), 8B9 (black), and 13A3 (light gray) bound to activated human T cells, which were pre-incubated with one of the following anti-TIM3 antibodies: 3G4, 8B9, 9F6, 17C3, 17C8, and 8C4. Figure 24B In this process, activated human T cells are pre-incubated with either 14H7 or 23B3 antibodies, and then the binding of the 13A3 antibody is measured. Figure 24A and 24B These two figures provide the geometric mean fluorescence intensity of the binding of anti-TIM3 antibody to activated human T cells.

[0370] Figure 25 The amino acid residues essential for the binding of anti-TIM3 monoclonal antibodies 13A3, 3G4, 17C3, and 8B9 to human TIM3 are shown. The signal sequence and transmembrane domain are underlined.

[0371] Figure 26A and 26BSome anti-TIM3 antibodies have shown that they 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 This demonstrates how certain anti-TIM3 antibodies can block the binding of hTIM3-Fc to PS-liposomes, such as through... Figure 26A The results shown are from the PS-hTIM3 “tandem” blocking assay. The upper part displays data for anti-TIM3 antibodies 3G4, 13A3, 17C3, and 17C8. The lower part displays data for anti-TIM3 antibodies 8B9, 8C4, and 9F6. The tables on the right side of each figure provide the PS response time and whether hTIM3-Fc binds to PS-liposomes.

[0372] Figure 27 A summary of the functional activities of various anti-TIM3 antibodies (e.g., TIM3.5 (13A3), TIM3.4 (3G4), TIM3.2 (17C3), TIM3.9 (17C8), 9F6, TIM3.8 (8B9 with S61P substitution in VH), and TIM3.6 (8C4)) is presented. Data for binding assays, T-cell assays, TIL assays, and PS-TIM3 blocking assays are provided.

[0373] Figure 28 A list of all SEQ ID numbers is provided, along with a description of the sequences represented by those SEQ ID numbers.

[0374] Figure 29A and 29B The study demonstrated the antitumor activity of combined administration of antiPD1 and antiTIM3 antibodies in a CT26 colorectal tumor mouse model. Figure 29A The tumor volume of mice (n=10 per group) treated with (i) control IgG (top left panel), (ii) RMT3-23 anti-TIM3 antibody alone (top right panel), (iii) RMP1-14 anti-PD1 antibody alone (bottom left panel), and (iv) a combination of RMT3-23 anti-TIM3 and RMP1-14 anti-PD1 antibody (bottom right panel) is shown at various time points after tumor implantation. Figure 29B The changes in mean tumor volume as a function of time (days after tumor implantation) are shown 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 antibodies.

[0375] Figure 30 A list of common hTIM3 peptides used to map epitopes of anti-TIM3 antibodies (13A3 and 3G4) using hydrogen / deuterium exchange mass spectrometry (HDX-MS) is shown. Each bar represents a peptic peptide. Circled residues (i.e., N99, T145, and N172) indicate glycosylation sites.

[0376] Figure 31 The images show the human TIM3 binding regions of anti-TIM3 antibodies (13A3 and 3G4) identified using HDX-MX. The top image shows the binding region of the 13A3 anti-TIM3 antibody. The bottom image shows the binding region of the 3G4 anti-TIM3 antibody.

[0377] Figure 32A and 32B The results of Scatchard analysis show the binding of TIM3.18.IgG1.3 to CHO cells that ectopically express human or cynomolgus monkey TIM3. Figure 32A Showing 125 I-TIM3 Ab standard curve. Figure 32B The amounts of TIM3.18.IgG1.3 antibody that bind to CHO cells expressing human (left panel) and cynomolgus monkey (right panel) TIM3 are shown. Estimated affinity values ​​and the number of TIM3 molecules expressed on the surface of CHO cells are provided below each figure. These values ​​were calculated as described in Example 4.

[0378] Figure 33 The results of a Scatchard analysis of the binding of TIM3.18.IgG1.3 to activated Th1 cells from two separate donors are shown (left and right panels). 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 34A and 34B The results showed that TIM3.18.IgG1.3 and TIM3.18.IgG1.3 Fab enhanced the proliferation of Th1 T cells in a polarized Th1 / irradiated CHO-OKT3 co-culture assay. Figure 34A Th1 cell proliferation is shown under multiple concentrations of TIM3.18.IgG1.3, 13A3 ("13A3-g4"), no antibody, or allotype control antibody (hIgG1.1 or hIgG4). Each column of antibody represents a different concentration. Figure 34BTh1 cell proliferation is shown at multiple concentrations of TIM3.18.IgG1.3 Fab, without antibody (“ab”), or with the allotype control antibody IgG1.3 (“G1.3”). Each column of TIM3.18 antibody represents a different concentration.

[0380] Figure 35 This study demonstrates 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 comparative purposes. CHO cells untreated with any antibody (“Ab-free”) or treated with the isotype control IgG1.3 antibody (“IgG1.3”) served as negative controls. The horizontal dashed line indicates proliferation relative to the negative control.

[0381] Figure 36 The results show that the anti-TIM3 antibody TIM3.18.IgG1.3 (black circle) enhanced the proliferation of Th1 T cells in combination with nivolumab in a polarized Th1 / irradiated CHO-OKT3-PD-L1 co-culture assay. CHO cells untreated with any antibody (hollow square) or treated with the allotype control IgG1.3 antibody (gray square) served as negative controls.

[0382] Figure 37 The study demonstrated that the anti-TIM3 antibody TIM3.18.IgG1.3 enhanced interferon-γ secretion from tumor-infiltrating lymphocytes (TILs) of renal cell carcinoma stimulated with irradiated CHO-OKT3 cells. TILs stimulated with CHO cells served as negative controls, either in the presence of isotype control antibodies (hIgG4 or hIgG1.1f) or in the absence of antibodies. Each column of TIM3.18 antibody represents a different concentration of the antibody.

[0383] Figure 38 The study showed that the anti-TIM3 antibody TIM3.18.IgG1.3 enhanced interferon-γ secretion in tumor-associated lymphoid tissue (TILs) of breast cancer stimulated with irradiated CHO-OKT3 cells. Assays without antibody or with an allotype control antibody were used as controls. The horizontal dashed line indicates interferon-γ secretion above the negative control.

[0384] Figure 39 Histograms showing the expression of CD163, CD206, and TIM3 on M0 macrophages in the AlloMLR (mixed lymphocyte response) assay are presented, with results shown in [the table / image / etc.]. Figure 40 Each histogram shows the fluorescence intensity of (1) CD163, CD206 or TIM3, (2) the isotype control antibody (i.e., the negative control), and (3) the background fluorescence ("unstained") (i.e., the negative control).

[0385] Figure 40 Cell proliferation was shown in the AlloMLR assay performed with or without anti-TIM3 antibody TIM3.18.IgG1.3, isotype control, or no antibody.

[0386] Figure 41 This is a band 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.

[0387] Figure 42 The structure of the TIM3:TIM3.18 Fab complex, determined by crystallography, is shown. Fab fragments are shown as bands. TIM3 is represented by a white surface, and Fab contact residues are represented by dark gray.

[0388] Figure 43 This is a diagram illustrating an assay for measuring the potential internalization effect of anti-TIM3 antibodies. The left diagram provides a schematic of the assay. The right diagram illustrates the calculation of the internalization value.

[0389] Figure 44 The results show that anti-TIM3 antibody 13A3 (bottom left) and some of its variants (D101E – top left; N60Q – top right) do not trigger receptor (i.e., TIM3)-mediated internalization.

[0390] Figure 45A and 45B The description of anti-TIM3 antibody 13A3 is shown. Figure 45A ) and 3G4 Figure 45B A banding diagram of epitopes for each antibody is shown. The amino acid sequence of the epitope for each antibody is provided below the banding diagram. Different patterns identified specific regions of the anti-TIM3 antibody corresponding to specific epitopes. Invention Details

[0392] To make this specification easier to understand, certain terms are defined first. Further definitions are given throughout the detailed specification.

[0393] It should be noted that the term "a" or "an" entity refers to one or more of the same 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" are used interchangeably herein.

[0394] Furthermore, the term “and / or” as used herein is considered to mean that each of the two specified features or components has or does not have the specific disclosure of the other. Therefore, the term “and / or” as used herein in phrases such as “A and / or B” is intended to include “A and B”, “A or B”, “A only”, and “B only”. Similarly, the term “and / or” as used in phrases 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; A only; B only; and C only.

[0395] It should be understood that wherever the language "contains" or "includes" is used to describe an aspect, similar aspects described as "composed of" and / or "substantially composed of" are also provided.

[0396] 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 in connection with this disclosure. 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 for many of the terms used in this disclosure to those skilled in the art.

[0397] Units, prefixes, and symbols are represented in the form accepted by the Système International de Unites (SI). Numerical ranges include the numbers defining the range. Unless otherwise specified, nucleotide sequences are written from left to right in a 5' to 3' direction. Amino acid sequences are written from left to right in the direction from amino to carboxyl. The headings provided herein are not intended to limit the various aspects of this disclosure, all of which can be obtained by referring to the entire specification. Therefore, the terms defined immediately below are more fully defined by reference to the entire specification.

[0398] The term “about” is used herein to mean approximately, roughly, presumably, or within a range. When the term “about” is used in conjunction with a numerical range, it adjusts the range by extending boundaries above and below the stated numerical value. Typically, the term “about” can adjust for values ​​above or below the stated value by, for example, 10% variance.

[0399] 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 for TIM3 includes phosphatidylserine (TIM3-L). TIM3 is also known as hepatitis A virus cell receptor 2 (HAVCR2), T-cell immunoglobulin mucin receptor 3, TIM-3, TIMD3, TIMD-3, kidney injury molecule 3, KIM-3, and CD366. The term "TIM3" includes any variant or isoform of TIM3 naturally expressed in cells. Therefore, the antibodies described herein are cross-reactive with TIM3 in species other than humans (e.g., cynomolgus monkey TIM3). Alternatively, the antibodies may be specific to human TIM3 and not exhibit any cross-reactivity with other species. TIM3 or any variants and isoforms thereof may be isolated from cells or tissues that naturally express them, or may be recombinantly generated using techniques well known in the art and / or those described herein.

[0400] Two isoforms of human TIM3 have been identified. Isoform 1 (accession number NP_116171; SEQ ID NO:286) consists of 301 amino acids and represents the canonical sequence. Isoform 2 (accession number 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. Amino acid residues 132-142 also differ from the canonical sequence described above.

[0401] The following are the amino acid sequences of two known human TIM3 isoforms.

[0402] (A) Human TIM3 isoform 1 (accession number NP_116171; SEQ ID NO:286; encoded by the nucleotide sequence with accession number NM_032782.4; SEQ ID NO:288); Figure 25 ):

[0403] MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAE TQTLGSLPDINLTQISTLANELRDSRLANDLRDSGATIRIGIYIGAGICAGLALALIFGALIFKWYSHSKEKIQNLSLISLANLPPSGLANAVAEGIRSEENIYTIEENVYEVEEPNEYYCYVSSRQQPSQPLGCRFAMP

[0404] (B) Human TIM3 isoform 2 (accession number AAH20843; SEQ ID NO: 287; encoded by the nucleotide sequence with accession number BC020843.1; SEQ ID NO: 289):

[0405] MFSHLPFDCVLLLLLLLLTRS SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPGEWTFACHLYE

[0406] The signal sequences of isoforms 1 and 2 correspond to amino acids 1-21 (underlined). Therefore, 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:

[0407] SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGATIRIG (SEQ ID NO: 290).

[0408] The cynomolgus monkey TIM3 protein consists of the following amino acid sequence (including the signal sequence):

[0409] MFSHLPFDCVLLLLLLLLTRSSEVEYIAEVGQNAYLPCSYTPAPPGNLVPVCWGKGACPVFDCSNVVLRTENRDVNDRTSGRYWLKGDFHKGDVSLTIENVTLADSGVYCCRIQIPGIMNDEKHNLKLVVIKPAKVTPAPTLQRDLTSA FPRMLTTGEHGPAETQTPGSLPDVNLTQIFTLTNELRDSGATIRTAIYIAAGISAGLALALIFGALIFKWYSHSKEKTQNLSLISLANIPPSGLANAVAEGIRSEENIYTIEEDVYEVEEPNEYYCYVSSGQQPSQPLGCRFAMP(SEQ ID NO:360)

[0410] In some implementations, the term "antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated VH herein) and a heavy chain constant region (abbreviated CH herein). In some 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 VL herein) and a light chain constant region. The light chain constant region consists of a single domain (abbreviated CL herein). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of both the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The heavy chain may or may not have a C-terminal lysine. Unless otherwise stated herein, amino acids in the variable regions are numbered using the Kabat numbering system, while amino acids in the constant regions are numbered using the EU system.

[0411] In some embodiments, the “IgG antibody” used herein, such as human IgG1, IgG2, IgG3, and IgG4 antibodies, has the structure of naturally occurring IgG antibodies, i.e., it has the same number of heavy and light chains and disulfide bonds as naturally occurring IgG antibodies of the same subclass. For example, an anti-TIM3 IgG1, IgG2, IgG3, or IgG4 antibody consists of two heavy chains (HC) and two light chains (LC), wherein the two HCs and LCs are linked by the same number and positions of disulfide bonds present in naturally occurring IgG1, IgG2, IgG3, and IgG4 antibodies, respectively (unless the antibody has been mutated to modify the disulfide bonds).

[0412] Antibodies typically bind to their corresponding antigens with high affinity, which is achieved by 10 -5 Up to 10 -11 M or a smaller dissociation constant (K) D ) reflects. It is generally considered that anything greater than approximately 10 -4 M of K D This indicates non-specific binding. As used herein, an antibody that "specifically binds" to an antigen refers to an antibody that binds to both the antigen and substantially the same antigen with high affinity, meaning it has a 10... -7 M or smaller, 10 -8 M or smaller, 5x10 -9 M or smaller or in 10 -8 M and 10 -10 K between M or smaller D However, it does not bind with high affinity to unrelated antigens. An antigen is considered "substantially identical" to a given antigen if it exhibits high sequence identity; for example, if it exhibits at least 80%, 90%, 95%, 97%, or 99% sequence identity with the given antigen. For instance, in some embodiments, antibodies that specifically bind to human TIM3 may also be cross-reactive with TIM3 antigens from certain primate species (e.g., cynomolgus monkey TIM3), but not with TIM3 antigens from other species or with antigens that are not TIM3.

[0413] Immunoglobulins can be derived from any commonly known isotype, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG isotypes are subclassed in some species: in humans, IgG1, IgG2, IgG3, and IgG4, and in mice, IgG1, IgG2a, IgG2b, and IgG3. In some embodiments, the anti-TIM3 antibody described herein belongs to the IgG1 subtype. Immunoglobulins (e.g., IgG1) exist in several allotypes, differing from each other at most in a few amino acids. For example, “antibody” includes naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies; and fully synthetic antibodies.

[0414] As used herein, the term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human TIM3). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments covered by the term "antigen-binding portion" of an antibody such as the anti-TIM3 antibody described herein include (i) Fab fragments (fragments cleaved by papain) or fragments derived from V... L V H (ii) similar monovalent fragments composed of LC and CH1 domains; (iii) F(ab')2 fragments (fragments derived from pepsin cleavage) or similar divalent fragments containing two Fab fragments linked by disulfide bonds through a hinge region; H (iv) The Fd fragment composed of the CH1 domain; L and V H The Fv segment, composed of structural domains, (v) is composed of V H The dAb fragment consists of domains (Ward et al., (1989) Nature 341:544-546); (vi) separate complementarity-determining regions (CDRs) and (vii) combinations of two or more separate CDRs, which may optionally be connected by a synthetic linker. Furthermore, although the two domains V of the Fv fragment... L and V H Encoded by different genes, but linked together using synthetic linkers via recombination methods, thus forming a single protein chain, where V... L District and V HRegions pair to form monovalent molecules (called single-chain Fvs (scFvs); see, for example, 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 term "antigen-binding moiety" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the utility of the fragments is screened in the same manner as for intact antibodies. Antigen-binding moieties can be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0415] A “bispecific” or “bifunctional antibody” is an artificial hybrid antibody with two distinct heavy / light chain pairs and two distinct binding sites. Bispecific antibodies can be generated by a variety of methods, including hybridoma fusion or Fab fragment linkage. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

[0416] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies, i.e., the individual antibodies contained in the population are substantially similar and bind to the same epitopes (e.g., the antibody exhibits a single binding specificity and affinity), except for possible variants that may arise during the production of monoclonal antibodies, which are typically present in small amounts. The modifier "monoclonal" indicates the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced 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 optional constant regions derived from human germline immunoglobulin sequences. In some embodiments, human monoclonal antibodies are produced by hybridoma, comprising fusing B cells obtained from a transgenic nonhuman animal (e.g., a transgenic mouse) into immortalized cells, wherein the transgenic nonhuman animal has a genome containing human heavy chain transgenes and light chain transgenes.

[0417] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, generated, or isolated in a recombinant manner, such as (a) antibodies isolated from transgenic or transchromosomal animals (e.g., mice) of the human immunoglobulin gene or from hybridomas prepared therefrom, (b) antibodies isolated from transformed host cells intended to express antibodies, such as from transfectomas, (c) antibodies isolated from recombinant combined human antibody libraries, and (d) antibodies prepared, expressed, generated, or isolated by any other means involving splicing a human immunoglobulin gene sequence into other DNA sequences. Such recombinant human antibodies contain variable and constant regions utilizing a specific human germline immunoglobulin sequence encoded by the germline gene, but including subsequent rearrangements and mutations, such as those occurring during antibody maturation. As is known in the art (see, for example, Lonberg (2005) Nature Biotech. 23(9): 1117-1125), the variable region contains an antigen-binding domain encoded by multiple genes that rearrange to form antibodies specific to foreign antigens. In addition to rearrangement, the variable region can be further modified by multiple single-amino acid changes (called somatic mutations or hypermutations) to increase the antibody's affinity for foreign antigens. In a further response to the antigen, the constant region will change (i.e., isotype conversion). Therefore, nucleic acid molecules encoding light and heavy chain immunoglobulin polypeptides that have rearranged and mutated in response to antigens cannot have sequence identity with the original nucleic acid molecule, but are substantially identical or similar (i.e., at least 80% identity).

[0418] A "human" antibody (HuMAb) is an antibody having a variable region, wherein both the framework region and the CDR region are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, that constant region is also derived from a human germline immunoglobulin sequence. Anti-TIM3 antibodies described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced through random or site-specific mutagenesis in vitro or somatic mutations in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which a CDR sequence derived from another mammalian species, such as a mouse, is grafted onto a human framework region sequence. The terms "human" antibody and "fully human" antibody are used synonymously.

[0419] "Humanized" antibodies are antibodies in which some, most, or all of the amino acids outside the CDR domain of a non-human antibody are replaced by corresponding amino acids derived from human immunoglobulins. In some embodiments of the humanized form of the antibody, some, most, or all of the amino acids outside the CDR domain have been replaced by amino acids from human immunoglobulins, while some, most, or all of the amino acids within one or more CDR regions remain unchanged. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are permitted, as long as they do not eliminate the antibody's ability to bind to a specific antigen. "Humanized" antibodies retain antigen specificity similar to that of the original antibody.

[0420] "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 mouse antibody and the constant region is derived from a human antibody.

[0421] 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).

[0422] "Allotype" refers to naturally occurring variants within a specific isotype group that differ in some amino acids (see, for example, Jefferis et al. (2009) mAbs 1:1). The anti-TIM3 antibody described herein can be any allotype. As used herein, antibodies of the isotypes referred to as "IgG1f", "IgG1.1f", or "IgG1.3f" are IgG1, IgG1.1, and IgG1.3 antibodies of the isotype "f", respectively, 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.

[0423] The phrases “antibody that recognizes an antigen” and “antibody that is specific to an antigen” are used interchangeably with the term “antibody that binds specifically to an antigen” in this document.

[0424] As used in this article, “isolated antibody” is intended to refer to an antibody that is essentially free of other proteins and cellular material.

[0425] As used in this article, "antibody that inhibits the binding of TIM3-L to TIM3" refers to an antibody that inhibits the binding of TIM3 to its ligand, such as phosphatidylserine, for example, in binding assays using human TIM3-transfected CHO cells or activated T cells expressing TIM3, which have EC 50Approximately 1 μg / mL or less, such as approximately 0.9 μg / mL or less, approximately 0.85 μg / mL or less, approximately 0.8 μg / mL or less, approximately 0.75 μg / mL or less, approximately 0.7 μg / mL or less, approximately 0.65 μg / mL or less, approximately 0.6 μg / mL or less, approximately 0.55 μg / mL or less, approximately 0.5 μg / mL or less, approximately 0.45 μg / mL or less, approximately 0.4 μg / mL or less, approximately 0.35 μg / mL or less, approximately 0.3 μg / mL or less, approximately 0.25 μg / mL or less, approximately 0.2 μg / mL or less, approximately 0.15 μg / mL or less, approximately 0.1 μg / mL or less, or approximately 0.05 μg / mL or less, in methods known in the art, such as the FACS-based binding assay described herein.

[0426] "Effective functions" refer to the interaction between the antibody's Fc region and an Fc receptor or ligand, or the resulting biochemical event. Exemplary "effective 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 typically require the Fc region to bind to a binding domain (e.g., an antibody variable domain).

[0427] "Fc receptors" or "FcRs" are receptors that bind to the Fc region of immunoglobulins. FcRs that bind IgG antibodies comprise 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). Several properties of human FcγRs are known in the art. Most innate effector cell types co-express one or more activating FcγRs and one inhibitory FcγRIIB, while natural killer (NK) cells selectively express one activating Fc receptor (FcγRIII in mice and FcγRIIIA in humans), but do not express the inhibitory FcγRIIB in mice or humans. Human IgG1 binds to most Fc receptors and is considered equivalent to mouse IgG2a in terms of the type of activating Fc receptor it binds to.

[0428] The “Fc region” (fragment crystallizable region), or “Fc domain”, or simply “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 (C1q) of the classical complement system. Therefore, the Fc region contains the constant regions of the antibody, excluding the first constant region immunoglobulin domains (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 antibody heavy chains; the IgM and IgE Fc regions contain three heavy chain constant domains (CH domains 2-4) on each polypeptide chain. For IgG, the Fc region contains the hinge between the immunoglobulin domains CH2 and CH3, and between the CH1 and CH2 domains. Although the definition of the Fc region boundary of the immunoglobulin heavy chain 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 C-terminus of the heavy chain, where the numbering is based on 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 acids 447 or 446 of IgG (if the C-terminal lysine residue is absent) or 445 (if the C-terminal glycine and lysine residues are absent). As used herein, the Fc region can be a native sequence Fc, including any allotype or variant Fc (e.g., non-naturally occurring Fc). Fc can also refer to the isolated region or, in the case of a protein polypeptide containing Fc, the region itself, such as "binding protein containing the Fc region" or "Fc fusion protein" (e.g., antibody or immunoadhesin).

[0429] A “natural sequence Fc region” or “natural sequence Fc” contains an amino acid sequence identical to that of an Fc region found in nature. Natural sequence human Fc regions include the natural sequence human IgG1 Fc region; the natural sequence human IgG2 Fc region; the natural sequence human IgG3 Fc region; and the natural sequence human IgG4 Fc region, as well as their naturally occurring variants. Natural sequence Fc includes various allotypes of Fc (see, for example, Jefferis et al. (2009) mAbs 1:1).

[0430] The term "epitope" or "antigenic determinant" refers to a site on an antigen (e.g., TIM3) where an immunoglobulin or antibody specifically binds, for example, as defined by a particular method used to identify it. Epitopes can be formed from consecutive amino acids (typically linear epitopes) or from discontinuous amino acids juxtaposed in the ternary folding of a protein (typically conformational epitopes). Epitopes formed from consecutive amino acids are generally, but not always, retained upon exposure to denaturing solvents, while epitopes formed from ternary folds are generally 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 consecutive 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 epitopes include techniques in the art and described herein, such as X-ray crystallography, antigen mutation analysis, two-dimensional nuclear magnetic resonance, and HDX-MS (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, edited by GEMorris (1996)).

[0431] The term "epitope mapping" refers to the process of identifying molecular determinants used for antibody-antigen recognition.

[0432] When referring to two or more antibodies, the term "binding to the same epitope" means that the antibody binds to the same segment of amino acid residues, as determined by a given method. Techniques for determining whether an antibody binds to the "same epitope of TIM3" using the antibodies described herein include, for example, epitope mapping methods, such as X-ray analysis of crystals of antigen:antibody complexes to provide atomic resolution of the epitope; and hydrogen / deuterium exchange mass spectrometry (HDX-MS). Other methods monitor the binding of antibodies to antigen fragments or mutant variations of the antigen, where loss of binding due to modification of amino acid residues within the antigen sequence is generally considered an indicator of epitope composition. Additionally, computer-combined methods for epitope mapping can also be used. These methods depend on the ability of the target antibody to affinity-isolate specific short peptides from a combined phage display peptide library. Antibodies with the same VH and VL or the same CDR1, 2, and 3 sequences are expected to bind to the same epitope.

[0433] "An antibody that competes with another antibody for binding to the 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 for binding to a target, i.e., whether and to what extent one antibody inhibits the binding of the other antibody to the target. In some embodiments, one antibody competes with another and inhibits the binding of the other 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 that is incubated with the target first). Competition assays can be performed as described in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi:10.1101 / pdb.prot4277 or Ed Harlow and David Lane, Chapter 11 of “Using Antibodies,” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999. If two antibodies block each other, the antibodies are “cross-competitive,” blocking at least 50% in both ways, i.e., in the competition assay, regardless of whether one antibody or the other antibody is first contacted with the antigen.

[0434] Competitive binding assays for determining whether two antibodies compete or cross-competitively bind include, for example, competitive binding to T cells expressing TIM3 via flow cytometry, as described in the examples. Other methods include: SPR (e.g., BIACORE), solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competitive 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 labeling sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Press (1988)); solid-phase direct labeling RIA using 1-125 labeling (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid-phase direct biotin-avidin EIA (Cheung et al., Virology) 176:546(1990)); and directly labeled RIA. (Moldenhauer et al., Scand. J. Immunol. 32:77(1990)).

[0435] As used herein, the terms “specific binding,” “selective binding,” “selective binding,” and “specific binding” refer to antibody binding to an epitope on a predetermined antigen. Typically, antibodies (i) bind at a concentration of approximately less than 10... -7 M, if approximately less than 10 -8 M, 10 -9 M or 10 -10 M or even lower equilibrium dissociation constant (K) D ) combination, for example, when using a predetermined antigen (e.g., recombinant human TIM3) as the analyte and an antibody as the ligand, in The antibody is identified in the 2000 instrument by, for example, surface plasmon resonance (SPR) technology, or by Scatchard analysis of antibody binding to antigen-positive cells, and (ii) binds to the predetermined antigen with an affinity at least twice that of a non-specific antigen (e.g., BSA, casein) other than or closely related antigens. Therefore, "antibody that specifically binds to human TIM3" refers to an antibody that binds to 10 -7 M or smaller, such as approximately less than 10 -8 M, 10 -9 M or 10 -10 M or even lower K D Antibodies that bind to soluble or cell-bound human TIM3. Antibodies that "cross-react with cynomolgus monkey TIM3" refer to those formulated with 10... -7 M or smaller, such as approximately less than 10 -8 M, 10 -9 M or 10 -10 M or even lower K D Antibodies that bind to TIM3 in cynomolgus monkeys. 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.

[0436] As used in this article, the term "k" assoc "or"k a "k" refers to the association rate of a specific antibody-antigen interaction, while, as used herein, the term "k" refers to the rate of association of a specific antibody-antigen interaction. dis "or"k d "K" refers to the dissociation rate of a specific antibody-antigen interaction. As used herein, the term "K" is... D " refers to the dissociation constant, which is derived from k d With k a The ratio (i.e., k) d / k a The Kc of the antibody is expressed as a molar concentration (M). The Kc of the antibody can be determined using methods well-known in the art. D Value. Determine antibody K. D Available methods for determining the value include surface plasmon resonance, biosensor systems (such as...) (System) or flow cytometry and Scatchard analysis.

[0437] As used herein, the term "high affinity" for IgG antibodies refers to a K+ affinity for the target antigen. D 10 -8 M or smaller, 10 - 9 M or smaller or 10 -10 M or smaller antibodies. However, the "high affinity" binding can vary for other antibody isotypes. For example, the "high affinity" binding for the IgM isotype is characterized by K... D 10 -10 M or smaller or 10 -8 M or smaller antibodies.

[0438] In the context of in vitro or in vivo assays using antibodies or their antigen-binding fragments, the term "EC" is used. 50 "Refers to the concentration of an antibody or its antigen-binding moiety that induces a 50% maximum response, i.e., the 50% maximum response is half between the maximum response and the baseline."

[0439] As used herein, the term "naturally occurring" applies to objects that can be found in nature. For example, polypeptide or polynucleotide sequences present in organisms (including viruses) are naturally occurring, meaning they can be isolated from nature and have not been intentionally modified by humans.

[0440] A "polypeptide" is a chain containing at least two consecutively linked amino acid residues, with 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 contain one or more polypeptides.

[0441] As used herein, the term "nucleic acid molecule" is intended to include both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded and can be cDNA.

[0442] "Conservative amino acid substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are already defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with 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, the predicted non-essential amino acid residue in the anti-TIM3 antibody is substituted with another amino acid residue from the same side chain family. Methods for identifying conserved substitutions of nucleotides and amino acids that do not eliminate antigen binding are well known in the art (see, for example, 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)).

[0443] For nucleic acids, the term "substantial homology" means that when two nucleic acids or their designated sequences are optimally aligned and compared, the two nucleic acids or their designated sequences are identical in at least about 80%, at least about 90% to 95%, or at least about 98% to 99.5% of their nucleotides with appropriate nucleotide insertions or deletions. Alternatively, substantial homology exists when the segment hybridizes with a complementary sequence of the strand under selected hybridization conditions.

[0444] For peptides, the term "fundamental homology" means that when two peptides or their designated sequences are best aligned and compared, the two peptides or their designated sequences are identical in 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, with appropriate amino acid insertions or deletions.

[0445] The percentage of identity between two sequences is a function of the number of common positions shared by the sequences (i.e., % homology = number of common positions / total number of positions x 100), taking into account the number of gaps and the length of each gap, which need to be introduced for optimal alignment of the two sequences. Mathematical algorithms can be used to perform sequence comparisons and determine the percentage of identity between two sequences, as described in the following non-limiting examples.

[0446] The percentage of identity between two nucleotide sequences can be determined using the GAP procedure in the GCG software package (available from worldwideweb.gcg.com), using the NWSgapdna.CMP matrix and nick weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. Alternatively, the E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) algorithm, incorporated into the ALIGN procedure (version 2.0), can be used to determine the percentage of identity between two nucleotide or amino acid sequences using a PAM120 weighted residue table, nick length penalty of 12, and nick penalty of 4. In addition, the Needleman and Wunsch (J.Mol.Biol.(48):444-453(1970)) algorithm of the GAP program included in the GCG software package (available from http: / / www.gcg.com) can be used to determine the percentage of identity between two amino acid sequences using a Blossum 62 matrix or a 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.

[0447] The nucleic acid and protein sequences described herein can be further used as "query sequences" to search public databases for, for example, to identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J.Mol.Biol.215:403-10. A BLAST nucleotide search can be performed using the NBLAST program with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. A BLAST protein search can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecules described herein. For gap alignments for comparative purposes, gap BLAST can be used as described in Altschul et al., (1997) Nucleic AcidsRes.25(17):3389-3402. When using the BLAST and gap BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. See worldwideweb.ncbi.nlm.nih.gov.

[0448] Nucleic acids can be present in whole cells, cell lysates, or in partially purified or substantially pure forms. When a nucleic acid is purified from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other parts of chromosomes) or proteins, using standard techniques including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other methods well-known in the art, the nucleic acid is “isolated” or “presents as substantially pure.” See F. Ausubel et al., eds., Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

[0449] Nucleic acids, such as cDNA, can be mutated using standard techniques to provide gene sequences. For coding sequences, these mutations can affect the amino acid sequence as needed. In particular, DNA sequences substantially homologous to or derived from the natural V, D, J, constant regions, switches, and other such sequences described herein are considered (where “derived” means a sequence that is identical to or derived from another sequence).

[0450] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop in which an additional DNA segment can be linked. Another type of vector is a viral vector, in which an additional DNA segment can be linked to a viral genome. Some vectors are capable of autonomous replication in the host cells to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and free mammalian vectors). After introduction into a host cell, other vectors (e.g., non-free mammalian vectors) can be integrated into the host cell's genome and thus replicate along with the host genome. Furthermore, some vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Typically, expression vectors useful in recombinant DNA technology are in plasmid form. 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 are also included, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which have equivalent functions.

[0451] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell containing nucleic acids not naturally present in the cell, and may be a cell into which a recombinant expression vector has been introduced. It should be understood that these terms are intended not only to refer to the specific object 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 actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0452] "Immune response" is as understood in the art and generally refers to a biological response in vertebrates against foreign substances or abnormalities such as cancer cells, which protects the organism from these substances and the diseases they cause. An immune response is mediated by the action of one or more cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules, said soluble macromolecules being produced by any of these cells or by the liver (including antibodies, cytokines, and complement), which result in the selective targeting, binding, disruption, destruction, 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. Immune responses include, for example, the activation or suppression of T cells, such as effector T cells, Th cells, CD4+, etc. + Cells, CD8 + T cells or Treg cells, or any other cells that activate or suppress the immune system, such as NK cells.

[0453] "Immunomotor" or "immunomodulatory agent" refers to a substance, such as a substance that targets components of a signaling pathway, which can participate in regulating, modulating, or modifying an immune response. "Regulating," "modifying," or "modifying" an immune response refers to any alteration in the activity of cells of the immune system or 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 changes that may occur in the immune system. Inhibitory and stimulatory immunomodulators have been identified, some of which can have an enhancing function in the tumor microenvironment. In some embodiments, immunomodulators target molecules on the surface of T cells. "Immunomotor target" or "target of immunomodulation" is a molecule, such as a cell surface molecule, that is targeted by binding to a substance, agent, part, compound, or molecule and whose activity is altered by said binding. Immunomotor targets include, for example, receptors ("immunomotor receptors") and receptor ligands ("immunomotor ligands") on the cell surface.

[0454] "Immunotherapy" refers to the treatment of a subject who has a disease, is at risk of developing a disease, or has a relapse of a disease by means of methods including inducing, enhancing, suppressing, or otherwise altering the immune system or immune response.

[0455] "Immunostimulatory therapy" or "immunostimulatory treatment" refers to a therapy that causes an increase (induction or enhancement) in the immune response of a subject, for example, to treat cancer.

[0456] "Enhancing the endogenous immune response" refers to increasing the potency or efficacy of the immune response present in the subject. For example, this can be achieved by overcoming mechanisms that suppress the endogenous host immune response or by stimulating mechanisms that enhance the endogenous host immune response.

[0457] "T effect" eff ") cells refer to T cells with cell lysis 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 cells. eff Cells, such as CD4 + and CD8 + T eff Cells and Th1 cells.

[0458] Enhanced agonist activity of T cell co-stimulatory receptors and / or enhanced antagonist activity of inhibitory receptors can lead to an increased ability to stimulate an immune response or the immune system. This increased ability to stimulate an immune response or the immune system can be measured by assays that measure immune responses (e.g., changes in cytokine or chemokine release, cell lysis activity (determined directly on target cells or indirectly by detecting CD107a or granzyme)). 50 The increase in the multiplier or maximum activity level and proliferation is reflected in the ability to stimulate an immune response or immune system activity, which can be increased by at least 10%, 30%, 50%, 75%, 2 times, 3 times, 5 times or more.

[0459] As used herein, the term "link" refers to the association of two or more molecules. Linkages can be covalent or non-covalent. The link can also be a gene link (i.e., a recombination fusion). Such links can be achieved using a variety of techniques recognized in the art, such as chemical conjugation and recombinant protein production.

[0460] As used herein, “administration” means the physical introduction of a composition containing a therapeutic agent into a subject using a variety of methods and delivery systems known to those skilled in the art. Different routes of administration of the anti-TIM3 antibody described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral administration routes, such as by injection or infusion. As used herein, the phrase “parenteral administration” refers to a method of administration other than enteral and local administration, typically by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, subcutaneous, subcutaneous, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. Alternatively, the antibody described herein may be administered via non-parenteral routes, such as local, epidermal, or mucosal administration routes, such as intranasal, oral, vaginal, rectal, sublingual, or local administration. It can also be administered, for example, once, multiple times, and / or over one or more extended periods.

[0461] As used herein, the term "T cell-mediated response" refers to a response mediated by T cells, including effector T cells (e.g., CD8+). + T cells and helper T cells (e.g., CD4 cells) ... + (Cells). T cell-mediated responses include, for example, T cell cytotoxicity and proliferation.

[0462] As used herein, the term "cytotoxic T lymphocyte (CTL) response" refers to an immune response induced by cytotoxic T cells. CTL responses are primarily mediated by CD8+ cells. + T-cell mediated.

[0463] As used herein, the terms “inhibition” or “blocking” (e.g., referring to the inhibition / blocking of TIM3-L binding to TIM3 on cells) are used interchangeably and include partial and complete inhibition / blocking. In some embodiments, the 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%, for example, as determined further herein. In some embodiments, the anti-TIM3 antibody inhibits the binding of TIM3-L to TIM3 by no more than 50%, such as about 40%, 30%, 20%, 10%, 5%, or 1%, for example, as determined further herein.

[0464] As used herein, the phrase “inhibition of tumor growth” includes any measurable reduction in tumor growth, such as inhibition of tumor growth by at least about 10%, for example, 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%.

[0465] As used in this article, “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 via the lymphatic system or bloodstream.

[0466] As used herein, the term “treatment” means any type of intervention performed on a subject or the process of administering or applying an active agent to a subject in order to reverse, alleviate, improve, suppress, slow, or prevent the progression, development, severity, or recurrence of disease-related symptoms, complications, symptom patterns, or biochemical indicators, or to improve overall survival. Treatment may be for subjects with the disease or for subjects without the disease (e.g., for prevention).

[0467] "Hematologic malignancies" include lymphoma, leukemia, myeloma, or lymphoid malignancies, as well as cancers of the spleen and lymph nodes. Exemplary lymphomas include B-cell lymphoma (B-cell hematologic cancer) and T-cell lymphoma. B-cell lymphomas include Hodgkin's lymphoma and most non-Hodgkin's lymphomas. Non-limiting examples of B-cell lymphomas include diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphocytic lymphoma (overlapping with chronic lymphocytic leukemia), mantle cell lymphoma (MCL), Burkitt lymphoma, mediastinal large B-cell lymphoma, Waldenstrom's macroglobulinemia, marginal zone B-cell lymphoma, marginal zone splenic lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma, and lymphomatoid granulomatosis. Non-limiting examples of T-cell lymphomas include extranodal T-cell lymphoma, cutaneous T-cell lymphoma, anaplastic large cell lymphoma, and angioimmunoblastic T-cell lymphoma. Hematologic 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. Hematologic malignancies also include myelomas, such as, but not limited to, multiple myeloma and smoldering multiple myeloma. The term hematologic malignancies includes other hematologic and / or B-cell or T-cell-related cancers.

[0468] The term "effective amount" or "effective dose" is defined as an amount sufficient to achieve or at least partially achieve the desired effect. A "therapeutic effective amount" or "therapeutic effective dose" of a drug or therapeutic agent means any amount of the drug, when administered alone or in combination with another therapeutic agent, that promotes disease remission, wherein said disease remission is demonstrated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods, or prevention of impairment or disability due to disease distress. A therapeutic effective amount or dose of a drug includes a "preventive effective amount" or "preventive 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 or relapsing into the disease, that inhibits the occurrence or relapse of the disease. The ability of a therapeutic agent to promote disease remission or inhibit the occurrence or relapse of the disease can be assessed using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems, predicting the efficacy of the therapeutic agent in humans, or by determining the activity of the agent in an in vitro assay.

[0469] For example, an anticancer agent is a drug that promotes cancer regression in a subject. In some implementations, a therapeutically effective amount of the drug promotes cancer regression to the point of elimination. "Promoting cancer regression" means that the administration of an effective amount of the drug, alone or in combination with an anticancer 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 disease distress, or other ways of alleviating the patient's disease symptoms. Additionally, the terms "effective" and "efficacy" in relation to treatment include both pharmaceutical efficacy and physiological safety. Pharmaceutical efficacy refers to the drug's ability to promote cancer regression in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (adverse effects) at the cellular, organ, and / or organismal level due to the administration of the drug.

[0470] As an example of treating tumors, a therapeutically effective dose or amount 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% compared to an untreated subject. In some embodiments, a therapeutically effective dose or amount of the drug completely inhibits cell growth or tumor growth, i.e., inhibits cell growth or tumor growth by 100%. The ability of the compound to inhibit tumor growth can be assessed using the assays described below. Alternatively, this property of the composition can be assessed by examining the ability of the compound to inhibit cell growth, and this inhibitory effect can be measured in vitro using assays known to a skilled craftsman. In some embodiments described herein, tumor regression can be observed and last for at least about 20 days, at least about 40 days, or at least about 60 days.

[0471] The term "patient" includes human and other mammalian subjects who receive preventative or therapeutic treatment.

[0472] As used herein, the term "subject" includes any human or non-human animal. For example, the methods and compositions described herein can be used to treat a subject with cancer. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0473] As used in this article, the term "weight-based" amount or dose refers to a dose administered to a patient based on their weight. For example, when a patient weighing 60 kg requires 3 mg / kg of anti-TIM3 antibody, an appropriate amount of anti-TIM3 antibody (i.e., 180 mg) can be calculated and administered.

[0474] For the purposes of this disclosure, the term "fixed dose" means that two or more different antibodies (e.g., anti-TIM3 antibody and a second antibody, such as PD-1 or PD-L1 antibody) are present in the composition in a specific (fixed) proportion 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 implementations, 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, or about 1:1. The ratio of primary antibody (e.g., anti-TIM3 antibody) to secondary antibody is approximately 80 mg to 200 mg, approximately 200:1, approximately 180:1, approximately 160:1, approximately 140:1, approximately 120:1, approximately 100:1, approximately 90:1, approximately 80:1, approximately 70:1, approximately 60:1, approximately 50:1, approximately 40:1, approximately 30:1, approximately 20:1, approximately 15:1, approximately 10:1, approximately 9:1, approximately 8:1, approximately 7:1, approximately 6:1, approximately 5:1, approximately 4:1, approximately 3:1, or approximately 2:1 mg. For example, a 2:1 ratio of anti-TIM3 antibody to PD-1 antibody (e.g., nivolumab) could mean that a vial or injection contains approximately 480 mg of anti-TIM3 antibody and 240 mg of anti-PD-1 antibody, or approximately 2 mg / ml of anti-TIM3 antibody and 1 mg / ml of anti-PD-1 antibody.

[0475] Regarding the methods and dosages described herein, the term "flat dose" refers to the dose administered to a patient regardless of their weight or body surface area (BSA). Therefore, a flat dose is not provided in mg / kg, but rather in an absolute amount of the drug (e.g., anti-TIM3 antibody). For example, a person weighing 60 kg and a person weighing 100 kg would receive the same dose of antibody (e.g., 480 mg anti-TIM3 antibody).

[0476] As used herein, the terms “ug” and “uM” are used interchangeably with “μg” and “μM”, respectively.

[0477] The various aspects described herein will be further described in detail in the following subsections.

[0478] I. Anti-human TIM3 antibody

[0479] This document describes antibodies, such as fully human antibodies, characterized by specific functional features or properties. For example, the antibody specifically binds to human TIM3, and more specifically, binds to a specific domain (e.g., a functional domain) within the extracellular domain of human TIM3. In some embodiments, the antibody specifically binds to a site on TIM3 that binds to TIM3-L. In some embodiments, the antibody is an antagonistic antibody, i.e., it inhibits or suppresses the T-cell inhibitory activity of TIM3 on cells such as T cells. In some embodiments, the anti-TIM3 antibody cross-reacts with TIM3 from one or more non-human primates, such as cynomolgus monkey TIM3. In some embodiments, the antibody specifically binds to the extracellular region of both human TIM3 and cynomolgus monkey TIM3. In some embodiments, the antibody binds to human TIM3 with high affinity.

[0480] The anti-TIM3 antibody described in this article exhibits one or more of the following functional properties:

[0481] (a) Bound to soluble and / or membrane-bound human TIM3;

[0482] (b) Binding to soluble and / or membrane-bound cyno TIM3;

[0483] (c) Inducing or stimulating an immune response;

[0484] (d) Inducing or stimulating T cell activation, such as Th1 cell activation (as demonstrated by enhanced cytokine secretion and / or proliferation);

[0485] (e) Inducing or stimulating T cell proliferation (e.g., CD4+) + CD8 + T cells (Th1 cells or TILs), for example, in the co-culture assay described in the example embodiments;

[0486] (f) Inducing or stimulating the production of IFN-γ from T cells, such as Th1 cells or tumor-infiltrating lymphocytes (TILs), such as TILs from human renal, lung, pancreatic, or breast cancer tumors, as determined in the assays described in the examples;

[0487] (g) Blocking or inhibiting the binding of human TIM3 to PtdSer, for example, as determined in the assay described in the examples;

[0488] (h) When it binds to TIM3 on the cell, it does not internalize or downregulate cell surface TIM3;

[0489] (i) binding to the human TIM3 extracellular domain (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);

[0490] (j) Competitively or cross-blocking the binding of antibodies to human TIM3 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), as determined in the assays described in the examples;

[0491] (k) Binding to human TIM3, but not to human TIM3 having amino acid substitutions with 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, as numbered in SEQ ID NO:286. Figure 25 );and

[0492] (l) Integrate with human TIM3 region 49 VPVCWGKGACPVFE 62 (SEQ ID NO:367) and 111 RIQIPGIMNDEKFNLKL 127 (SEQ ID NO:368), as determined by HDX-MS;

[0493] (m) Having a heavy chain variable region and / or a light chain variable region interacting 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

[0494] (n) Competitively or cross-blocking 13A3 or TIM3.18.IgG1.3 binding to human TIM3, for example, as described in the examples.

[0495] In some implementations, the anti-TIM3 antibody described herein uses high affinity, for example, K... D 10 -7 M or smaller, 10 -8 M or smaller, 10 -9 M or smaller, 10 -10 M or smaller, 10 -11 M or smaller, 10 -12 M or smaller, 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 human TIM3. In some embodiments, the anti-TIM3 antibody binds to soluble human TIM3, for example, as determined by BIACORE (e.g., as described in the examples), in K D 10 -7 M or smaller, 10 -8 M or smaller, 10 -9 M (1nM) or smaller, 10 -10 M or smaller, 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 antibody binds to (e.g., cell membrane-bound) human TIM3, such as on activated human T cells, as determined by flow cytometry and a Scatchard plot, to K. D 10 -7 M or smaller, 10 -8 M or smaller, 10 -9 M (1nM) or smaller, 5x10 -10 M or smaller, 10 -10 M or smaller, 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. In some embodiments, the anti-TIM3 antibody binds to (e.g., cell membrane-bound) human TIM3, such as on activated human T cells, for example, as determined by flow cytometry, to EC2. 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 antibody described herein is, for example, in K D 10 -7 M or smaller, 10 -8 M or smaller, 10 -9 M or smaller, 10 -10 M or smaller, 10 -11 M or smaller, 10 -12 M or smaller, 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, for example, as determined by BIACORE (e.g., as described in the examples), in K D 10 -7 M or smaller, 10 -8 M or smaller, 10 -9 M (1nM) or smaller, 10 -10 M or smaller, 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-binding. Anti-TIM3 antibodies can bind to membrane-bound TIM3 in cynomolgus monkeys, for example, via EC-1. 50100 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, for example, as determined by flow cytometry (e.g., as described in the examples). In some embodiments, the anti-TIM3 antibody binds to (e.g., cell membrane bound) cyno TIM3, such as on activated human T cells, for example, as determined by flow cytometry and Scatchard plot, in K D 10 -7 M or smaller, 10 -8 M or smaller, 10 -9 M (1nM) or smaller, 5x10 -10 M or smaller, 10 -10 M or smaller, 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.

[0496] In some embodiments, the anti-TIM3 antibodies described herein stimulate or enhance the immune response, for example, by activating T cells in a tumor. For example, anti-TIM3 antibodies can activate or co-stimulate cells, as demonstrated by enhanced cytokine (e.g., IFN-γ) secretion and / or enhanced proliferation, which can be caused by inhibition of TIM3-mediated T cell suppressive activity. In some embodiments, T cell activation or co-stimulation induced by TIM3 antibodies occurs in the presence of CD3 stimulation. In some embodiments, anti-TIM3 antibodies increase IFN-γ secretion by a factor of 50%, 100% (i.e., 2-fold), 3-fold, 4-fold, 5-fold, or more, optionally up to a maximum of 10-fold, 30-fold, or 100-fold, as measured, for example, on primary human T cells and / or T cells expressing human TIM3 (e.g., tumor-infiltrating lymphocytes (TILs)).

[0497] In some embodiments, the anti-TIM3 antibody is administered, for example, at a concentration 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 in EC2. 50 Inhibits the binding of phosphatidylserine to human TIM3 on cells expressing human TIM3, such as CHO cells or activated T cells.

[0498] In some embodiments, the anti-TIM3 antibody described herein binds to an epitope, such as a conformational epitope, in the extracellular portion of human TIM3, for example, in the Ig-like domain of the extracellular region, namely amino acids 22 to 202 of SEQ ID NO:286. Figure 25 In some embodiments, the anti-TIM3 antibody binds to epitopes located in amino acids 22 to 120 of the extracellular domain of human TIM3 (SEQ ID NO: 286) or in amino acids 1 to 99 of mature human TIM3 (SEQ ID NO: 290) (see Examples). In some embodiments, the anti-TIM3 antibody binds to or targets an epitope within a region comprising 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 Examples). Figure 25 In some embodiments, the anti-TIM3 antibody binds to or targets an epitope within a region comprising 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 also). Figure 25 In some embodiments, the anti-TIM3 antibody binds to or targets an epitope (CPVFECG, SEQ ID NO: 296) within the region consisting of amino acids 58-64 of human TIM3 having SEQ ID NO: 286; and binds to or targets an epitope (RIQIPGIMND, SEQ ID NO: 298; see also) within the region consisting of amino acids 111-120 of human TIM3 having SEQ ID NO: 286. Figure 25 In some embodiments, the anti-TIM3 antibody binds to or targets an epitope within a region comprising 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 also) Figure 25 ).

[0499] In some embodiments, the anti-TIM3 antibody binds to an epitope substantially identical to that of 13A3, namely, an epitope (or a region of human TIM3) containing one or more amino acid residues C58, P59, F61, E62, C63, R111, and D120 of SEQ ID NO:286. Figure 25In some embodiments, the anti-TIM3 antibody binds to an epitope (or a region of human TIM3) containing one or more 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 to or only binds with significantly reduced affinity to human TIM3 proteins wherein one or more amino acid residues C58, P59, F61, E62, C63, R111, and D120 of SEQ ID NO:286 (e.g., substituted with non-conserved amino acids) are changed to another amino acid. In some embodiments, the anti-TIM3 antibody does not significantly bind to or only binds with significantly reduced affinity to human TIM3 proteins wherein one or more amino acid residues C58, P59, F61, E62, C63, D104, R111, Q113, and D120 of SEQ ID NO:286 (e.g., substituted with non-conserved amino acids) are changed to another amino acid.

[0500] In some embodiments, the anti-TIM3 antibody binds to an epitope substantially identical to that of 3G4, i.e., the epitope (or region of human TIM3) contains one or more amino acid residues of SEQ ID NO:286: C58, P59, V60, F61, E62, C63, G116, and M118. Figure 25 In some embodiments, the anti-TIM3 antibody binds to an epitope (or a region of human TIM3) containing one or more amino acid residues of SEQ ID NO:286, namely C58, P59, V60, F61, E62, C63, D104, G116, and M118. Figure 25 In some embodiments, the anti-TIM3 antibody does not significantly bind to or only binds with significantly reduced affinity to human TIM3 proteins wherein one or more amino acid residues C58, P59, V60, F61, E62, C63, G116, and M118 of SEQ ID NO:286 (e.g., substituted with non-conserved amino acids) are changed to another amino acid. In some embodiments, the anti-TIM3 antibody does not significantly bind to or only binds with significantly reduced affinity to human TIM3 proteins wherein one or more amino acid residues C58, P59, V60, F61, E62, C63, D104, G116, and M118 of SEQ ID NO:286 (e.g., substituted with non-conserved amino acids) are changed to another amino acid.

[0501] In some embodiments, the anti-TIM3 antibody binds to an epitope substantially identical to that of 17C3, namely, an epitope (or region of human TIM3) containing one or more amino acid residues of SEQ ID NO:286, namely C58, P59, V60, F61, E62, C63, G64, and G116. Figure 25 In some embodiments, the anti-TIM3 antibody binds to an epitope (or a region of human TIM3) containing one or more 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 significantly bind to or only binds with significantly reduced affinity to human TIM3 proteins wherein one or more amino acid residues C58, P59, V60, F61, E62, C63, G64, and G116 of SEQ ID NO:286 (e.g., substituted with non-conserved amino acids) are changed to another amino acid. In some embodiments, the anti-TIM3 antibody does not significantly bind to or only binds with significantly reduced affinity to human TIM3 proteins wherein one or more amino acid residues C58, P59, V60, F61, E62, C63, G64, D104, and G116 of SEQ ID NO:286 (e.g., substituted with non-conserved amino acids) are changed to another amino acid.

[0502] In some embodiments, the anti-TIM3 antibody binds to an epitope substantially the same as 8B9, namely, an epitope (or region of human TIM3) containing one or more 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 a region of human TIM3) containing one or more 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 substantially the same as 8B9, namely, an epitope (or region of human TIM3) containing one or more 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 significantly bind to or only binds to human TIM3 proteins with significantly reduced affinity, wherein one or more amino acid residues of SEQ ID NO:286 are L48, W78, S80, R81, W83, G86, D87, and R89. Figure 25(For example, by substitution of non-conserved amino acids) the human TIM3 protein changed to another amino acid. In some embodiments, the anti-TIM3 antibody does not significantly bind or only binds with 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 (For example, by substitution of non-conserved amino acids) the human TIM3 protein is changed to another amino acid. In some embodiments, the anti-TIM3 antibody does not significantly bind or only binds with 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 (For example, human TIM3 protein that is changed to another amino acid by replacing it with a non-conservative amino acid.)

[0503] In some embodiments, the anti-TIM3 antibody competes for (or inhibits) binding to human TIM3 with anti-TIM3 antibodies containing a CDR or variable region as described herein, such as 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 at least 50%, 60%, 70%, 80%, 90%, or 100% of antibodies 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3, or any of TIM3.2 to TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24, and TIM3.25 binding to human TIM3. 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 inhibit the binding of anti-TIM3 antibodies to human TIM3 by at least 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, the anti-TIM3 antibody inhibits at least 50%, 60%, 70%, 80%, 90%, or 100% of the 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, and 1 3A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3 or any of TIM3.2 to TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24 and TIM3.25 inhibit the binding of anti-TIM3 antibodies to human TIM3 by at least 50%, 60%, 70%, 80%, 90% or 100% (e.g., competing in both directions).

[0504] 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:

[0505] (1) Binds to soluble human TIM3, for example, with K D10 nM or less (e.g., 0.01 nM to 10 nM), for example, as determined by Biacore, for example, as described in the examples;

[0506] (2) Binding to soluble cynomolgus monkey TIM3, for example, with K D 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;

[0507] (3) Binding to membrane-bound human TIM3, for example, EC 50 1 ug / mL or less (e.g., 0.01 ug / mL to 1 ug / mL) in combination, for example, as determined by flow cytometry (e.g., as described in the examples);

[0508] (4) Binding to membrane-bound human TIM3, for example, with K D 1 nM or less (e.g., 0.01 nM to 10 nM) in combination, for example, as determined by Scatchard analysis, for example, as described in the examples;

[0509] (5) Binding to membrane-bound cynomolgus monkey TIM3, for example, with EC 50 20 ug / mL or less (e.g., 0.01 ug / mL to 20 ug / mL) in combination, for example, as determined by flow cytometry (e.g., as described in the examples);

[0510] (6) Binding to membrane-bound cynomolgus monkey TIM3, for example, with K D 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;

[0511] (7) (e.g., by blocking or reducing the inhibitory effect of TIM3) induce or enhance T cell activation, as demonstrated by (i) increased IFN-γ production in T cells expressing TIM3 (e.g., Th1 cells or TILs) and / or (ii) enhanced proliferation of T cells expressing TIM3 (e.g., Th1 cells or TILs), for example, as described in the examples.

[0512] (8) Stimulate T cell proliferation in a mixed lymphocyte response (MLR) assay, for example, as described in the examples;

[0513] (9) Inhibit the binding of phosphatidylserine to TIM3, for example, as determined by the PS-hTIM3 "tandem" blocking assay, for example, as described in the examples;

[0514] (10) When bound to TIM3 on the cell, it is not internalized or downregulated;

[0515] (11) Binding 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), for example, as described in the examples;

[0516] (12) Compared to binding with wild-type human TIM3, it has reduced binding with one or more of the following 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 (as indicated in SEQ ID NO:286). Figure 25 The binding of human TIM3 substituted with another amino acid, for example, as described in the examples;

[0517] (13) An antibody that competes with an antibody containing any of the VH and VL domains 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, for example, as described in the examples;

[0518] (14) Combine with human TIM3 area 49 VPVCWGKGACPVFE 62 (SEQ ID NO:367) and 111 RIQIPGIMNDEKFNLKL 127 (SEQ ID NO:368), as measured by HDX-MS, for example, as described in the examples;

[0519] (15) Having a heavy chain variable region and / or a light chain variable region interacting 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; according to SEQ ID NO:286). Figure 25 )); and / or

[0520] (16)(a) Compared to binding to wild-type human TIM3, it has reduced binding to 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids therein: C58, P59, F61, E62, C63, R111, D120, and optionally D104 and Q113 (according to SEQ ID NO:286). Figure 25 (a) Human TIM3 substituted with another amino acid; (b) bound 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) competitive or cross-blocking of 13A3 or TIM3.18.IgG1.3 binding to human TIM3, for example, as described in the examples;

[0521] (17) K D 5x10 -8 M, 2x10 -8 M, 10 -8 M or 5x10 -9 M or smaller binding hTIM3-IgV, as determined by the method described in Example 22;

[0522] (18) K D 10 -7 M, 2x10 -8 M or 10 -8 M or smaller binds to hTIM3-IgV, as determined by the method described in Example 22;

[0523] (19) K D 5x10 -7 M, 2x10-8 M or 10 -8 M or smaller bound to cynomolgus monkey TIM3-ECD, as determined by the method described in Example 22; and / or

[0524] (20) K D 8x10 -8 M, 5x10 -8 M or 10 -8 M or smaller bound to hTIM3-ECD, as determined by the method described in Example 22.

[0525] Therefore, an antibody exhibiting one or more of these functional properties (e.g., biochemical, immunochemical, cellular, physiological, or other biological activities), as determined according to methods known in the art and those described herein, is understood to show a statistically significant difference in said specific activity compared to what is seen in the absence of the antibody (e.g., or when a control antibody of unrelated specificity is present). In some embodiments, an increase in a measurement parameter (e.g., T cell proliferation, cytokine production) induced by the anti-TIM3 antibody in a given assay achieves a statistically significant increase of at least 10%, for example, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% (i.e., 2-fold), 3-fold, 5-fold, or 10-fold; and in some embodiments, the antibody described herein can increase the measurement parameter, for example, by more than 92%, 94%, 95%, 97%, 98%, 99%, 100% (i.e., 2-fold), 3-fold, 5-fold, or 10-fold relative to the same assay performed in the absence of the antibody. Conversely, in a given assay, a reduction in a measurement parameter induced by an anti-TIM3 antibody (e.g., tumor volume, binding of TIM3-L to human TIM3) achieves a statistically significant reduction of at least 10%, such as at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, and in some embodiments, the antibody described herein can reduce the measurement parameter, for example, by more than 92%, 94%, 95%, 97%, 98%, or 99%, relative to the same assay performed without the antibody.

[0526] Standard assays for assessing the binding ability of antibodies to TIM3 of various species are known in the art, including, for example, ELISA, Western blotting, and RIA. Suitable assays are described in detail in the examples. Antibody binding kinetics (e.g., binding affinity) can also be assessed by standard assays known in the art, such as Biacore analysis. Assays for assessing the effects of antibodies on the functional properties of TIM3 (e.g., ligand binding, T cell proliferation, cytokine production) are further described in detail below and in the examples.

[0527] In some implementations, the anti-TIM3 antibody is not a natural antibody or is not a naturally occurring antibody. For example, the anti-TIM3 antibody has different post-translational modifications than naturally occurring antibodies, such as having more, fewer, or different types of post-translational modifications.

[0528] In some embodiments, the anti-TIM3 antibody does not possess agonist activity, as determined, for example, in the cross-linking of the anti-TIM3 antibody in a CHO-OKT3-CD32:T cell co-culture assay, wherein the antibody does not enhance activity above that of anti-TIM3 alone. In some embodiments, the anti-TIM3 antibody blocks the interaction between TIM3 and its ligand without promoting agonist activity.

[0529] In some implementations, anti-TIM3 antibodies enhance IL-12 production in monocytes or dendritic cells treated with LPS.

[0530] In some implementations, anti-TIM3 antibodies, through combination therapy, revitalize tumor-infiltrating CD8 cells that co-express PD-1 and TIM3. + T cells, thus avoiding CD8 + T cell depletion.

[0531] II. Example of an anti-TIM3 antibody

[0532] Specifically, the anti-TIM3 antibody described herein is an isolated antibody and structurally characterized as described herein, having a CDR and / or variable region sequence 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, such as 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) with their variable region or CDR sequence. See PCT / US2017 / 041946, which is incorporated herein 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 NO: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 NO: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 sequence of 9F6 is shown in SEQ ID NO: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 NO:60 and 418. The VL amino acid sequences of the mutant forms of 13A3, 8B9, 9F6, and 23B3 are the sequences of the corresponding non-mutant 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 The document provides a summary of the SEQ ID NO identities.

[0533] Therefore, this article provides isolated antibodies or their antigen-binding moieties 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 NO:34-40, 112-121, 364, 410-412, 449 and 456.

[0534] This article also provides isolated antibodies or their antigen-binding portions, 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 NO:60-63, 417, 418 and 450.

[0535] This article provides isolated anti-human TIM3 antibodies or their antigen-binding moiety, comprising:

[0536] (a) Sequences containing the heavy chain variable region and the light chain variable region of SEQ ID NO:34 and 60, respectively;

[0537] (b) Sequences containing the heavy chain variable region and the light chain variable region of SEQ ID NO:35 and 61, respectively;

[0538] (c) Sequences containing the heavy chain variable region and the light chain variable region of SEQ ID NO:36 and 61, respectively;

[0539] (d) Sequences containing the heavy chain variable region and the light chain variable region of SEQ ID NO:37 and 60, respectively;

[0540] (e) Sequences containing the heavy chain variable region and the light chain variable region of SEQ ID NO:38 and 61, respectively;

[0541] (f) Sequences containing the heavy chain variable region and the light chain variable region of SEQ ID NO:38 and 62, respectively;

[0542] (g) Contains the heavy chain variable region and light chain variable region sequences of SEQ ID NO:38 and 63, respectively;

[0543] (h) contains the heavy chain variable region and light chain variable region sequences of SEQ ID NO:39 and 60, respectively;

[0544] (i) Sequences containing the heavy chain variable region and the light chain variable region of SEQ ID NO:40 and 61, respectively;

[0545] (j) contains the heavy chain variable region and the light chain variable region sequences of SEQ ID NO:121 and 63, respectively;

[0546] (k) contains the heavy chain variable region and light chain variable region sequences of SEQ ID NO:120 and 61, respectively;

[0547] (l) Contains the heavy chain variable region and light chain variable region sequences of SEQ ID NO:112 and 60, respectively;

[0548] (m) contains the heavy chain variable region and light chain variable region sequences of SEQ ID NO:113 and 60, respectively;

[0549] (n) contains the heavy chain variable region and light chain variable region sequences of SEQ ID NO:114 and 60, respectively;

[0550] (o) contains the heavy chain variable region and light chain variable region sequences of SEQ ID NO:115 and 60, respectively;

[0551] (p) contains the heavy chain variable region and light chain variable region sequences of SEQ ID NO:116 and 60, respectively;

[0552] (q) contains the heavy chain variable region and light chain variable region sequences of SEQ ID NO:117 and 60, respectively;

[0553] (r) contains the heavy chain variable region and light chain variable region sequences of SEQ ID NO:118 and 60, respectively;

[0554] (s) respectively contain the heavy chain variable region and the light chain variable region sequences of SEQ ID NO:119 and 60;

[0555] (t) contains the heavy chain variable region and light chain variable region sequences of SEQ ID NO:364 and 60, respectively;

[0556] (u) contains the heavy chain variable region and light chain variable region sequences of SEQ ID NO:410 and 417, respectively;

[0557] (v) Contains the heavy chain variable region and light chain variable region sequences of SEQ ID NO:411 and 60, respectively;

[0558] (w) contains the heavy chain variable region and light chain variable region sequences of SEQ ID NO:411 and 418, respectively;

[0559] (x) contains the heavy chain variable region and light chain variable region sequences of SEQ ID NO:412 and 60, respectively;

[0560] (y) contains the heavy chain variable region and light chain variable region sequences of SEQ ID NO:449 and 450, respectively;

[0561] (z) contains the heavy chain variable region and light chain variable region sequences of SEQ ID NO:449 and 63, respectively;

[0562] (aa) contains the heavy chain variable region and light chain variable region sequences of SEQ ID NO:456 and 450, respectively; or

[0563] (bb) contains the heavy chain variable region and light chain variable region sequences of SEQ ID NO:456 and 63, respectively.

[0564] Anti-TIM3 antibodies may comprise heavy chain CDR1, CDR2, and CDR3 of 13A3, 8B9, 8C4, 17C3, 9F6, 3G4, 17C8, 14H7, 23B3, or any of TIM3.2 to TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24, or TIM3.25, and light chain CDR1, CDR2, and CDR3, or combinations thereof. The amino acid sequences of VH CDR1 of 13A3, 8B9, 8C4, and 17C3 are shown in SEQ ID NO:41-44, respectively. The amino acid sequences of VH CDR1 of 9F6, 3G4, 17C8, 14H7, and 23B3 are shown in SEQ ID NO:45. The amino acid sequence of VH CDR1 of the mutated 13A3 antibody (i.e., TIM3.10-TIM3.18) is identical to that of the non-mutated 13A3 antibody, i.e., SEQ ID NO:41. The amino acid sequence of VH CDR1 of the mutated 8B9 antibody (i.e., TIM3.8) is identical to that of the non-mutated 8B9 antibody, i.e., SEQ ID NO:42. The amino acid sequence of VH CDR1 of the mutated 9F6 antibody (i.e., TIM3.7) is identical to that of the non-mutated 9F6 antibody, i.e., SEQ ID NO:45. The amino acid sequence of VH CDR1 of the mutated 23B3 antibody (i.e., TIM3.25) is identical to that of the non-mutated 23B3 antibody, i.e., SEQ ID NO:45.

[0565] The amino acid sequences of VH CDR2 for 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 VH CDR2 for the mutant 13A3 antibodies TIM3.10, TIM3.17, and TIM3.18 are shown in SEQ ID NO:122. The amino acid sequences of VH CDR2 for the mutant 13A3 antibodies TIM3.11 and TIM3.12 are shown in SEQ ID NO:123 and 124, respectively. The amino acid sequences of VH CDR2 for the mutant 13A3 antibodies TIM3.13 and TIM3.16 are the same as the amino acid sequences of VH CDR2 for the non-mutated 13A3 antibodies, i.e., SEQ ID NO:46. The amino acid sequence of the mutant 8B9 antibody (i.e., TIM3.8) VH CDR2 is shown in SEQ ID NO:125. The amino acid sequence of the mutant 9F6 antibody (i.e., TIM3.7) VH CDR2 is identical to that of the non-mutated 9F6 antibody, i.e., SEQ ID NO:50. The amino acid sequence of the mutant 23B3 antibody (i.e., TIM3.25) VHCDR2 is identical to that of the non-mutated 23B3 antibody, i.e., SEQ ID NO:415.

[0566] The amino acid sequences of 13A3, 8B9, 8C4, 17C3, 9F6, 3G4, 17C8, 14H7, and 23B3 VH CDR3 are shown in SEQ ID NO:53-59, 414, and 416, respectively. The amino acid sequences of the mutant 13A3 antibodies TIM3.10 to TIM3.12 VH CDR3 are the sequences of the non-mutated 13A3 antibodies, i.e., SEQ ID NO:53. The amino acid sequences of the mutant 13A3 antibodies TIM3.13 and TIM3.18 VH CDR3 are shown in SEQ ID NO:126. The amino acid sequences of the mutant 13A3 antibodies TIM3.15 and TIM3.17 VH CDR3 are shown in SEQ ID NO:128. The amino acid sequences of the mutant 13A3 antibodies TIM3.14 and TIM3.16 VH CDR3 are shown in SEQ ID NO:127 and 129, respectively. The amino acid sequence of the mutated 8B9 antibody (i.e., TIM3.8) VH CDR3 is the same as that of the non-mutated 8B9 antibody, i.e., SEQ ID NO:54. The amino acid sequence of the mutated 9F6 antibody (i.e., TIM3.7) VH CDR3 is the same as that of the non-mutated 9F6 antibody, i.e., SEQ ID NO:57. The amino acid sequence of the mutated 23B3 antibody (i.e., TIM3.25) VH CDR3 is the same as that of the non-mutated 23B3 antibody, i.e., SEQ ID NO:416.

[0567] The amino acid sequences of 13A3, 8B9, 8C4, 17C3, 3G4, 17C8, 14H7, and 23B3 VL CDR1 are shown in SEQ ID NO: 64. The amino acid sequence of 9F6 VL CDR1 is shown in SEQ ID NO: 64 and 65. The amino acid sequences of 13A3, 8B9, 8C4, 17C3, 3G4, 17C8, 14H7, and 23B3 VL CDR2 are shown in SEQ ID NO: 66. The amino acid sequence of 9F6 VL CDR2 is shown in SEQ ID NO: 66 and 67. The amino acid sequences of 13A3, 17C3, and 3G4 VL CDR3 are shown in SEQ ID NO: 68. The amino acid sequences of 8B9, 8C4, 17C8, and 14H7 VL CDR3 are shown in SEQ ID NO: 69. The amino acid sequence of 9F6 VL CDR3 is shown in SEQ ID NO: 69. The amino acid sequences of 23B3 VL CDR3 are shown in SEQ ID NOs: 69, 70, and 71. The amino acid sequences of the mutant antibodies 13A3, 8B9, 9F6, and 23B3 VL CDR are those of the corresponding non-mutated antibodies. Figure 16 The SEQ ID NO table of the anti-TIM3 antibody CDR described herein is provided.

[0568] The Kabat system is used to describe CDR regions (Kabat, EA 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 among schemes known as the EU index or EU numbering system, which is based on the sequence number 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 Chothia, IMGT, Martin (enhanced Chothia), or AHo numbering schemes.

[0569] Assuming that each of these antibodies binds to human TIM3, and that antigen-binding specificity is primarily provided by CDR1, 2, and 3 regions, then it is possible to “mix and match” VH CDR1, 2, and 3 sequences and VL CDR1, 2, and 3 sequences, for example, Figure 16 Those sequences (i.e., CDRs from different antibodies can be mixed and matched, although each antibody must contain VH CDR1, 2, and 3 and VL CDR1, 2, and 3) to create other anti-TIM3 binding molecules described herein. TIM3 binding to such “mixed and matched” antibodies can be tested using binding assays (e.g., ELISA) described above and in the examples. In some embodiments, when mixing and matching VH CDR sequences, CDR1, CDR2, and / or CDR3 sequences from a specific VH sequence are replaced with structurally similar CDR sequences. Similarly, when mixing and matching VL CDR sequences, CDR1, CDR2, and / or CDR3 sequences from a specific VL sequence are replaced with structurally similar CDR sequences. It will be apparent to those skilled in the art that new VH and VL sequences can be created by replacing one or more VH and / or VL CDR region sequences with structurally similar sequences from the CDR sequences of the monoclonal antibodies 13A3, 8B9, 8C4, 17C3, 9F6, 3G4, 17C8, 14H7, 23B3, and any of TIM3.2 to TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24, and TIM3.25 disclosed herein.

[0570] This article provides isolated anti-human TIM3 antibodies or their antigen-binding moiety, containing

[0571] (a) Heavy chain variable region CDR1, containing an amino acid sequence selected from SEQ ID NO:41-45 and 469;

[0572] (b) Heavy chain variable region CDR2, comprising an amino acid sequence selected from SEQ ID NO:46-52, 122-125, 413, 415 and 470;

[0573] (c) Heavy chain variable region CDR3, comprising an amino acid sequence selected from SEQ ID NO:53-59, 126-129, 414, 416 and 471;

[0574] (d) The light chain variable region CDR1 contains an amino acid sequence selected from SEQ ID NO:64-65 and 472;

[0575] (e) The light chain variable region CDR2, comprising an amino acid sequence selected from SEQ ID NO: 66-67 and 473; and

[0576] (f) Light chain variable region CDR3, containing an amino acid sequence selected from SEQ ID NO:68-71, 419 and 474;

[0577] The antibody specifically binds to human TIM3.

[0578] 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 regions CDR1, CDR2, and CDR3 contain:

[0579] (a)SEQ ID NO:41, 46, 53;

[0580] (b)SEQ ID NO:42, 47, 54;

[0581] (c)SEQ ID NO:43, 48, 55;

[0582] (d)SEQ ID NO:44, 49, 56;

[0583] (e)SEQ ID NO:45, 50, 57;

[0584] (f)SEQ ID NO:45, 51, 58;

[0585] (g)SEQ ID NO:45, 52, 59;

[0586] (h)SEQ ID NO:41, 122, 53;

[0587] (i)SEQ ID NO:41, 123, 53;

[0588] (j)SEQ ID NO:41, 124, 53;

[0589] (k)SEQ ID NO:41, 46, 126;

[0590] (l)SEQ ID NO:41, 46, 127;

[0591] (m)SEQ ID NO:41, 46, 128;

[0592] (n)SEQ ID NO:41, 46, 129;

[0593] (o) SEQ ID NO: 41, 122, 128;

[0594] (p) SEQ ID NO: 41, 122, 126;

[0595] (q) SEQ ID NO: 45, 413, 414;

[0596] (r)SEQ ID NO:45, 415, 416; or

[0597] (s)SEQ ID NO:469, 470 and 471,

[0598] The antibody specifically binds to human TIM3.

[0599] 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 regions CDR1, CDR2, and CDR3 contain:

[0600] (a)SEQ ID NO: 64, 66, 68;

[0601] (b)SEQ ID NO: 64, 66, 69;

[0602] (c)SEQ ID NO: 65, 67, 70;

[0603] (d)SEQ ID NO: 64, 66, 71;

[0604] (e)SEQ ID NO: 64, 66, 419; or

[0605] (f) SEQ ID NO:472, 473, 474,

[0606] The antibody specifically binds to human TIM3.

[0607] In some implementations, the anti-TIM3 antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0608] (a1) The heavy chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:41, 46 and 53 respectively, and the light chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:64, 66 and 68 respectively;

[0609] (a2) The heavy chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:41, 122 and 53 respectively, and the light chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:64, 66 and 68 respectively;

[0610] (a3) The heavy chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:41, 123 and 53 respectively, and the light chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:64, 66 and 68 respectively;

[0611] (a4) The heavy chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:41, 124 and 53 respectively, and the light chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:64, 66 and 68 respectively;

[0612] (a5) The heavy chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:41, 46 and 126 respectively, and the light chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:64, 66 and 68 respectively;

[0613] (a6) The heavy chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:41, 46 and 127 respectively, and the light chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:64, 66 and 68 respectively;

[0614] (a7) The heavy chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:41, 46 and 128 respectively, and the light chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:64, 66 and 68 respectively;

[0615] (a8) The heavy chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:41, 46 and 129 respectively, and the light chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:64, 66 and 68 respectively;

[0616] (a9) The heavy chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:41, 122 and 128 respectively, and the light chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:64, 66 and 68 respectively;

[0617] (a10) The heavy chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:41, 122 and 126 respectively, and the light chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:64, 66 and 68 respectively;

[0618] (b1) The heavy chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:42, 47 and 54 respectively, and the light chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:64, 66 and 69 respectively;

[0619] (b2) The heavy chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:42, 125 and 54 respectively, and the light chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:64, 66 and 69 respectively;

[0620] (c) The heavy chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:43, 48 and 55 respectively, and the light chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:64, 66 and 69 respectively;

[0621] (d) The heavy chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:44, 49 and 56 respectively, and the light chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:64, 66 and 68 respectively;

[0622] (e1) The heavy chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:45, 50 and 57 respectively, and the light chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:64, 66 and 69 respectively;

[0623] (e2) The heavy chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:45, 50 and 57 respectively, and the light chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:64, 66 and 71 respectively;

[0624] (e3) The heavy chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:45, 50 and 57 respectively, and the light chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:65, 67 and 70 respectively;

[0625] (f) The heavy chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:45, 51 and 58 respectively, and the light chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:64, 66 and 68 respectively;

[0626] (g) The heavy chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:45, 52 and 59 respectively, and the light chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:64, 66 and 69 respectively;

[0627] (h) The heavy chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:45, 413 and 414 respectively, and the light chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:64, 66 and 69 respectively;

[0628] (i1) The heavy chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:45, 415 and 416 respectively, and the light chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:64, 66 and 68 respectively;

[0629] (i2) The heavy chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:45, 415 and 416 respectively, and the light chain variable regions CDR1, CDR2 and CDR3 contain SEQ ID NO:64, 66 and 419 respectively;

[0630] (j1) The heavy chain variable regions CDR1, CDR2, and CDR3 contain SEQ ID NO: 469, 470, and 471, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 contain SEQ ID NO: 472, 473, and 474, respectively; or

[0631] (j2) The heavy chain variable regions CDR1, CDR2, and CDR3 contain SEQ ID NO:469, 470, and 471, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 contain SEQ ID NO:64, 66, and 71, respectively.

[0632] The antibody specifically binds to human TIM3.

[0633] The VH domain or one or more of its CDRs 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 of its CDRs 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 (which may be absent except for the C-terminal lysine (K), or except for the C-terminal glycine and lysine (GK)) and the full-length light chain combine to form a full-length antibody.

[0634] The VH domains described herein can be fused with a constant domain of naturally occurring or modified human IgG, such as IgG1, IgG2, IgG3, or IgG4, for example, as further described herein. For example, the VH domain may comprise an amino acid sequence of any VH domain described herein fused with a constant region of human IgG, such as IgG1, which is, for example, the following wild-type human IgG1 constant domain amino acid sequence:

[0635] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:291)

[0636] Or an allotype of SEQ ID NO:291, having the following amino acid sequence:

[0637]

[0638] The VH domain of an anti-TIM3 antibody may contain an amino acid sequence of any VH domain described herein fused to a constant domain without effector function, such as the following amino acid sequence of the constant domain of human IgG1 without effector function:

[0639]

[0640] or

[0641]

[0642] For example, IgG1 allotypes contain K97R, D239E, and / or L241M (underlined in bold above) and are numbered according to SEQ ID NO:277, 294, and 295. In the full-length heavy chain region, for example, 8C4 (SEQ ID NO:3) and according to EU numbers, 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) as numbered in SEQ ID NO:277, 294, and 295, or at L234, A235, G237, A330, and P331 according to EU numbers. In some embodiments, the constant region of the anti-TIM3 antibody contains one or more mutations or substitutions at amino acids L117A, A118E, G120A, A213S, and P214S of SEQ ID NO:291, or at L234A, L235E, G237A, A330S, and P331S according to EU numbers. The constant region of the anti-TIM3 antibody may also contain one or more mutations or substitutions in L117A, A118E, and G120A of SEQ ID NO:291, or in L234A, L235E, and G237A according to EU numbers.

[0643] Alternatively, the VH domain of the anti-TIM3 antibody may comprise an amino acid sequence of any VH domain described herein fused to a human IgG4 constant region, for example, the human IgG4 constant region being the following human IgG4 amino acid sequence or a variant thereof:

[0644] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:292, including S228P).

[0645] The VL domains described herein can be fused to constant domains of the human κ or λ light chain. For example, the VL domain of an anti-TIM3 antibody may comprise an amino acid sequence of any VL domain described herein fused to the following human IgG1κ light chain amino acid sequence:

[0646] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:278)

[0647] In some embodiments, the heavy chain constant region contains a lysine or another amino acid at the C-terminus, for example, it contains the following last amino acid in the heavy chain: LSPGK (SEQ ID NO: 279). In some embodiments, the heavy chain constant region lacks one or more amino acids at the C-terminus and has, for example, the C-terminal sequence LSPG (SEQ ID NO: 280) or LSP (SEQ ID NO: 281).

[0648] The exemplary amino acid sequences of the heavy and light chains correspond to SEQ ID NO:1-28, 72-111, 301-354 of the heavy chain and SEQ ID NO:29-30 and 32-33 of the light chain.

[0649] This article provides isolated anti-human TIM3 antibodies or their antigen-binding moiety, comprising:

[0650] (a1) Contains heavy chain and light chain sequences of SEQ ID NO:301 (or 302) and 29, respectively;

[0651] (a2) Contains heavy chain and light chain sequences of SEQ ID NO:1 (or 8) and 29, respectively;

[0652] (a3) Contains heavy chain and light chain sequences of SEQ ID NO:15 (or 22) and 29, respectively;

[0653] (a4) Contains heavy chain and light chain sequences of SEQ ID NO:303 (or 304) and 29, respectively;

[0654] (a5) contains heavy chain and light chain sequences of SEQ ID NO:72 (or 82) and 29, respectively;

[0655] (a6) contains heavy chain and light chain sequences of SEQ ID NO:92 (or 102) and 29, respectively;

[0656] (a7) Contains heavy chain and light chain sequences of SEQ ID NO:305 (or 306) and 29, respectively;

[0657] (a8) contains the heavy chain and light chain sequences of SEQ ID NO:73 (or 83) and 29, respectively;

[0658] (a9) contains heavy chain and light chain sequences of SEQ ID NO:93 (or 103) and 29, respectively;

[0659] (a10) contains the heavy chain and light chain sequences of SEQ ID NO:307 (or 308) and 29, respectively;

[0660] (a11) contains the heavy chain and light chain sequences of SEQ ID NO:74 (or 84) and 29, respectively;

[0661] (a12) contains the heavy chain and light chain sequences of SEQ ID NO:94 (or 104) and 29, respectively;

[0662] (a13) contains heavy chain and light chain sequences of SEQ ID NO:309 (or 310) and 29, respectively;

[0663] (a14) contains the heavy chain and light chain sequences of SEQ ID NO:75 (or 85) and 29, respectively;

[0664] (a15) contains the heavy chain and light chain sequences of SEQ ID NO:95 (or 105) and 29, respectively;

[0665] (a16) contains the heavy chain and light chain sequences of SEQ ID NO:311 (or 312) and 29, respectively;

[0666] (a17) contains the heavy chain and light chain sequences of SEQ ID NO:76 (or 86) and 29, respectively;

[0667] (a18) contains the heavy chain and light chain sequences of SEQ ID NO:96 (or 106) and 29, respectively;

[0668] (a19) contains the heavy chain and light chain sequences of SEQ ID NO:313 (or 314) and 29, respectively;

[0669] (a20) contains the heavy chain and light chain sequences of SEQ ID NO:77 (or 87) and 29, respectively;

[0670] (a21) contains heavy chain and light chain sequences of SEQ ID NO:97 (or 107) and 29, respectively;

[0671] (a22) contains the heavy chain and light chain sequences of SEQ ID NO:315 (or 316) and 29, respectively;

[0672] (a23) contains the heavy chain and light chain sequences of SEQ ID NO:78 (or 88) and 29, respectively;

[0673] (a24) contains the heavy chain and light chain sequences of SEQ ID NO:98 (or 108) and 29, respectively;

[0674] (a25) contains the heavy chain and light chain sequences of SEQ ID NO:317 (or 318) and 29, respectively;

[0675] (a26) contains the heavy chain and light chain sequences of SEQ ID NO:79 (or 89) and 29, respectively;

[0676] (a27) contains heavy chain and light chain sequences of SEQ ID NO:99 (or 109) and 29, respectively;

[0677] (a28) contains the heavy chain and light chain sequences of SEQ ID NO:319 (or 320) and 29, respectively;

[0678] (a29) contains the heavy chain and light chain sequences of SEQ ID NO:349 (or 350) and 29, respectively;

[0679] (a30) contains the heavy chain and light chain sequences of SEQ ID NO:351 (or 352) and 29, respectively;

[0680] (a31) contains heavy chain and light chain sequences of SEQ ID NO:353 (or 354) and 29, respectively;

[0681] (b1) Contains heavy chain and light chain sequences of SEQ ID NO:321 (or 322) and 30, respectively;

[0682] (b2) Contains heavy chain and light chain sequences of SEQ ID NO:2 (or 9) and 30, respectively;

[0683] (b3) Contains heavy chain and light chain sequences of SEQ ID NO:16 (or 23) and 30, respectively;

[0684] (b4) Contains heavy chain and light chain sequences of SEQ ID NO:323 (or 324) and 30, respectively;

[0685] (b5) Contains heavy chain and light chain sequences of SEQ ID NO:80 (or 90) and 30, respectively;

[0686] (b6) Contains heavy chain and light chain sequences of SEQ ID NO: 100 (or 110) and 30, respectively;

[0687] (b7) Contains heavy chain and light chain sequences of SEQ ID NO:325 (or 326) and 30, respectively;

[0688] (c1) Contains heavy chain and light chain sequences of SEQ ID NO:327 (or 328) and 30, respectively;

[0689] (c2) Contains heavy chain and light chain sequences of SEQ ID NO:3 (or 10) and 30 respectively;

[0690] (c3) Contains heavy chain and light chain sequences of SEQ ID NO:17 (or 24) and 30, respectively;

[0691] (c4) Contains heavy chain and light chain sequences of SEQ ID NO:329 (or 330) and 30, respectively;

[0692] (d1) contains heavy chain and light chain sequences of SEQ ID NO:331 (or 332) and 29, respectively;

[0693] (d2) Contains heavy chain and light chain sequences of SEQ ID NO:4 (or 11) and 29, respectively;

[0694] (d3) contains the heavy chain and light chain sequences of SEQ ID NO:18 (or 25) and 29, respectively;

[0695] (d4) Contains heavy chain and light chain sequences of SEQ ID NO:333 (or 334) and 29, respectively;

[0696] (e1.1) Contains heavy chain and light chain sequences of SEQ ID NO:335 (or 336) and 32, respectively;

[0697] (e1.2) Contains heavy chain and light chain sequences of SEQ ID NO:335 (or 336) and 33, respectively;

[0698] (e1.3) contains the heavy chain and light chain sequences of SEQ ID NO:335 (or 336) and 31, respectively;

[0699] (e2) Contains heavy chain and light chain sequences of SEQ ID NO:5 (or 12) and 33, respectively;

[0700] (e3) contains heavy chain and light chain sequences of SEQ ID NO:19 (or 26) and 33, respectively;

[0701] (e4) Contains heavy chain and light chain sequences of SEQ ID NO:337 (or 338) and 33, respectively;

[0702] (e5) contains heavy chain and light chain sequences of SEQ ID NO:81 (or 91) and 33, respectively;

[0703] (e6) contains heavy chain and light chain sequences of SEQ ID NO: 101 (or 111) and 33, respectively;

[0704] (e7) Contains heavy chain and light chain sequences of SEQ ID NO:339 (or 340) and 33, respectively;

[0705] (f1) contains heavy chain and light chain sequences of SEQ ID NO:341 (or 342) and 29, respectively;

[0706] (f2) Contains heavy chain and light chain sequences of SEQ ID NO:6 (or 13) and 29, respectively;

[0707] (f3) contains heavy chain and light chain sequences of SEQ ID NO:20 (or 27) and 29, respectively;

[0708] (f4) contains heavy chain and light chain sequences of SEQ ID NO:343 (or 344) and 29, respectively;

[0709] (g1) contains heavy chain and light chain sequences of SEQ ID NO:345 (or 346) and 30, respectively;

[0710] (g2) contains heavy chain and light chain sequences of SEQ ID NO:7 (or 14) and 30, respectively;

[0711] (g3) contains the heavy chain and light chain sequences of SEQ ID NO:21 (or 28) and 30, respectively;

[0712] (g4) contains the heavy chain and light chain sequences of SEQ ID NO:347 (or 348) and 30, respectively;

[0713] (h1) contains heavy chain and light chain sequences of SEQ ID NO:386 (or 387) and 408, respectively;

[0714] (h2) Contains heavy chain and light chain sequences of SEQ ID NO:388 (or 389) and 408, respectively;

[0715] (h3) contains heavy chain and light chain sequences of SEQ ID NO:390 (or 391) and 408, respectively;

[0716] (h4) contains the heavy chain and light chain sequences of SEQ ID NO:392 (or 393) and 408, respectively;

[0717] (i1.1) contains the heavy chain and light chain sequences of SEQ ID NO:394 (or 395) and 29, respectively;

[0718] (i1.2) contain heavy chain and light chain sequences of SEQ ID NO:394 (or 395) and 409, respectively;

[0719] (i2) Contains heavy chain and light chain sequences of SEQ ID NO:396 (or 397) and 29, respectively;

[0720] (i3) Contains heavy chain and light chain sequences of SEQ ID NO:398 (or 399) and 29, respectively;

[0721] (i4) contains heavy chain and light chain sequences of SEQ ID NO:400 (or 401) and 29, respectively;

[0722] (i5) contains heavy chain and light chain sequences of SEQ ID NO:402 (or 403) and 29, respectively;

[0723] (i6) contains heavy chain and light chain sequences of SEQ ID NO:404 (or 405) and 29, respectively;

[0724] (i7) contains heavy chain and light chain sequences of SEQ ID NO:406 (or 407) and 29, respectively;

[0725] (j1) contains heavy chain and light chain sequences of SEQ ID NO:451 (or 461) and 453, respectively;

[0726] (j2) contains heavy chain and light chain sequences of SEQ ID NO:452 (or 462) and 453, respectively;

[0727] (k1) contains the heavy chain and light chain sequences of SEQ ID NO:454 (or 463) and 33, respectively;

[0728] (k2) contains the heavy chain and light chain sequences of SEQ ID NO:455 (or 464) and 33, respectively;

[0729] (l1) Contains heavy chain and light chain sequences of SEQ ID NO:457 (or 465) and 453 respectively;

[0730] (l2) Contains heavy chain and light chain sequences of SEQ ID NO:458 (or 466) and 453 respectively;

[0731] (m1) contains the heavy chain and light chain sequences of SEQ ID NO:459 (or 467) and 33, respectively; or

[0732] (m2) contains the heavy chain and light chain sequences of SEQ ID NO:460 (or 468) and 33, respectively.

[0733] The antibody specifically binds to human TIM3.

[0734] In some embodiments, the anti-TIM3 antibody comprises a combination of heavy and light chain sequences, such as those described herein 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 a disulfide bond linking each light chain to each heavy chain.

[0735] Heavy and light chains containing at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, or 70% of the same amino acid sequence as any of the heavy or light chains shown herein, such as SEQ ID NO: 1-33, 72-111, 301-354, 386-409, 451, 452, 453, 454, 455, 457, 458, 459, 460, and 461-468, may be used to form anti-human TIM3 antibodies with the desired characteristics, such as those further described herein. Exemplary variants are those that contain, for example, allogeneic variations in the constant domain and / or mutations (e.g., mutations disclosed herein) in the variable or constant regions. Heavy and light chains containing an amino acid sequence that differs from any of the heavy or light chains (or their variable regions) shown herein by a maximum of 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) can be used to form anti-human TIM3 antibodies with the desired characteristics, such as those further described herein.

[0736] In some embodiments, the above-mentioned antibodies 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:

[0737] (1) Binds to soluble human TIM3, for example, with a KD 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;

[0738] (2) Binding to soluble cynomolgus monkey TIM3, for example, with a KD 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;

[0739] (3) Human TIM3 bound to the membrane, for example, at EC50 1 ug / mL or less (e.g., 0.01 ug / mL to 1 ug / mL), for example, as determined by flow cytometry (e.g., as described in the examples);

[0740] (4) Binding to membrane-bound human TIM3, for example, with K D 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;

[0741] (5) Binding to membrane-bound cynomolgus monkey TIM3, for example, at EC50 20 ug / mL or less (e.g., 0.01 ug / mL to 20 ug / mL), for example, as determined by flow cytometry (e.g., as described in the examples);

[0742] (6) Binding to membrane-bound cynomolgus monkey TIM3, for example, with K D 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;

[0743] (7) (e.g., by blocking or reducing the inhibitory effect of TIM3) induce or enhance T cell activation, as demonstrated by (i) increased IFN-γ production in T cells expressing TIM3 (e.g., Th1 cells or TILs) and / or (ii) enhanced proliferation of T cells expressing TIM3 (e.g., Th1 cells or TILs), for example, as described in the examples.

[0744] (8) Stimulate T cell proliferation in a mixed lymphocyte response (MLR) assay, for example, as described in the examples;

[0745] (9) Inhibit the binding of phosphatidylserine to TIM3, for example, as determined by the PS-hTIM3 “tandem” blocking assay, for example, as described in the examples;

[0746] (10) When bound to TIM3 on the cell, it is not internalized or downregulated;

[0747] (11) Binding 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), for example, as described in the examples;

[0748] (12) Compared to binding with wild-type human TIM3, it has reduced binding with one or more of the following 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 (as indicated in SEQ ID NO:286). Figure 25 The binding of human TIM3 substituted with another amino acid, for example, as described in the examples;

[0749] (13) An antibody that competes with an antibody containing any of the VH and VL domains 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, for example, as described in the examples;

[0750] (14) Combine with human TIM3 area 49 VPVCWGKGACPVFE 62 (SEQ ID NO:367) and 111 RIQIPGIMNDEKFNLKL 127 (SEQ ID NO:368), as measured by HDX-MS, for example, as described in the examples;

[0751] (15) Having a heavy chain variable region and / or a light chain variable region interacting 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; according to SEQ ID NO:286). Figure 25 ));

[0752] (16)(a) Compared to binding to wild-type human TIM3, it has reduced binding to 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids therein: C58, P59, F61, E62, C63, R111, D120, and optionally D104 and Q113 (according to SEQ ID NO:286). Figure 25 (a) Human TIM3 substituted with another amino acid; (b) bound 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) competitive or cross-blocking of 13A3 or TIM3.18.IgG1.3 binding to human TIM3, for example, as described in the examples.

[0753] (17) K D 5x10 -8 M, 2x10 -8 M, 10 -8 M or 5x10 -9 M or smaller binds to hTIM3-IgV, as determined by the method described in Example 22;

[0754] (18) K D 10 -7 M, 2x10 -8 M or 10 -8 M or smaller binds to hTIM3-IgV, as determined by the method described in Example 22;

[0755] (19) K D 5x10 -7 M, 2x10-8 M or 10 -8 M or smaller bound to cynomolgus monkey TIM3-ECD, as determined by the method described in Example 22; and / or

[0756] (20) K D 8x10 -8 M, 5x10 -8 M or 10 -8 M or smaller binds to hTIM3-ECD, as determined by the method described in Example 22.

[0757] Such antibodies include, for example, human antibodies, humanized antibodies, or chimeric antibodies.

[0758] In some implementations, the anti-TIM3 antibody described herein binds to a conformational epitope.

[0759] In some embodiments, the anti-TIM3 antibody described herein binds to amino acid residues in the following region of the mature human TIM3 extracellular domain (SEQ ID NO: 290):

[0760] SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLN GDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMND (SEQ ID NO:299) corresponds to amino acid residues 1-99 of the extracellular domain of mature human TIM3 (SEQ ID NO:290) or amino acids 22 to 120 of human TIM3 having SEQ ID NO:286.

[0761] In some embodiments, the anti-TIM3 antibody described herein binds to amino acid residues in 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).

[0762] In some embodiments, the anti-TIM3 antibody described herein binds to amino acid residues in 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).

[0763] In some embodiments, the anti-TIM3 antibody described herein binds to amino acid residues in the following region 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).

[0764] In some embodiments, the binding of the anti-TIM3 antibody to wild-type and mutant human TIM3 has the same pattern as one or more antibodies 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3, and TIM3.2 to TIM3.18. In some embodiments, the anti-TIM3 antibody binds to amino acid residues in the following region of the extracellular domain of mature human TIM3 (SEQ ID NO: 290): CPVFECG (SEQ ID NO: 296), WTSRYWLNGDFRKGDVSLTIENVTLAD (SEQ ID NO: 297), and / or RIQIPGIMND (SEQ ID NO: 298).

[0765] In some implementations, the anti-TIM3 antibody binds (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, for example, as described in the examples. In some embodiments, the anti-TIM3 antibody interacts with regions of amino acid residues 40-62 and 111-127 of hTIM3, but does not significantly interact with other regions, such as the N-terminal region of amino acid residue Y40, the region between amino acid residues E62 and R111, and the C-terminal region of amino acid residue L127, as determined by HDX-MS, for example, as described in the examples.

[0766] In some embodiments, the anti-TIM3 antibody, relative to binding to wild-type human TIM3, targets one or more of the following 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 (as numbered in SEQ ID NO:286). Figure 25 Human TIM3 substituted with another amino acid exhibits reduced binding to this antibody (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, for example, as described in the examples.

[0767] In some implementations, the binding of anti-TIM3 antibodies to wild-type and mutant human TIM3 follows a pattern similar to TIM3.18.IgG1.3 or 13A3, i.e., the antibody:

[0768] (i) Combining (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, a peptide having a sequence located at the N-terminus of 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 at the C-terminus of amino acid residue 127, as determined by HDX-MS (e.g., as described in the examples);

[0769] (ii) Cannot bind to such human TIM3 or binds to such human TIM3 with significantly reduced binding, 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 (according to SEQ ID NO:286 number ( Figure 25 )); and / or

[0770] (iii) Having a heavy chain variable region and / or a light chain variable region interacting 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; according to SEQ ID NO:286). Figure 25 )).

[0771] 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.

[0772] As further discussed herein, the heavy chain constant region of the anti-TIM3 antibody described herein can be any isotype, such as IgG1, IgG2, IgG3, and IgG4, or combinations thereof and / or modifications thereof. The anti-TIM3 antibody may have effector functionality or may have reduced or no effector functionality. In some embodiments, the anti-TIM3 antibody includes a modified heavy chain constant region that provides enhanced properties to the antibody.

[0773] 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, 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 NO:29-33 and 453.

[0774] III. Antibodies with specific strain sequences

[0775] In some implementations, 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.

[0776] As described herein, a human antibody specific to TIM3 has been prepared, comprising a heavy chain variable region. This heavy chain variable region is a product of or derived from the human lineage VH 4-39, VH 4-59, VH 1-46, VH 3-11, VH 4-17, VH 3-10, VH 6-19, VH 6-13, VH 4-23, VH JH4b, VH JH5b and / or VH JH6b genes. JH6b gene. Therefore, this document provides isolated monoclonal antibodies or their antigen-binding moieties comprising a heavy chain variable region selected from or derived from the human VH germline gene of VH 4-39, VH 4-59, VH 1-46, VH 3-11, VH 4-17, VH 3-10, VH6-19, VH 6-13, VH 4-23, VHJH4b, VHJH5b, VHJH6b, and any combination thereof.

[0777] Human antibodies specific to TIM3 have been prepared, comprising a light chain variable region that is a product of or derived from the human germline VK gene, specifically the VK JK5, VK JK4, VK JK3, VK L18, and / or VK JK1 gene. Therefore, this document provides isolated monoclonal antibodies or their antigen-binding portions comprising a light chain variable region that is a product of or derived from the human VK germline gene selected from VK A27, VK JK5, VK JK4, VK JK3, VK L18, VK JK1, and any combination thereof.

[0778] The anti-TIM3 antibodies described herein include those that contain both a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is a product of or derived from one of the human VH genes listed above, and the light chain variable region is a product of or derived from one of the human VK genes listed above, as shown in the figure.

[0779] As used herein, human antibodies comprise heavy chain variable regions and light chain variable regions, which, if obtained from a system using human germline immunoglobulin genes, are “products” or “derived from” specific germline sequences. Such systems include immunizing transgenic mice carrying human immunoglobulin genes with a target antigen or screening libraries of human immunoglobulin genes displayed on bacteriophages with a target antigen. Human antibodies that are “products” or “derived from” human germline immunoglobulin sequences can be identified by comparing the amino acid sequence of the human antibody with the amino acid sequence of a human germline immunoglobulin and selecting the human germline immunoglobulin sequence that is most closely related to the sequence of the human antibody (i.e., has the highest percentage identity). Human antibodies that are “products” or “derived from” specific human germline immunoglobulin sequences may contain amino acids different from the compared germline sequence due to 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 the amino acid residues that identify the human antibody as human when compared with germline immunoglobulin sequences from other species (e.g., mouse germline sequences). In some cases, the human antibody may 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, the difference between a human antibody derived from a specific human germline sequence and the amino acid sequence encoded by the human germline immunoglobulin gene does not exceed 10 amino acids. In some cases, the human antibody may show a difference of no more than 5, or even no more than 4, 3, 2, or 1 amino acid compared to the amino acid sequence encoded by the germline immunoglobulin gene.

[0780] IV. Homoantibodies

[0781] This document covers antibodies having heavy chain variable regions and light chain variable regions comprising amino acid sequences homologous to the amino acid sequences of the anti-TIM3 antibodies described herein, and wherein the antibodies retain the desired functional properties of the anti-TIM3 antibodies described herein.

[0782] For example, isolated anti-TIM3 antibodies or their antigen-binding moieties may contain heavy chain variable regions and light chain variable regions, wherein:

[0783] (a) The heavy chain variable region comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the same amino acid sequence as the amino acid sequence selected from SEQ ID NO: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 variations (i.e., amino acid substitution, addition or deletion) relative to the amino acid sequence selected from SEQ ID NO: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;

[0784] (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 the amino acid sequence selected from SEQ ID NO: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 variations (i.e., amino acid substitutions, additions, or deletions) relative to the amino acid sequence selected from SEQ ID NO: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;

[0785] (c) The antibody specifically binds to human TIM3, and

[0786] (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 properties:

[0787] (1) Binds to soluble human TIM3, for example, with K D 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;

[0788] (2) Binding to soluble cynomolgus monkey TIM3, for example, with K D 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;

[0789] (3) Binding to membrane-bound human TIM3, for example, EC 501 ug / mL or less (e.g., 0.01 ug / mL to 1 ug / mL), for example, as determined by flow cytometry (e.g., as described in the examples);

[0790] (4) Binding to membrane-bound human TIM3, for example, with K D 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;

[0791] (5) Binding to membrane-bound cynomolgus monkey TIM3, for example, at EC50 20 ug / mL or less (e.g., 0.01 ug / mL to 20 ug / mL), for example, as determined by flow cytometry (e.g., as described in the examples);

[0792] (6) Binding to membrane-bound cynomolgus monkey TIM3, for example, with K D 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;

[0793] (7) (e.g., by blocking or reducing the inhibitory effect of TIM3) induce or enhance T cell activation, as demonstrated by (i) increased IFN-γ production in T cells expressing TIM3 (e.g., Th1 cells or TILs) and / or (ii) enhanced proliferation of T cells expressing TIM3 (e.g., Th1 cells or TILs), for example, as described in the examples.

[0794] (8) Stimulate T cell proliferation in a mixed lymphocyte response (MLR) assay, for example, as described in the examples;

[0795] (9) Inhibit the binding of phosphatidylserine to TIM3, for example, as determined by the PS-hTIM3 “tandem” blocking assay, for example, as described in the examples;

[0796] (10) When bound to TIM3 on the cell, it is not internalized or downregulated;

[0797] (11) Binding 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), for example, as described in the examples;

[0798] (12) Compared to binding with wild-type human TIM3, it has reduced binding with one or more of the following 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 (as indicated in SEQ ID NO:286). Figure 25 The binding of human TIM3 substituted with another amino acid, for example, as described in the examples;

[0799] (13) An antibody that competes with an antibody containing any of the VH and VL domains 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, for example, as described in the examples;

[0800] (14) Combine with human TIM3 area 49 VPVCWGKGACPVFE 62 (SEQ ID NO:367) and 111 RIQIPGIMNDEKFNLKL 127 (SEQ ID NO:368), as measured by HDX-MS, for example, as described in the examples;

[0801] (15) Having a heavy chain variable region and / or a light chain variable region interacting 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; according to SEQ ID NO:286). Figure 25 ));

[0802] (16)(a) Compared to binding to wild-type human TIM3, it has reduced binding to 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids therein: C58, P59, F61, E62, C63, R111, D120, and optionally D104 and Q113 (according to SEQ ID NO:286). Figure 25 (a) Human TIM3 substituted with another amino acid; (b) bound 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) competitive or cross-blocking of 13A3 or TIM3.18.IgG1.3 binding to human TIM3, for example, as described in the examples;

[0803] (17) K D 5x10 -8 M, 2x10 -8 M, 10 -8 M or 5x10 -9 M or smaller binding hTIM3-IgV, as determined by the method described in Example 22;

[0804] (18) K D 10 -7 M, 2x10 -8 M or 10 -8 M or smaller binding hTIM3-IgV, as determined by the method described in Example 22;

[0805] (19) K D 5x10 -7 M, 2x10 -8 M or 10 -8 M or smaller bound to cynomolgus monkey TIM3-ECD, as determined by the method described in Example 22; and / or

[0806] (20) K D 8x10 -8 M, 5x10 -8 M or 10 -8 M or smaller bound to hTIM3-ECD, as determined by the method described in Example 22.

[0807] 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 properties listed in (1) to (20) above. The antibody may be, for example, a human antibody, a humanized antibody, or a chimeric antibody.

[0808] The isolated anti-TIM3 antibody or its antigen-binding moiety may contain both heavy and light chains, wherein:

[0809] (a) The heavy chain comprises an amino acid sequence in which at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence is identical to the 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 (TIM3.20 HC IgG1f and IgG1.3f), 454, 455, 463, 464 (TIM3.21 HC IgG1f and IgG1.3f). The amino acid sequences 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 IgG1.1f 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 relative to 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 IgG1.1f and IgG1.3f). IgG.1f and IgG1.3f), 454, 455, 463, 464 (TIM3.21 HC IgG1f and IgG1.3f), 457, 458, 465, 466 (TIM3.22 HC IgG1f and IgG1.3f), and 459, 460, 467, 468 (TIM3.23 HC The amino acid sequence of IgG1f and IgG1.3f contains 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25 or 1-50 amino acid variations (i.e., amino acid substitutions, additions or deletions), provided that, in some embodiments, if the sequence is a heavy chain sequence without effector function, the IgG1.1 constant region is not modified with mutations that render the heavy chain without effector function (e.g., R214, A234, E235, A237, S330 and S331 are not modified), and the IgG1.3 constant region is not modified with R214, A234 and E235, wherein optionally, the heavy chain variable region contains the CDR sequence of one of the anti-TIM3 antibodies described herein;

[0810] (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 a CDR sequence of one of the anti-TIM3 antibodies described herein;

[0811] (c) The antibody specifically binds to human TIM3, and

[0812] (d) Antibodies exhibiting functional properties 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or all of the following:

[0813] (1) Binds to soluble human TIM3, for example, with K D 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;

[0814] (2) Binding to soluble cynomolgus monkey TIM3, for example, with K D 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;

[0815] (3) Binding to membrane-bound human TIM3, for example, EC 50 1 ug / mL or less (e.g., 0.01 ug / mL to 1 ug / mL), for example, as determined by flow cytometry (e.g., as described in the examples);

[0816] (4) Binding to membrane-bound human TIM3, for example, with K D 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;

[0817] (5) Binding to membrane-bound cynomolgus monkey TIM3, for example, at EC50 20 ug / mL or less (e.g., 0.01 ug / mL to 20 ug / mL), for example, as determined by flow cytometry (e.g., as described in the examples);

[0818] (6) Binding to membrane-bound cynomolgus monkey TIM3, for example, with K D 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;

[0819] (7) (e.g., by blocking or reducing the inhibitory effect of TIM3) induce or enhance T cell activation, as demonstrated by (i) increased IFN-γ production in T cells expressing TIM3 (e.g., Th1 cells or TILs) and / or (ii) enhanced proliferation of T cells expressing TIM3 (e.g., Th1 cells or TILs), for example, as described in the examples.

[0820] (8) Stimulate T cell proliferation in a mixed lymphocyte response (MLR) assay, for example, as described in the examples;

[0821] (9) Inhibit the binding of phosphatidylserine to TIM3, for example, as determined by the PS-hTIM3 “tandem” blocking assay, for example, as described in the examples;

[0822] (10) When bound to TIM3 on the cell, it is not internalized or downregulated on the cell surface;

[0823] (11) Binding 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), for example, as described in the examples;

[0824] (12) Compared to binding with wild-type human TIM3, it has reduced binding with one or more of the following 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 (as indicated in SEQ ID NO:286). Figure 25 The binding of human TIM3 substituted with another amino acid, for example, as described in the examples;

[0825] (13) An antibody that competes with an antibody containing any of the VH and VL domains 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, for example, as described in the examples;

[0826] (14) Combine with human TIM3 area 49 VPVCWGKGACPVFE 62 (SEQ ID NO:367) and 111 RIQIPGIMNDEKFNLKL 127 (SEQ ID NO:368), as measured by HDX-MS, for example, as described in the examples;

[0827] (15) Having a heavy chain variable region and / or a light chain variable region interacting 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; according to SEQ ID NO:286). Figure 25 ));

[0828] (16)(a) Compared to binding to wild-type human TIM3, it has reduced binding to 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids therein: C58, P59, F61, E62, C63, R111, D120, and optionally D104 and Q113 (according to SEQ ID NO:286). Figure 25 (a) Human TIM3 substituted with another amino acid; (b) bound 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) competitive or cross-blocking of 13A3 or TIM3.18.IgG1.3 binding to human TIM3, for example, as described in the examples;

[0829] (17) K D 5x10 -8 M, 2x10 -8 M, 10 -8 M or 5x10 -9 M or smaller binding hTIM3-IgV, as determined by the method described in Example 22;

[0830] (18) K D 10 -7 M, 2x10 -8 M or 10 -8 M or smaller binding hTIM3-IgV, as determined by the method described in Example 22;

[0831] (19) K D 5x10 -7 M, 2x10 -8 M or 10 -8 M or smaller bound to cynomolgus monkey TIM3-ECD, as determined by the method described in Example 22; and / or

[0832] (20) K D 8x10 -8 M, 5x10 -8 M or 10 -8 M or smaller bound to hTIM3-ECD, as determined by the method described in Example 22.

[0833] Anti-TIM3 antibodies are also provided that contain CDR1, VH CDR2, VH CDR3, VL CDR1, VLCDR2, and / or VL CDR3 that differ from the corresponding 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, wherein the difference is in alterations to 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, or 1-5 amino acids (i.e., amino acid substitution, addition, or deletion). In some embodiments, the anti-TIM3 antibody comprises a change of 1-5 amino acids in each of the 1, 2, 3, 4, 5, or 6 CDRs, 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. In some implementations, the anti-TIM3 antibody comprises a total of 1-5 amino acid changes in all CDRs, relative to the CDRs in 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.

[0834] In some embodiments, the anti-TIM3 antibody comprises VH and VL CDRs composed of those 13A3s, wherein one or more amino acids in one or more CDRs are among other anti-TIM3 antibodies disclosed herein.

[0835] For example, in some embodiments, the anti-TIM3 antibody comprises a VH CDR1 modified with one or more amino acids 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, for example, S or none; X2 is any amino acid, for example, R or none; X3 is any amino acid, for example, S, R or D; X4 is any amino acid, for example, Y or H; X5 is any amino acid, for example, W or M; and X6 is any amino acid, for example, G, N, S or H.

[0836] In some embodiments, the anti-TIM3 antibody comprises a VH CDR2 containing one or more amino acid modifications relative to SIYYSGFTYYNPSLKS (SEQ ID NO:46), and may contain, for example, the following degenerate sequence: X1X2X3X4GX5X6X7X8YX9X10X 11 X 12 X 13 X 14 (SEQ ID NO:283), wherein X1 is any amino acid, for example, S, Y, I, or F; X2 is any amino acid, for example, Y, H, N, or S; X3 is any amino acid, for example, Y, P, G, T, S, or N; X4 is any amino acid, for example, S, T, R, or G; X5 is any amino acid, for example, F, S, or D; X6 is any amino acid, for example, S, T, or I; X7 is any amino acid, for example, I or none; X8 is any amino acid, for example, Y, N, or I; X9 is any amino acid, for example, N, Q, S, or A; X10 is any amino acid, for example, P, S, Q, or D; X11 is any amino acid, for example, S or K; X12 is any amino acid, for example, L, F, or V; X13 is any amino acid, for example, K or Q; and X14 is any amino acid, for example, S or G.

[0837] In some embodiments, the anti-TIM3 antibody comprises VH CDR3 containing one or more amino acid modifications relative to GGPYGDYAHWFDP (SEQ ID NO:53), and may include, for example, the following degenerate sequence:

[0838] 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, for example, D, E, G, or none; X2 is any amino acid, for example, F, G, R, or none; X3 is any amino acid, for example, Y, M, I, or none; X4 is any amino acid, for example, G, S, V, or none; X5 is any amino acid, for example, G, T, R, S, or none; X6 is any amino acid, for example, G, S, or none; X7 is any amino acid, for example, M, N, W, or none; X8 is any amino acid, for example, Y, S, E, N, or none; X9 is any amino acid, for example, Y, or none; X10 is... X11 is any amino acid, for example, F, P, or Y; X12 is any amino acid, for example, Y or F; X13 is any amino acid, for example, G or none; X14 is any amino acid, for example, Y or none; X15 is any amino acid, for example, A or none; X16 is any amino acid, for example, H or none; X17 is any amino acid, for example, W or none; X18 is any amino acid, for example, F, M or none; X19 is any amino acid, for example, D or E; and X20 is any amino acid, for example, P, I, V, Y, or L.

[0839] In some embodiments, the anti-TIM3 antibody comprises VL CDR1 containing the amino acid sequence shown in SEQ ID NO:64 or SEQ ID NO:65.

[0840] In some embodiments, the anti-TIM3 antibody comprises VL CDR2 containing the amino acid sequence shown in SEQ ID NO:66 or SEQ ID NO:67.

[0841] In some embodiments, the anti-TIM3 antibody comprises a VL CDR3 modified with one or more amino acids relative to QQYGSSPIT (SEQ ID NO:68), and may comprise, for example, the following degenerate sequence: QQX1X2SX3X4X5X6 (SEQ ID NO:285), wherein X1 is any amino acid, for example, F or Y; X2 is any amino acid, for example, N or G; X3 is any amino acid, for example, Y or S; X4 is any amino acid, for example, P or none; X5 is any amino acid, for example, I, R, L or none; and X6 is any amino acid, for example, T or none.

[0842] Having the VH and VL regions 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, for example, SEQ ID NO:34-40, 112-121, 364 and 410-412, and SEQ ID NO:60-63, 417 and 418, or SEQ ID NO:1-28, 72-111, 349-352, 388-391, 396-399 or 402-405, and SEQ ID Antibodies with sequences of homology to the heavy and light chains of NO:29-33, 408 or 409, or CDR, can be obtained by mutagenesis (e.g., site-directed mutagenesis or PCR-mediated mutagenesis) encoding nucleic acid molecules of 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 by testing the function retained by the encoded altered antibody (i.e., the function shown in (1) to (16) above) using the functional assays described herein.

[0843] V. Antibodies with conserved modifications

[0844] Anti-TIM3 antibodies may 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 a specific amino acid sequence or its conserved modification based on the anti-TIM3 antibody described herein (e.g., 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3 or any of TIM3.2 to TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24, and TIM3.25), and wherein the antibody retains the functional properties of the anti-TIM3 antibody described herein. Therefore, isolated anti-TIM3 antibodies or their antigen-binding portions may 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:

[0845] (a) The heavy chain variable region CDR3 sequence comprises an amino acid sequence selected from amino acid sequences SEQ ID NO:53-59, 126-129, 414, 416 and 471 and their conserved modifications, said conserved modifications being, for example, 1, 2, 3, 4, 5, 1-2, 1-3, 1-4 or 1-5 conserved amino acid substitutions, wherein optionally said heavy chain variable region comprises the CDR sequence of one of the anti-TIM3 antibodies described herein;

[0846] (b) The light chain variable region CDR3 sequence comprises an amino acid sequence selected from amino acid sequences SEQ ID NO:68-71, 419 and 474 and their conserved modifications, said conserved modification being, for example, 1, 2, 3, 4, 5, 1-2, 1-3, 1-4 or 1-5 conserved amino acid substitutions, wherein optionally said light chain variable region comprises the CDR sequence of one of the anti-TIM3 antibodies described herein;

[0847] (c) The antibody specifically binds to human TIM3, and

[0848] (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:

[0849] (1) Binds to soluble human TIM3, for example, with K D 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;

[0850] (2) Binding to soluble cynomolgus monkey TIM3, for example, with K D 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;

[0851] (3) Binding to membrane-bound human TIM3, for example, EC 50 1 ug / mL or less (e.g., 0.01 ug / mL to 1 ug / mL), for example, as determined by flow cytometry (e.g., as described in the examples);

[0852] (4) Binding to membrane-bound human TIM3, for example, with K D 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;

[0853] (5) Binding to membrane-bound cynomolgus monkey TIM3, for example, with EC 5020 ug / mL or less (e.g., 0.01 ug / mL to 20 ug / mL), for example, as determined by flow cytometry (e.g., as described in the examples);

[0854] (6) Binding to membrane-bound cynomolgus monkey TIM3, for example, with K D 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;

[0855] (7) (e.g., by blocking or reducing the inhibitory effect of TIM3) induce or enhance T cell activation, as demonstrated by (i) increased IFN-γ production in T cells expressing TIM3 (e.g., Th1 cells or TILs) and / or (ii) enhanced proliferation of T cells expressing TIM3 (e.g., Th1 cells or TILs), for example, as described in the examples.

[0856] (8) Stimulate T cell proliferation in a mixed lymphocyte response (MLR) assay, for example, as described in the examples;

[0857] (9) Inhibit the binding of phosphatidylserine to TIM3, for example, as determined by the PS-hTIM3 “tandem” blocking assay, for example, as described in the examples;

[0858] (10) When bound to TIM3 on the cell, it is not internalized or downregulated on the cell surface;

[0859] (11) Binding 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), for example, as described in the examples;

[0860] (12) Compared to binding with wild-type human TIM3, it has reduced binding with one or more of the following 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 (as indicated in SEQ ID NO:286). Figure 25 The binding of human TIM3 substituted with another amino acid, for example, as described in the examples;

[0861] (13) An antibody that competes with an antibody containing any of the VH and VL domains 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, for example, as described in the examples;

[0862] (14) Combine with human TIM3 area 49 VPVCWGKGACPVFE 62 (SEQ ID NO:367) and 111 RIQIPGIMNDEKFNLKL 127 (SEQ ID NO:368), as measured by HDX-MS, for example, as described in the examples;

[0863] (15) Having a heavy chain variable region and / or a light chain variable region interacting 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; according to SEQ ID NO:286). Figure 25 ));

[0864] (16)(a) Compared to binding to wild-type human TIM3, it has reduced binding to 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids therein: C58, P59, F61, E62, C63, R111, D120, and optionally D104 and Q113 (according to SEQ ID NO:286). Figure 25 (a) Human TIM3 substituted with another amino acid; (b) bound 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) competitive or cross-blocking of 13A3 or TIM3.18.IgG1.3 binding to human TIM3, for example, as described in the examples;

[0865] (17) K D 5x10 -8 M, 2x10 -8 M, 10 -8 M or 5x10 -9 M or smaller binding hTIM3-IgV, as determined by the method described in Example 22;

[0866] (18) K D 10 -7 M, 2x10 -8 M or 10 -8 M or smaller binding hTIM3-IgV, as determined by the method described in Example 22;

[0867] (19) K D 5x10 -7 M, 2x10 -8 M or 10 -8 M or smaller bound to cynomolgus monkey TIM3-ECD, as determined by the method described in Example 22; and / or

[0868] (20) K D 8x10 -8 M, 5x10 -8 M or 10 -8 M or smaller bound to hTIM3-ECD, as determined by the method described in Example 22.

[0869] In some embodiments, the heavy chain variable region CDR2 sequence comprises an amino acid sequence selected from amino acid sequences SEQ ID NO:46-52, 122-125, 413, 415 and 470 and their conserved modifications, such as 1, 2, 3, 4, 5, 1-2, 1-3, 1-4 or 1-5 conserved amino acid substitutions; and the light chain variable region CDR2 sequence comprises an amino acid sequence selected from amino acid sequences SEQ ID NO:66-67 and 473 and their conserved modifications, such as 1, 2, 3, 4, 5, 1-2, 1-3, 1-4 or 1-5 conserved amino acid substitutions.

[0870] In some embodiments, the heavy chain variable region CDR1 sequence comprises an amino acid sequence selected from amino acid sequences SEQ ID NO:41-45 and 469 and their conserved modifications, such as 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, or 1-5 conserved amino acid substitutions; and the light chain variable region CDR1 sequence comprises an amino acid sequence selected from amino acid sequences SEQ ID NO:64-65 and 472 and their conserved modifications, such as 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, or 1-5 conserved amino acid substitutions.

[0871] 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 properties listed in (1) to (20) above. Such antibodies may be, for example, human antibodies, humanized antibodies, or chimeric antibodies.

[0872] Conserved amino acid substitutions may be made in portions of the antibody, either outside the CDR or in addition to the CDR. For example, conserved amino acid modifications may be made in the framework region or the Fc region. The variable region or heavy or light chain may 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 conserved amino acid substitutions relative to the anti-TIM3 antibody sequence provided herein. In some embodiments, the anti-TIM3 antibody comprises a combination of conserved and non-conserved amino acid modifications.

[0873] VI. Antibodies that bind to the same epitope or compete for binding

[0874] Antibodies that compete with one or more of the specific anti-TIM3 antibodies described herein (e.g., antibodies 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3 and / or TIM3.2-TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24 and TIM3.25) for binding to human TIM3 are also provided. Such competitive antibodies can be identified in standard TIM3 binding assays based on their ability to competitively inhibit 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, IM3.24 and TIM3.25 from binding to human TIM3. The standard TIM3 binding assay includes TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, IM3.24 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, multiple 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 known as the “blocking antibody”) inhibits the binding of the labeled (second) antibody, it can be said that the first antibody inhibits the binding of the second antibody to the target on the plate, or that it competes with the second antibody for binding to the target. Additionally or alternatively, Biacore analysis can be used to assess the competitive ability of the antibodies. The ability of the test antibody to inhibit the binding of the described anti-TIM3 antibody to TIM3 demonstrates that the test antibody can compete with the described antibody for binding to human TIM3.

[0875] Therefore, this document provides 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%, for example, as determined by ELISA or FACS (e.g., using the assay methods described in the following paragraphs).

[0876] An exemplary competitive experiment to determine, for example, whether the first antibody blocks the binding of the second antibody (i.e., "competing with it") can be performed as described in the examples or as follows: Activated human T cells are prepared as follows: Peripheral blood mononuclear cells (PBMCs) are isolated from human whole blood using a Ficoll gradient and activated for 3 days with 10 μg / mL phytohemagglutinin (PHA-L) (USBiol#P3370-30) and 200 IU / mL recombinant IL-2 (Peprotech#200-02). The activated T cells are resuspended in FACS buffer (PBS containing 5% fetal bovine serum) and 10 samples are collected per well. 5 Cells were seeded at a density of 1,000 cells per well in 96-well plates. The plates were placed on ice, and unconjugated primary antibody was added at concentrations ranging from 0 to 50 μg / mL (triple titration starting from the highest concentration of 50 μg / mL). Irrelevant IgG was used as an isotype control for the primary antibody, and was added at the same concentration (triple titration starting from the highest concentration of 50 μg / mL). Samples pre-incubated with 50 μg / mL of unlabeled secondary antibody could be included as a positive control for complete blockade (100% inhibition), and samples without antibody in the first incubation could be used as a negative control (no competition; 0% inhibition). After incubation for 30 minutes, labeled, for example, biotinylated secondary antibody was added at a concentration of 2 μg / mL per well without washing. The samples were incubated on ice for another 30 minutes. Unbound antibodies were removed by washing cells with FACS buffer. The labeled secondary antibody binding cells was detected using a reagent for detecting the label, such as streptavidin (Invitrogen, catalog number S21388) for detecting biotinylate-conjugated antibodies. Samples were obtained on a FACS Calibur Flow Cytometer (BD, San Jose) and used... Software (Tree Star, Inc., Ashland, OR) analysis was used. Results can be expressed as % inhibition (i.e., subtracting the amount of labeled material at each concentration from 100% and dividing by the amount of labeled material obtained without blocking antibody). Typically, the same experiment is then performed in reverse order, i.e., the first antibody is the second antibody, and the second antibody is the first antibody.

[0877] In some implementations, an antibody at least partially (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) or completely (100%) blocks the binding of another antibody to a target (e.g., human TIM3 or a portion thereof), regardless of whether inhibition occurs when one or the other antibody is the first antibody. When antibodies compete with each other in two ways (i.e., in a competitive experiment in which the first antibody is added first and in a competitive experiment in which the second antibody is added first), the first and second antibodies “reciprocally block” each other from binding to the target.

[0878] In some implementations, 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), for example, as determined by a given epitope mapping technique. Techniques for determining that an antibody binds to the “same epitope on TIM3” as described herein include, for example, epitope mapping methods, such as X-ray analysis of the antigen:antibody complex crystals, which provides atomic resolution of the epitope. Other methods monitor the binding of the antibody to an antigen fragment or a mutant variant of the antigen, and loss of binding due to modification of amino acid residues within the antigen sequence is generally considered an indicator of epitope composition (see [link to relevant documentation]). Figure 25 Alternatively, computer-based combined methods for epitope mapping can be used. The method can also depend on the ability of the target antibody to affinity-isolate specific short peptides (in their native three-dimensional or denatured form) from the combined phage-displayed peptide library. The peptide is then treated as a guide corresponding to an epitope definition for screening the peptide library. Computer algorithms have also been developed for epitope mapping, which have been shown to map conformationally discontinuous epitopes.

[0879] Antibodies that compete with or bind to the same epitope as the anti-TIM3 antibody described herein can be identified using methods known in the art. For example, mice can be immunized with the human TIM3 described herein to generate hybridomas, and the resulting monoclonal antibodies can be screened for their ability to compete with the antibody described herein for binding to human TIM3. Mice can also be immunized with a smaller TIM3 fragment containing the epitope that binds to the antibody. The epitope or region containing the epitope can be located, for example, by screening for binding to a series of overlapping peptides across TIM3. Alternatively, the method used by Jespers et al., Biotechnology 12:899, 1994, can be used to guide the selection of antibodies having the same epitope and therefore having properties similar to the anti-TIM3 antibody described herein. Using phage display, the heavy chain of the anti-TIM3 antibody is first paired with a (human) light chain library to select TIM3-binding antibodies, and then a new light chain is paired with a (human) heavy chain library to select (human) TIM3-binding antibodies having the same epitope or epitope region as the anti-TIM3 antibody described herein. Alternatively, variants of the antibodies described herein can be obtained by mutagenesis of the cDNA encoding the heavy and light chains of the antibody.

[0880] As described by Cunningham and Wells (1989) Science 244:1081-1085, some other form of alanine scanning mutagenesis or point mutagenesis of amino acid residues in TIM3 can also be used to obtain TIM3 antibody binding characteristics.

[0881] The binding properties of an antibody can also be determined by evaluating its binding 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, for example in direct ELISA, competitive ELISA (where the ability of the peptide to prevent the antibody from binding to TIM3 already bound to the wells of a microtiter plate is evaluated), or on a microarray.

[0882] The binding signature of anti-TIM3 antibodies can also be obtained through MS-based protein footprinting, such as hydrogen / deuterium exchange mass spectrometry (HDX-MS) and rapid photochemical oxidation (FPOP) of proteins. 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 specifically incorporated herein 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 specifically incorporated herein by reference.

[0883] The binding characteristics of anti-TIM3 antibodies can also be obtained through structural methods, such as X-ray crystallography (e.g., WO2005 / 044853), molecular modeling, and nuclear magnetic resonance (NMR) spectroscopy, including NMR determination of the HD exchange rate of unstable amide hydrogen in TIM3 when TIM3 is free and when it is bound to the target antibody in the complex (Zinn-Justin et al. (1992) Biochemistry 31, 11335-11347; Zinn-Justin et al. (1993) Biochemistry 32, 6884-6891).

[0884] Regarding X-ray crystallography, crystallization can be performed using any method known 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), pendant drop vapor diffusion (e.g., McPherson (1976) J. Biol. Chem. 251:6300-6303), seeding, and dialysis. Protein preparations with concentrations of at least about 1 mg / mL or about 10 mg / mL to about 20 mg / mL are preferred. Crystallization is best achieved in a precipitant solution containing about 10% to about 30% (w / v) 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. It is also desirable to include a protein stabilizer, such as glycerol at a concentration of about 0.5% to about 20%. Suitable salts, such as sodium chloride, lithium chloride, or sodium citrate, at concentrations ranging from about 1 mM to about 1000 mM, may also be desired in the precipitant solution. The precipitant is buffered to a pH of about 3.0 to about 5.0. Specific buffers useful in the precipitant solution can vary and are well known in the art (Scopes, Protein Purification: Principles and Practice, 3rd 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 temperature range, including 2°C, 4°C, 8°C, and 26°C.

[0885] Antibody-antigen crystals can be studied using well-known X-ray diffraction techniques and refined using computer software such as X-PLOR (Yale University, 1992, published by Molecular Simulations, Inc.); see, for example, Blundell & Johnson (1985) Meth. Enzymol. 114 & 115, eds. H. W. Yckoff 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, eds. Carter & Sweet; Roversi et al. (2000) Acta Cryst. D56:1313-1323), the disclosures of which are incorporated herein by reference in their entirety.

[0886] Anti-TIM3 antibodies can bind to the same epitopes as any anti-TIM3 antibody having the amino acid sequence described herein, as determined by epitope mapping techniques such as those described herein.

[0887] This document includes antibodies that bind to human TIM3 and optionally to cynomolgus monkey TIM3, said antibodies having similar binding characteristics to the anti-TIM3 antibodies described herein and identified by one of the methods used in the examples.

[0888] In some embodiments, the anti-TIM3 antibody described herein binds to the extracellular portion of human TIM3 (e.g., in the Ig-like domain or IgV domain of the extracellular region), specifically amino acids 22 to 130 of SEQ ID NO:286. Figure 25 Epitopes in, for example, conformational epitopes. In some embodiments, the anti-TIM3 antibody binds to epitopes located at amino acids 22 to 120 of the extracellular domain of human TIM3 (SEQ ID NO: 286) or at 1 to 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 a region consisting of amino acids 58-64 of human TIM3 having SEQ ID NO: 286, said region corresponding to amino acid residues 37-43 of mature human TIM3 (CPVFECG, SEQ ID NO: 296; see Examples). Figure 25In some embodiments, the anti-TIM3 antibody binds to or binds to an epitope in a region consisting of amino acids 111-120 of human TIM3 having SEQ ID NO: 286, said region corresponding to amino acid residues 90-99 of mature human TIM3 (RIQIPGIMND, SEQ ID NO: 298; see also) Figure 25 In some embodiments, the anti-TIM3 antibody binds to or binds to the region consisting of amino acids 58-64 (CPVFECG, SEQ ID NO: 296) of human TIM3 having SEQ ID NO: 286 and amino acids 111-120 (RIQIPGIMND, SEQ ID NO: 298; see also) of human TIM3 having SEQ ID NO: 286. Figure 25 Epitopes in the region composed of ) . In some embodiments, the anti-TIM3 antibody binds to or binds to amino acids 78-89 (WTSRYWLNGDFR, SEQ ID NO:297) of human TIM3 having SEQ ID NO:286; see Figure 25 The epitope in the region composed of ) corresponds to amino acid residues 57-83 of mature human TIM3.

[0889] In some embodiments, the anti-TIM3 antibody binds to an epitope substantially identical to 13A3. In some embodiments, the anti-TIM3 antibody binds to one or more amino acid residues comprising SEQ ID NO:286: C58, P59, F61, E62, C63, R111, and D120. Figure 25 The epitope (or region of human TIM3) of SEQ ID NO:286. In some embodiments, the anti-TIM3 antibody binds to one or more amino acid residues comprising SEQ ID NO:286: C58, P59, F61, E62, C63, D104, R111, Q113, and D120. Figure 25 The anti-TIM3 antibody binds to the epitope (or region of human TIM3) of SEQ ID NO:286 with significantly reduced affinity or with significantly reduced affinity to human TIM3 proteins in which one or more amino acid residues C58, P59, F61, E62, C63, R111, and D120 (e.g., substituted with non-conserved amino acids) are changed to another amino acid. In some embodiments, the anti-TIM3 antibody binds to the anti-TIM3 antibody with significantly reduced affinity or with significantly reduced affinity to human TIM3 proteins in which one or more amino acid residues C58, P59, F61, E62, C63, D104, R111, Q113, and D120 (e.g., substituted with non-conserved amino acids) are changed to another amino acid.

[0890] In some embodiments, the anti-TIM3 antibody binds to an epitope substantially identical to that of 3G4. In some embodiments, the anti-TIM3 antibody binds to one or more amino acid residues comprising SEQ ID NO:286: C58, P59, V60, F61, E62, C63, G116, and M118. Figure 25 The epitope (or region of human TIM3) of SEQ ID NO:286. In some embodiments, the anti-TIM3 antibody binds to one or more amino acid residues comprising SEQ ID NO:286: C58, P59, V60, F61, E62, C63, D104, G116, and M118. Figure 25 The anti-TIM3 antibody binds to the epitope (or region of human TIM3) of SEQ ID NO:286 with significantly reduced affinity or with significantly reduced affinity to human TIM3 proteins in which one or more amino acid residues C58, P59, V60, F61, E62, C63, G116, and M118 (e.g., substituted with non-conserved amino acids) are changed to another amino acid. In some embodiments, the anti-TIM3 antibody binds to the anti-TIM3 antibody with significantly reduced affinity or with significantly reduced affinity to human TIM3 proteins in which one or more amino acid residues C58, P59, V60, F61, E62, C63, D104, G116, and M118 (e.g., substituted with non-conserved amino acids) are changed to another amino acid.

[0891] In some embodiments, the anti-TIM3 antibody binds to an epitope substantially identical to 17C3. In some embodiments, the anti-TIM3 antibody binds to one or more amino acid residues comprising SEQ ID NO:286: C58, P59, V60, F61, E62, C63, G64, and G116. Figure 25 The epitope (or region of human TIM3) of SEQ ID NO:286. In some embodiments, the anti-TIM3 antibody binds to one or more amino acid residues comprising SEQ ID NO:286: C58, P59, V60, F61, E62, C63, G64, D104, and G116. Figure 25Epitopes (or regions of human TIM3). In some embodiments, the anti-TIM3 antibody does not significantly bind to or only binds with significantly reduced affinity to human TIM3 proteins in which one or more amino acid residues C58, P59, V60, F61, E62, C63, G64, and G116 of SEQ ID NO:286 (e.g., substituted with non-conserved amino acids) are changed to another amino acid. In some embodiments, the anti-TIM3 antibody does not significantly bind to or only binds with significantly reduced affinity to human TIM3 proteins in which one or more amino acid residues C58, P59, V60, F61, E62, C63, G64, D104, and G116 of SEQ ID NO:286 (e.g., substituted with non-conserved amino acids) are changed to another amino acid.

[0892] In some embodiments, the anti-TIM3 antibody binds to an epitope substantially identical to 8B9. In some embodiments, the anti-TIM3 antibody binds to one or more amino acid residues comprising SEQ ID NO:286: L48, W78, S80, R81, W83, G86, D87, and R89. Figure 25 The epitope (or region of human TIM3) of SEQ ID NO:286. In some embodiments, the anti-TIM3 antibody binds to one or more amino acid residues comprising SEQ ID NO:286: L48, W78, S80, R81, W83, L84, G86, D87, and R89. Figure 25 The epitope (or region of human TIM3) of SEQ ID NO:286. 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 one or more amino acid residues comprising SEQ ID NO:286: L48, W78, S80, R81, W83, G86, D87, R89, and D104. Figure 25The anti-TIM3 antibody binds to the epitope (or region of human TIM3) of SEQ ID NO:286 with significantly reduced affinity or with significantly reduced affinity to human TIM3 proteins in which one or more amino acid residues L48, W78, S80, R81, W83, G86, D87, and R89 (e.g., substituted with non-conserved amino acids) are changed to another amino acid. In some embodiments, the anti-TIM3 antibody binds to the anti-TIM3 antibody with significantly reduced affinity or with significantly reduced affinity to human TIM3 proteins in which one or more amino acid residues L48, W78, S80, R81, W83, L84, G86, D87, and R89 (e.g., substituted with non-conserved amino acids) are changed to another amino acid. In some embodiments, the anti-TIM3 antibody does not significantly bind to or only binds to one or more amino acid residues L48, W78, S80, R81, W83, G86, D87, R89, and D104 of SEQ ID NO:286 with significantly reduced affinity. Figure 25 (For example, human TIM3 protein that has been modified by replacing a non-conserved amino acid with another amino acid.)

[0893] In some embodiments, the anti-TIM3 antibody competes with (or inhibits) binding to human TIM3 against the anti-TIM3 antibody containing a CDR or variable region described herein, wherein the anti-TIM3 antibody containing a CDR or variable region 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 at least 50%, 60%, 70%, 80%, 90%, or 100% of antibodies 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3, or any of TIM3.2 to TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24, and TIM3.25 binding to human TIM3. 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 inhibit the binding of anti-TIM3 antibodies to human TIM3 by at least 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, the anti-TIM3 antibody inhibits at least 50%, 60%, 70%, 80%, 90%, or 100% of the 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, and 13 A3, 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 inhibit the binding of the anti-TIM3 antibody to human TIM3 by at least 50%, 60%, 70%, 80%, 90%, or 100% (e.g., competing in both directions).

[0894] In some embodiments, the anti-TIM3 antibody competes with anti-TIM3 antibodies containing the CDR or variable regions of anti-TIM3 antibodies 13A3, 3G4, 17C3, 17C8, 9F6, 14H7, and 23B3 (and their variants) for binding to human TIM3. In some embodiments, the anti-TIM3 antibody does not compete with anti-TIM3 antibodies containing the CDR or variable regions of anti-TIM3 antibodies 8B9 and 8C4 (and their variants) for binding to human TIM3.

[0895] VII. Engineered and modified antibodies

[0896] Engineered and modified antibodies are also provided, which can be engineered using antibodies having one or more VH and / or VL sequences disclosed herein as starting materials. The modified antibodies 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 constant regions, for example, to alter the effector function of the antibody.

[0897] One type of variable region engineering that can be implemented is CDR grafting. Antibodies primarily interact with target antigens through amino acid residues located in the six heavy chain complementarity-determining regions (CDRs) and light chain complementarity-determining regions. Therefore, the amino acid sequences within the CDRs differ more from those outside the CDRs between different antibodies. Since the CDR sequence is responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a specific naturally occurring antibody by constructing expression vectors, wherein the expression vectors include a CDR sequence from a specific naturally occurring antibody, which is then grafted onto a framework region sequence 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. USA 86:10029-10033; Winter’s U.S. Patent No. 5,225,539; and Queen et al.’s U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370).

[0898] Therefore, some embodiments described herein relate to isolated monoclonal antibodies or their antigen-binding portions, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 sequences containing amino acid sequences selected from SEQ ID NO:41-45 and 469; SEQ ID NO:46-52, 122-125, 413, 415, and 470; and SEQ ID NO:53-59, 126-129, 414, 416, and 471, respectively; and the light chain variable region comprises CDR1, CDR2, and CDR3 sequences containing amino acid sequences selected from SEQ ID NO:64-65 and 472; SEQ ID NO:66-67 and 473; and SEQ ID NO:68-71, 419, and 474, respectively. Therefore, such antibodies contain the VHCDR 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 contain framework region sequences different from those of these antibodies.

[0899] Such frame sequences can be obtained from public DNA databases or publicly available references that include germline antibody gene sequences. For example, germline DNA sequences of human heavy chain variable region and light chain variable region genes can be found in the "VBase" human germline sequence database (available at www.mrc-cpe.cam.ac.uk / vbase), or in Kabat, E.A. et al. (1991), Sequences of Proteins of Immunological Interest, 5th ed., 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, JPL et al. (1994) "A Directory of Human Germ-line V HThe Segments Reveal a Strong Bias in their Usage (Eur. J. Immunol. 24: 827-836) contains the contents of each of them, which are explicitly incorporated into this paper by reference.

[0900] In some embodiments, the framework region sequence used for the anti-TIM3 antibody described herein is one that is structurally similar to the framework region sequence used for the anti-TIM3 antibody described herein. VH CDR1, 2, and 3 sequences, as well as VLCDR1, 2, and 3 sequences, can be grafted onto a framework region having the same sequence as that found in the germline immunoglobulin gene from which the framework region sequence originates, or CDR sequences can be grafted onto a framework region containing one or more mutations compared to that germline sequence. For example, it has been found that, in certain circumstances, it is beneficial to mutate residues within the framework region to maintain or enhance the antigen-binding capacity of the antibody (see, for example, U.S. Patents 5,530,101; 5,585,089; 5,693,762; and 6,180,370 to Queen et al.).

[0901] The engineered anti-TIM3 antibodies described herein include antibodies in which the framework region residues within the VH and / or VL have been modified, for example to improve antibody properties, such as a mutation at amino acid 107 in 9F6. Typically, such framework region modifications are performed to reduce the immunogenicity of the antibody. For example, one approach is to “reverse mutation” one or more framework region residues to the corresponding germline sequence. More specifically, antibodies that have undergone somatic mutations may contain framework region residues different from the germline sequence from which the antibody originated. Such residues can be identified by comparing the antibody framework region sequence to the germline sequence from which the antibody originated. To revert the framework region sequence to its germline conformation, the somatic mutation can be “reverse mutation” to the germline sequence by, for example, site-directed mutagenesis or PCR-mediated mutagenesis. This invention also intends to include such “reverse mutation” 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, thereby reducing the potential immunogenicity of the antibody. This method is also known as "deimmunization" and is described in more detail in U.S. Patent Publication No. 20030153043 by Carr et al.

[0902] Another type of variable region modification involves mutating amino acid residues in the VH and / or VL CDR1, CDR2, and / or CDR3 regions to improve one or more binding properties (e.g., affinity) of the target antibody. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation, and the effect on antibody binding or other targeted function can be assessed in in vitro or in vivo assays as described herein and provided in the examples. In some embodiments, conserved modifications are introduced (as described above). The mutation can be an amino acid substitution, addition, or deletion. Furthermore, typically no more than 1, 2, 3, 4, or 5 residues are altered in the CDR region.

[0903] Therefore, this article also provides isolated anti-TIM3 monoclonal antibodies or their antigen-binding moieties, containing heavy chain variable regions with the following:

[0904] (a) VH CDR1 region containing an amino acid sequence selected from SEQ ID NO:41-45 and 469, or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions compared to SEQ ID NO:41-45 and 469;

[0905] (b) VH CDR2 region, which contains an amino acid sequence selected from SEQ ID NO: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 compared to SEQ ID NO:46-52, 122-125, 413, 415 and 470;

[0906] (c) VH CDR3 region, which contains an amino acid sequence selected from SEQ ID NO: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 compared to SEQ ID NO:53-59, 126-129, 414, 416 and 471;

[0907] (d) VL CDR1 region, which contains an amino acid sequence selected from SEQ ID NO:64-65 and 472, or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions compared to SEQ ID NO:64-65 and 472;

[0908] (e) VL CDR2 region containing an amino acid sequence selected from SEQ ID NO:66-67 and 473, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions compared to SEQ ID NO:66-67 and 473; and

[0909] (f) VL CDR3 region containing an amino acid sequence selected from SEQ ID NO:68-71, 419 and 474, or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions compared to SEQ ID NO:68-71, 419 and 474.

[0910] Methionine residues in the antibody CDR can be oxidized, leading to potential chemical degradation and thus reducing antibody efficacy. Therefore, the present invention also provides anti-TIM3 antibodies in which one or more methionine residues in the heavy chain CDR and / or light chain CDR are replaced with amino acid residues that do not undergo oxidative degradation. In some embodiments, methionine residues in the CDR of antibodies 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, 14H7, 23B3, or any of TIM3.2 to TIM3.18, TIM3.20, TIM3.21, TIM3.22, TIM3.23, TIM3.24, and TIM3.25 are replaced with amino acid residues that do not undergo oxidative degradation.

[0911] Similarly, the deamidation site can be removed from anti-TIM3 antibodies, particularly from CDRs.

[0912] The anti-TIM3 variable region described herein can be linked (e.g., covalently linked or fused) to Fc, such as IgG1, IgG2, IgG3, or IgG4 Fc, which can be any allotype or isoallotype, for example, 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, for example, Jefferies et al. (2009) mAbs 1:1).

[0913] In some implementations, the anti-TIM3 variable region described herein is linked to an effectorless or predominantly effectorless Fc, such as IgG1.

[0914] Generally, the variable regions described herein can be linked to Fc regions containing one or more modifications, which 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. Furthermore, 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. The residue numbers in the Fc region are Kabat EU index numbers.

[0915] The Fc region contains domains derived from the constant region of immunoglobulins, including fragments, analogs, variants, mutants, or derivatives of the constant region. 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 homologous to the C-terminal region of an immunoglobulin, and may include, alone or in combination, a CH1 domain, a hinge, a CH2 domain, a CH3 domain, or a CH4 domain.

[0916] Ig molecules interact with a variety of cellular receptors. For example, IgG molecules interact with three classes of Fcγ receptors (FcγRs) specific to IgG antibodies: FcγRI, FcγRII, and FcγRIII. It has been reported that the key sequences for IgG binding to FcγR receptors are located in the CH2 and CH3 domains. The serum half-life of an antibody is influenced by its ability to bind to Fc receptors (FcRs).

[0917] In some embodiments, the Fc region is a variant Fc region, for example, an Fc sequence that has been modified (e.g., by amino acid substitution, deletion and / or insertion) relative to the parental Fc sequence (e.g., an unmodified Fc polypeptide that is subsequently modified to produce a variant) to provide the desired structural features and / or biological activity.

[0918] Typically, variants of the constant region or portions thereof (e.g., CH1, CL, hinge, CH2, or CH3 domains) may 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 at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as the corresponding wild-type region or domain (CH1, CL, hinge, CH2, or CH3 domains, respectively), provided that the heavy chain constant region containing that particular variant retains the necessary biological activity.

[0919] For example, modifications can be made to the Fc region to produce Fc variants that, relative to the parental Fc, (a) increase or decrease antibody-dependent cell-mediated cytotoxicity (ADCC), (b) increase or decrease complement-mediated cytotoxicity (CDC), (c) increase or decrease affinity for C1q, and / or (d) increase or decrease affinity for the Fc receptor. Such Fc variants typically contain at least one amino acid modification in the Fc region. Combinations of amino acid modifications are considered particularly desirable. For example, the variant Fc region may include two, three, four, five, etc., substitutions therein (e.g., at specific Fc region positions identified herein).

[0920] The variant Fc region may also contain sequence alterations, where amino acids involved in disulfide bond formation are removed or replaced by other amino acids. Such removals can prevent reactions with other cysteine-containing proteins present in host cells used to generate the anti-TIM3 antibody described herein. Even with the removal of cysteine ​​residues, the single-chain Fc domain can still form a non-covalently bound dimer Fc domain. In some embodiments, the Fc region can be modified to make it 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, as this sequence can be recognized by digestive enzymes in E. coli, such as proline iminopeptidase. In some embodiments, one or more glycosylation sites in the Fc domain can be removed. Residues that are normally glycosylated (e.g., asparagine) can confer a cytolytic response. These residues can be deleted or replaced with non-glycosylated residues (e.g., alanine). In some embodiments, sites that interact with complement, such as C1q binding sites, can be removed from the Fc region. For example, the EKK sequence of human IgG1 can be deleted or replaced. In some embodiments, sites affecting binding to the Fc receptor may be removed, preferably sites other than the salvage receptor binding site. In some embodiments, the Fc region may be modified to remove ADCC sites. ADCC sites are known in the art. For information on ADCC sites in IgG1, see, for example, Molec. Immunol. 29(5):633-9 (1992). Specific examples of variant Fc domains are disclosed, for example, WO 97 / 34631 and WO 96 / 32478.

[0921] In some embodiments, the hinge region of the Fc is modified such that the number of cysteine ​​residues in the hinge region is altered, for example, increased or decreased. This method is further described in U.S. Patent No. 5,677,425 to Bodmer et al. Changing the number of cysteine ​​residues in the hinge region of the Fc can, for example, promote the assembly of light and heavy chains or increase or decrease antibody stability. In some embodiments, the Fc hinge region of the antibody is mutated to reduce the antibody's biological half-life. 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 binding to staphylococcal protein A (SpA) relative to the native Fc-hinge domain SpA binding. This method is further described in detail in U.S. Patent No. 6,165,745 to Ward et al.

[0922] In some embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to change 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 to give the antibody altered affinity for the effector ligand, but retain the antigen-binding ability of the parent 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. patents by Winter et al., 5,624,821 and 5,648,260.

[0923] 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 alter C1q binding and / or reduce or eliminate complement-dependent cytotoxicity (CDC). This method is described in more detail in U.S. Patent No. 6,194,551 to Idusogie et al.

[0924] In another instance, altering one or more amino acid residues at positions 231 and 239 changes the antibody's ability to fix complement. This method is further described in PCT Publication WO 94 / 29351 by Bodmer et al.

[0925] In yet another example, the Fc region can be modified by altering one or more amino acids at the following positions to reduce antibody-dependent cytotoxicity (ADCC) and / or decrease affinity for the Fcγ receptor: 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. Examples of substitutions include 236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D, and 332E. Examples of variants include 239D / 332E, 236A / 332E, 236A / 239D / 332E, 268F / 324T, 267E / 268F, 267E / 324T, and 267E / 268F7324T. Other modifications for enhancing FcγR and complement interactions include, but are not limited to, substitutions of 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.

[0926] Increased Fc modifications for binding to the Fcγ receptor include any one or more amino acid positions in the Fc region: 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, 3 Amino acid modifications at positions 05, 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, where the residues in the Fc region are numbered according to the EU index (WO00 / 42072).

[0927] Other Fc modifications that can be made to Fc are those used to reduce or eliminate binding to FcγR and / or complement proteins, thereby reducing or eliminating Fc-mediated effector functions such as ADCC, ADCP, and CDC. Exemplary modifications include, but are not limited to, substitutions, insertions, and deletions at positions 234, 235, 236, 237, 267, 269, 325, 328, 330, and / or 331 (e.g., 330 and 331), where the numbering is based on the EU index. Exemplary substitutions include, but are not limited to, 234A, 235E, 236R, 237A, 267R, 269R, 325L, 328R, 330S, and 331S (e.g., 330S and 331S), where the numbering is based on the EU index. Fc variants may include 236R / 328R. Other modifications used to reduce FcγR and complement interactions include substitutions for 297A, 234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 331S, 220S, 226S, 229S, 238S, 233P, and 234V, as well as removal of the glycosylation at position 297 by mutagenesis or enzymatic methods, or by methods produced in organisms, such as bacteria that produce non-glycosylated proteins. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology, 20:685-691.

[0928] Optionally, the Fc region may contain non-naturally occurring amino acid residues at additional and / or alternative locations known to those skilled in the art (see, for example, U.S. Patent Nos. 5,624,821; 6,277,375; 6,737,056; 6,194,551; 7,317,091; 8,101,720; PCX 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; WO 05 / 070963; WO 05 / 040217, WO 05 / 092925 and WO 06 / 020114).

[0929] Fc variants that enhance affinity for the inhibitory receptor FcγRIIb can also be used. Such variants can provide Fc fusion proteins with immunomodulatory activities associated with FcγRIIb cells, including, for example, B cells and monocytes. In some embodiments, the Fc variants provide selectively enhanced affinity for FcγRIIb relative to one or more activated receptors. Modifications for altering binding to FcγRIIb include one or more modifications selected from positions 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327, 328, 330, 331, and 332 according to E...

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: (a) the amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 are shown in SEQ ID NOs: 45, 413 and 414, respectively, and the amino acid sequences of the light chain CDR1, CDR2 and CDR3 are shown in SEQ ID NOs: 64, 66 and 69, respectively; (b) the amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 are shown in SEQ ID NOs: 45, 415 and 416, respectively, and the amino acid sequences of the light chain CDR1, CDR2 and CDR3 are shown in SEQ ID NOs: 64, 66 and 68, respectively; or (c) the amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 are shown in SEQ ID NOs: 45, 415 and 416, respectively, and the amino acid sequences of the light chain CDR1, CDR2 and CDR3 are shown in SEQ ID NOs: 64, 66 and 419, respectively.

2. The antibody according to claim 1, wherein the amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 are shown in SEQ ID NOs: 45, 413 and 414, respectively, and the amino acid sequences of the light chain CDR1, CDR2 and CDR3 are shown in SEQ ID NOs: 64, 66 and 69, respectively.

3. The antibody according to claim 1, wherein the amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 are shown in SEQ ID NOs: 45, 415, 416, respectively, and the amino acid sequences of the light chain CDR1, CDR2 and CDR3 are shown in SEQ ID NOs: 64, 66 and 68, respectively.

4. The antibody according to claim 1, wherein the amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 are shown in SEQ ID NOs: 45, 415 and 416, respectively, and the amino acid sequences of the light chain CDR1, CDR2 and CDR3 are shown in SEQ ID NOs: 64, 66 and 419, respectively.

5. The antibody according to claim 1, comprising a heavy chain variable region VH and a light chain variable region VL, wherein: (a) VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 410; and VL comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 417; (b) VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 411; and VL comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 418; or (c) VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 412; and VL comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:

60.

6. The antibody of claim 1, comprising a VH and a VL, wherein the VH comprises SEQ ID NO: 410 and the VL comprises SEQ ID NO:

417.

7. The antibody of claim 1, comprising a VH and a VL, wherein the VH comprises SEQ ID NO: 411 and the VL comprises SEQ ID NO:

418.

8. The antibody of claim 1, comprising a VH and a VL, wherein the VH comprises SEQ ID NO: 412 and the VL comprises SEQ ID NO:

60.

9. The antibody according to any one of claims 1-8, wherein the antibody is selected from IgG1, IgG2, IgG3, IgG4 and variants thereof.

10. The antibody according to claim 9, wherein the antibody is an IgG1 antibody.

11. The antibody according to claim 10, comprising an IgG1 Fc without effector function.

12. The antibody according to any one of claims 1 to 8, comprising: (a1) a heavy chain sequence comprising SEQ ID NO: 386 or 387 and a light chain sequence comprising SEQ ID NO: 408; (a2) a heavy chain sequence comprising SEQ ID NO: 388 or 389 and a light chain sequence comprising SEQ ID NO: 408; (a3) a heavy chain sequence comprising SEQ ID NO: 390 or 391 and a light chain sequence comprising SEQ ID NO: 408; (a4) a heavy chain sequence comprising SEQ ID NO: 392 or 393 and a light chain sequence comprising SEQ ID NO: 408; (b1) a heavy chain sequence comprising SEQ ID NO: 394 or 395 and a light chain sequence comprising SEQ ID NO: 29; (b2) a heavy chain sequence comprising SEQ ID NO: 394 or 395 and a light chain sequence comprising SEQ ID NO: 409; (b3) a heavy chain sequence comprising SEQ ID NO: 396 or 397 and a light chain sequence comprising SEQ ID NO: 29; (b4) a heavy chain sequence comprising SEQ ID NO: 398 or 399 and a light chain sequence comprising SEQ ID NO: 29; (b5) a heavy chain sequence comprising SEQ ID NO: 400 or 401 and a light chain sequence comprising SEQ ID NO: 29; (b6) a heavy chain sequence comprising SEQ ID NO: 402 or 403 and a light chain sequence comprising SEQ ID NO: 29; (b7) a heavy chain sequence comprising SEQ ID NO: 404 or 405 and a light chain sequence comprising SEQ ID NO: 29; or (b8) a heavy chain sequence comprising SEQ ID NO: 406 or 407 and a light chain sequence comprising SEQ ID NO:

29.

13. The antibody according to any one of claims 1-8, which has one or more of the following properties: (1) With an EC of 1 µg / mL or less 50 Binding to membrane-bound human TIM3 as determined by flow cytometry; (2) binding to membrane-bound cyno TIM3; (3) K D 5x10 -8 M, 2x10 -8 M, 1x10 -8 M or 5x10 -9 M or less binds to hTIM3-IgV; (4) K D 10 -7 M, 2x10 -8 M or 1x10 -8 M or less binds to hTIM3-IgV; (5) K D 5x10 -7 M, 2x10 -8 M or 1x10 -8 M or less binds to cynomolgus monkey TIM3-ECD; (6) K D 8x10 -8 M, 5x10 -8 M or 1x10 -8 M or less binds to hTIM3-ECD; or (7) Induce or enhance T cell activation.

14. A bispecific molecule comprising the antibody of any one of claims 1 to 13 linked to a molecule having a second binding specificity.

15. A nucleic acid molecule encoding the VH and VL of the antibody of any one of claims 6 to 8; or a nucleic acid molecule encoding the heavy chain and light chain of the antibody of claim 12.

16. An expression vector comprising the nucleic acid molecule of claim 15.

17. A cell transformed with the expression vector of claim 16.

18. An immunoconjugate comprising the antibody of any one of claims 1 to 13 linked to an agent.

19. A composition comprising the antibody of any one of claims 1 to 13, the bispecific molecule of claim 14, the nucleic acid molecule of claim 15, the expression vector of claim 16, the cell of claim 17 or the immunoconjugate of claim 18, and a carrier.

20. A composition comprising: (a) an expression vector comprising a nucleic acid molecule encoding the VH of the antibody or antigen-binding portion thereof of any one of claims 6 to 8, and an expression vector comprising a nucleic acid molecule encoding the VL of the antibody or antigen-binding portion thereof of any one of claims 6 to 8, or (b) an expression vector comprising a nucleic acid molecule encoding the heavy chain of the antibody according to claim 12, and an expression vector comprising a nucleic acid molecule encoding the light chain of the antibody according to claim 12.

21. A cell transformed with the composition of claim 20.

22. A kit comprising the antibody of any one of claims 1 to 13, the bispecific molecule of claim 14, the nucleic acid molecule of claim 15, the expression vector of claim 16, the cell of claim 17 or 21, the immunoconjugate of claim 18, or the composition of claim 19 or 20, and instructions for use.

23. A method for preparing an anti-TIM3 antibody, comprising expressing the antibody in the cell of claim 17 or 21, and isolating the antibody from the cell.

24. Use of the antibody of any one of claims 1 to 13, the bispecific molecule of claim 14, the nucleic acid molecule of claim 15, the expression vector of claim 16, the cell of claim 17 or 21, the immunoconjugate of claim 18 or the composition of claim 19 or 20 for the preparation of a medicament for treating cancer.

25. Use of the antibody of any one of claims 1 to 13 for the preparation of a kit for detecting the presence of T cell immunoglobulin and mucin domain-containing protein 3 (TIM3).

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