Combination therapy targeting PD-1, TIM-3, and LAG-3

Abstract

The present disclosure relates to a combination therapy that targets two or all of PD-1, TIM-3, and LAG-3 using antibodies specific for these targets in patients requiring immune enhancement. Also included in the present disclosure are compositions useful for the therapy. The therapy is useful for treating diseases such as cancer.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 481,973, filed on April 5, 2017, the entirety of which is incorporated herein by reference.

[0002] Sequence List This application is electronically filed in ASCII format and includes a sequence listing which is incorporated herein by reference in its entirety. An electronic copy of the sequence listing was created on 23 March 2018, named 022675_WO058_SL.txt, and has a size of 232,360 bytes. [Background technology]

[0003] PD-1, also known as programmed cell death protein 1 and CD279, is a 268-amino acid cell surface receptor belonging to the immunoglobulin superfamily. PD-1 is a member of the CD28 T cell regulatory factor family and is expressed in T cells, B cells, and macrophages. PD-1 binds to ligands PD-L1 (also known as B7 homolog) and PD-L2 (also known as B7-DC). PD-1 is a type I membrane protein whose structure includes an extracellular IgV domain, a transmembrane region, and an intracellular tail containing two phosphorylation sites. Known as an immune checkpoint protein, PD-1 functions as an inducible immunomodulatory receptor, playing a role, for example, in the negative regulation of the T cell response to antigen stimulation.

[0004] PD-L1 is the primary ligand for PD-1. When PD-L1 binds to PD-1, it inhibits T cell activity, reduces cytokine production, and suppresses T cell proliferation. Cancer cells expressing PD-L1 can utilize this mechanism to inactivate the antitumor activity of T cells via PD-L1 binding to the PD-1 receptor. Given its role in modulating the immune response, PD-1 is being studied as a potential target for immunotherapy, including the treatment of cancer and autoimmune diseases. Two anti-PD-1 antibodies, pembrolizumab and nivolumab, are approved in the United States and Europe for the treatment of certain cancers.

[0005] Other immune checkpoint proteins include TIM-3 (T cell immunoglobulin-mucin domain molecule 3) and LAG-3 (lymphocyte activation gene 3). TIM-3, also known as HAVCR2 (hepatitis A virus cell receptor 2) or CD366, is a member of the T cell immunoglobulin-mucin domain protein family. In humans, TIM-3 is encoded by the Havcr2 gene and is a 33 kDa type I glycoprotein with a distal IgV domain and a proximal mucin domain. It contains a conserved region of five Tyr residues in its intracellular domain, which is phosphorylated upon ligand binding. TIM-3 is expressed by a diverse range of different cells, including T cells, dendritic cells, macrophages, and natural killer (NK) cells, originating from both the adaptive and innate immune systems. TIM-3 expression is low in naive T cells but is highly upregulated when T cells are activated. In contrast to T cells, native cells such as dendritic cells, NK cells, and monocytes have high basal TIM-3 expression. TIM-3 associates with several predominantly heterogeneous ligands, including galectin-9, phosphatidylserine, CEACAM-1, and HMGB-1, although the precise roles of these ligands are currently unknown.

[0006] While TIM-3 is suggested to be a checkpoint inhibitor, there is relatively little evidence to support the idea that TIM-3 directly mediates T cell activation or cytokine secretion suppression in a manner similar to, for example, PD-1. Furthermore, in contrast to PD-1, TIM-3 appears to play a role in regulating cells of the innate immune system, particularly dendritic cells. Much of the functional data related to TIM-3 and its role in tumor immunity comes from in vivo studies using various antibodies. In most of these studies, due to insufficient validation of the antibodies, it is unclear whether the effects of TIM-3 antibodies are mediated by ligand binding inhibition or by agonist action against the target. Given its immunomodulatory properties, TIM-3 is being studied as a potential target for immunotherapy, including the treatment of cancer and autoimmune diseases. A single anti-TIM-3 antibody is currently in clinical development, but there are currently no approved anti-TIM-3 antibodies.

[0007] LAG-3, also known as CD223, is an immunoglobulin superfamily protein that functions as an immune checkpoint receptor. The mature protein is a 503-amino acid type I transmembrane protein with four extracellular Ig-like domains. It is expressed in various cell types, including activated T cells, regulatory T (Treg) cells, natural killer cells, B cells, and plasmacytoid dendritic cells. Information on LAG-3 sequence data, exon / intron configuration, and chromosomal localization indicates that it is closely related to CD4. Like CD4, LAG-3 binds to MHC class II molecules, but with higher affinity to a distinct site compared to CD4.

[0008] LAG-3 is a costimulatory receptor thought to regulate T cell proliferation, activation, and homeostasis in a manner similar to CTLA-4 and PD-1. Once a ligand binds to its extracellular domain, LAG-3 exerts its effects through subsequent signaling via its cytoplasmic domain. While MHC class II (MHCII) ligands are the most well-characterized ligands for LAG-3, other LAG-3 ligands, including LSECtin, have been described. LAG-3 lacks the classical ITIM or ITSM motif but possesses a conserved KIEELE motif (SEQ ID NO: 397), which is thought to be essential for achieving its inhibitory effect on T cell activity. The precise mechanism by which LAG-3 affects T cell activity is not fully understood. LAG-3 inhibits T cell growth by blocking activated T cells from entering the proliferative phase of the cell cycle, resulting in cell accumulation in the S phase. LAG-3 is also thought to play a role in enhancing the inhibitory activity of regulatory T cells and regulating dendritic cell function. Cancer cells have the ability to upregulate the expression of MHCII that binds to LAG-3 on effector T cells, thereby inhibiting its activity and inducing evasion of tumor immunity.

[0009] Given the crucial roles of PD-1, LAG-3, and TIM-3 as immunomodulators, novel and improved combination therapies targeting these receptors are needed to address cancer and certain disorders of the immune system. [Overview of the project] [Problems that the invention aims to solve]

[0010] This invention is based on the discovery that the immune-enhancing efficacy of anti-PD-1 antibodies, such as those described herein, is significantly increased when used in combination with anti-TIM-3 antibodies and / or anti-LAG-3 antibodies. The inventors have found that the combination therapy of this invention is particularly effective for treating cancer in human patients by activating the patient's own anti-cancer immunity. Compared to currently available cancer treatments, including antibody treatments, the combination therapy of this invention is intended to provide a superior clinical response. [Means for solving the problem]

[0011] Accordingly, the present invention provides a method for enhancing immunity in human patients in need, such as cancer patients, by administering to a patient (1) an anti-PD-1 antibody or its antigen-binding moiety that competes with an antibody selected from the group consisting of 12819.15384, 12748.15381, 12748.16124, 12865.15377, 12892.15378, 12796.15376, 12777.15382, 12760.15375 and 13112.15380 for binding to human PD-1, or that binds to the same human PD-1 epitope as the antibody; and (2) an anti-TIM-3 antibody or its antigen-binding moiety, and / or an anti-LAG-3 antibody or its antigen-binding moiety. In a particular embodiment, the method includes administering an anti-PD-1 antibody or its antigen-binding moiety, an anti-TIM-3 antibody or its antigen-binding moiety, and an anti-LAG-3 antibody or its antigen-binding moiety.

[0012] In some embodiments, the anti-PD-1 antibody is a) Residues V64, L128, P130, K131, and A132 of SEQ ID NO: 388; b) Residues V44 and T145 of SEQ ID NO: 388; c) Residues K131 and E136 of SEQ ID NO: 388; or d) Residues V44 and T145 of SEQ ID NO: 388 It binds to the human PD-1 epitope, including [specific component].

[0013] In some embodiments, the anti-PD-1 antibody is a) Residues 56-64, 69-90, and 122-140 of SEQ ID NO: 388; b) Residues 69-90 and 122-140 of Sequence ID No. 388; c) Residues 69-75 of sequence number 388; d) Residues 136-140 of SEQ ID NO: 388; or e) Residues 69-75 and 136-140 of SEQ ID NO: 388 It binds to the human PD-1 epitope, including [specific component].

[0014] In some embodiments, the anti-PD-1 antibody has the following characteristics: a) K₀ 750 pM or less D Binding to human PD-1; b) Kelvin of 7nM or less D It binds to cynomolgus monkey PD-1; c) K(s) 1nM or less D To bind to mouse PD-1; d) Not bind to rat PD-1; e) To increase IL-2 secretion in a whole blood assay of Staphylococcus enterotoxin B (SEB); f) To increase IFN-γ secretion in a unidirectional mixed lymphocyte reaction assay; g) Inhibit the interaction between PD-1 and PD-L1 by at least 60% at a concentration of 10 μg / ml in a flow cytometry competitive assay; h) Blocking at least 90% of the binding of PD-L1 and PD-L2 to PD-1 at a concentration of 10 μg / ml as determined by biolayer interferometry analysis; and i) inhibiting tumor growth in vivo; and having at least one of these properties. In a particular embodiment, the anti-PD-1 antibody has all of the above properties.

[0015] In some embodiments, the heavy chain complementarity-determining regions (H-CDRs) 1-3 and light chain complementarity-determining regions (L-CDRs) 1-3 of the anti-PD-1 antibody are a) Sequence numbers 228-233, respectively; b) Sequence numbers 238-243, respectively; c) Sequence numbers 248-253, respectively; d) Sequence numbers 258-263, respectively; e) Sequence numbers 268-273, respectively; f) Sequence numbers 278-283, respectively; g) Sequence IDs 288-293, respectively; or h) Sequence numbers 298-303, respectively It contains the amino acid sequence.

[0016] In some embodiments, the heavy and light chain variable domains of the anti-PD-1 antibody are a) Sequence IDs 226 and 227, respectively; b) Sequence IDs 236 and 237, respectively; c) Sequence IDs 236 and 392, respectively; d) Sequence IDs 246 and 247, respectively; e) Sequence IDs 256 and 257, respectively; f) Sequence IDs 266 and 267, respectively; g) Sequence IDs 276 and 277, respectively; h) Sequence IDs 286 and 287, respectively; or i) Sequence numbers 296 and 297, respectively It contains the amino acid sequence.

[0017] In some embodiments, the anti-PD-1 antibody is a) A heavy chain (HC) containing the amino acid sequences of SEQ ID NO: 226 and SEQ ID NO: 375, and a light chain (LC) containing the amino acid sequences of SEQ ID NO: 227 and SEQ ID NO: 379; b) HC containing the amino acid sequence of SEQ ID NO: 236 and SEQ ID NO: 375, and LC containing the amino acid sequence of SEQ ID NO: 237 and SEQ ID NO: 379; c) HC containing the amino acid sequence of SEQ ID NO: 236 and SEQ ID NO: 375, and LC containing the amino acid sequence of SEQ ID NO: 392 and SEQ ID NO: 379; d) HC containing the amino acid sequence of SEQ ID NO: 246 and SEQ ID NO: 375, and LC containing the amino acid sequence of SEQ ID NO: 247 and SEQ ID NO: 379; e) HC containing the amino acid sequences of SEQ ID NO: 256 and SEQ ID NO: 375, and LC containing the amino acid sequences of SEQ ID NO: 257 and SEQ ID NO: 379; f) HC containing the amino acid sequences of SEQ ID NO: 266 and SEQ ID NO: 375, and LC containing the amino acid sequences of SEQ ID NO: 267 and SEQ ID NO: 379; g) HC containing the amino acid sequences of SEQ ID NO: 276 and SEQ ID NO: 375, and LC containing the amino acid sequences of SEQ ID NO: 277 and SEQ ID NO: 379; h) HC containing the amino acid sequence of SEQ ID NO: 286 and SEQ ID NO: 375, and LC containing the amino acid sequence of SEQ ID NO: 287 and SEQ ID NO: 379; or i) HC containing the amino acid sequence of SEQ ID NO: 296 and SEQ ID NO: 375, and LC containing the amino acid sequence of SEQ ID NO: 297 and SEQ ID NO: 379 Includes.

[0018] In some embodiments, the anti-TIM-3 antibody is An antibody selected from the group consisting of 15086.17145, 15086.15086, 15086.16837, 15086.17144, 20131, 20293, 15105, 15107, 15109, 15174, 15175, 15260, 15284, 15299, 15353, 15354, 17244, 17245, 19324, 19416, 19568, 20185, 20300, 20362, and 20621 competes for binding to human TIM-3 or binds to the same human TIM-3 epitope as said antibody.

[0019] In some embodiments, the anti-TIM-3 antibody is a) Residues P50, V60, F61, E62, G64, R69, I117, M118, and D120 of SEQ ID NO: 389; b) Residues F61, R69, and I117 of SEQ ID NO: 389; or c) Residues P50, F61, E62, I117, M118, and D120 of SEQ ID NO: 389 It binds to the human TIM-3 epitope, which includes [specific component].

[0020] In some embodiments, the anti-TIM-3 antibody is a) Residues 62-67 of SEQ ID NO: 389; or b) Residues 114-117 of Sequence ID No. 389 It binds to the human TIM-3 epitope, which includes [specific component].

[0021] In some embodiments, the anti-TIM-3 antibody has the following characteristics: a) K(T) 23 nM or less when measured by surface plasmon resonance D To bind to human TIM-3; b) K(T) 22 nM or less when measured by surface plasmon resonance D To bind to TIM-3 in cynomolgus monkeys; c) Binding to human TIM-3 at an EC50 of 1.2 nM or less as measured by ELISA; d) Binding to cynomolgus monkey TIM-3 at an EC50 of 46 nM or less when measured by ELISA; e) To increase IFN-γ secretion in a unidirectional mixed lymphocyte reaction assay; f) To increase IFN-γ secretion in a bidirectional mixed lymphocyte reaction assay; g) Increasing TNF-α secretion in a unidirectional mixed lymphocyte reaction assay; h) Increasing TNF-α secretion from dendritic cells; and i) Inhibiting the interaction between TIM-3 and phosphatidylserine. It has at least one of the following. In a particular embodiment, the anti-TIM-3 antibody has at least the following characteristics: a), c), d), e), g), h), and i).

[0022] In some embodiments, the anti-TIM-3 antibodies H-CDR1~3 and L-CDR1~3 are a) Sequence numbers 8-13, respectively; b) Sequence numbers 18-23, respectively; c) Sequence numbers 28-33, respectively; d) Sequence numbers 38-43, respectively; e) Sequence numbers 48-53, respectively; f) Sequence numbers 58-63, respectively; g) Sequence IDs 68-73, respectively; h) Sequence numbers 78-83, respectively; i) Sequence numbers 88-93, respectively; j) Sequence numbers 98-103, respectively; k) Sequence numbers 108-113, respectively; l) Sequence numbers 118-123, respectively; m) Sequence numbers 128-133, respectively; n) Sequence numbers 138-143, respectively; o) Sequence numbers 148-153, respectively; p) Sequence numbers 158-163, respectively; q) Sequence numbers 168-173, respectively; r) Sequence numbers 178-183, respectively; s) Sequence numbers 188-193, respectively; t) Sequence numbers 198-203, respectively; u) Sequence numbers 208-213, respectively; or v) Sequence numbers 218-223, respectively It contains the amino acid sequence.

[0023] In some embodiments, the heavy and light chain variable domains of the anti-TIM-3 antibody are a) Sequence IDs 7 and 4, respectively; b) Sequence IDs 3 and 4, respectively; c) Sequence IDs 16 and 17, respectively; d) Sequence IDs 26 and 27, respectively; e) Sequence IDs 36 and 37, respectively; f) Sequence numbers 46 and 47, respectively; g) Sequence IDs 56 and 57, respectively; h) Sequence IDs 66 and 67, respectively; i) Sequence numbers 76 and 77, respectively; j) Sequence IDs 86 and 87, respectively; k) Sequence numbers 96 and 97, respectively; l) Sequence IDs 106 and 107, respectively; m) Sequence numbers 116 and 117, respectively; n) Sequence IDs 126 and 127, respectively; o) Sequence IDs 136 and 137, respectively; p) Sequence IDs 146 and 147, respectively; q) Sequence IDs 156 and 157, respectively; r) Sequence IDs 166 and 167, respectively; s) Sequence IDs 176 and 177, respectively; t) Sequence IDs 186 and 187, respectively; u) Sequence IDs 196 and 197, respectively; v) Sequence IDs 206 and 207, respectively; or w) Sequence numbers 216 and 217, respectively It contains the amino acid sequence.

[0024] In some embodiments, the anti-TIM-3 antibody is a) HC containing the amino acid sequence of SEQ ID NO: 7 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 4 and the amino acid sequence of SEQ ID NO: 378; b) HC containing the amino acid sequence of SEQ ID NO: 3 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 4 and the amino acid sequence of SEQ ID NO: 378; c) HC containing the amino acid sequence of SEQ ID NO: 16 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 17 and the amino acid sequence of SEQ ID NO: 378; d) HC containing the amino acid sequence of SEQ ID NO: 26 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 27 and the amino acid sequence of SEQ ID NO: 378; e) HCs containing the amino acid sequence of SEQ ID NO: 36 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LCs containing the amino acid sequence of SEQ ID NO: 37 and the amino acid sequence of SEQ ID NO: 378; f) HCs containing the amino acid sequence of SEQ ID NO: 46 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, as well as HCs containing the amino acid sequence of SEQ ID NO: 47 and the amino acid sequence of SEQ ID NO: 378; g) HC containing the amino acid sequence of SEQ ID NO: 56 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 57 and the amino acid sequence of SEQ ID NO: 378; h) HC containing the amino acid sequence of SEQ ID NO: 66 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 67 and the amino acid sequence of SEQ ID NO: 378; i) HCs containing the amino acid sequence of SEQ ID NO: 76 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LCs containing the amino acid sequence of SEQ ID NO: 77 and the amino acid sequence of SEQ ID NO: 378; j) HC containing the amino acid sequence of SEQ ID NO: 86 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 87 and the amino acid sequence of SEQ ID NO: 378; k) HC containing the amino acid sequence of SEQ ID NO: 96 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 97 and the amino acid sequence of SEQ ID NO: 378; l) HC containing the amino acid sequence of SEQ ID NO: 106 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 107 and the amino acid sequence of SEQ ID NO: 378; m) HC containing the amino acid sequence of SEQ ID NO: 116 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 117 and the amino acid sequence of SEQ ID NO: 378; n) HC containing the amino acid sequence of SEQ ID NO: 126 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 127 and the amino acid sequence of SEQ ID NO: 378; o) HC containing the amino acid sequence of SEQ ID NO: 136 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 137 and the amino acid sequence of SEQ ID NO: 378; p) HC containing the amino acid sequence of SEQ ID NO: 146 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 147 and the amino acid sequence of SEQ ID NO: 378; q) HC containing the amino acid sequence of SEQ ID NO: 156 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 157 and the amino acid sequence of SEQ ID NO: 378; r) HC containing the amino acid sequence of SEQ ID NO: 166 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 167 and the amino acid sequence of SEQ ID NO: 378; s) HC containing the amino acid sequence of SEQ ID NO: 176 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 177 and the amino acid sequence of SEQ ID NO: 378; t) HC containing the amino acid sequence of SEQ ID NO: 186 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 187 and the amino acid sequence of SEQ ID NO: 378; u) HC containing the amino acid sequence of SEQ ID NO: 196 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 197 and the amino acid sequence of SEQ ID NO: 378; v) HC containing the amino acid sequence of SEQ ID NO: 206 and the amino acid sequences of SEQ ID NO: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 378; or w) HC containing the amino acid sequence of SEQ ID NO: 216 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 378 Includes.

[0025] In some embodiments, the anti-LAG-3 antibody competes with an antibody selected from the group consisting of 15532, 15646, 15723, 15595, 15431, 15572, and 15011 for binding to human LAG-3, or binds to the same human LAG-3 epitope as the antibody.

[0026] In some embodiments, the anti-LAG-3 antibody is a) Residues H85, P86, A87, P89, S91, W92, and G93 of SEQ ID NO: 68; b) Residues A40, Q41, P43, P46, P49, D52, T62, Q64, H65, Q66, P67, D68, G93, P94, P96, R98, Y99, T100, V101, P106, G107, R119, E124, R129, G130, D131, S133, R137, P138, D143, R148, and R163 of SEQ ID NO: 68; c) Residues A40, Q41, P43, P46, P49, D52, T62, Q64, H65, Q66, P67, D68, P96, Y99, T100, V101, P106, G107, R119, E124, R129, G130, D131, S133, R137, P138, D143, R148, and R163 of SEQ ID NO: 68; or d) Residues G107, L109, R110, and S111 of SEQ ID NO: 68 It binds to the human LAG-3 epitope, including [specific component].

[0027] In some embodiments, the anti-LAG-3 antibody is a) Residues 98-105 of Sequence ID No. 68; b) Residues 78-105 and 123-131 of Sequence ID No. 68; c) Residues 23-30, 40-66, 88-105, 123-137, and 148-152 of SEQ ID NO: 68; or d) Residues 23-30, 40-66, 98-105, 118-137, and 148-161 of SEQ ID NO: 68 It binds to the human LAG-3 epitope, including [specific component].

[0028] In some embodiments, the anti-LAG-3 antibody has the following characteristics: a) At a concentration of 20 μg / mL, reduce the binding of human LAG-3 to human MHC class II on A375 cells by more than 85% compared to a negative control antibody, as determined by a flow cytometry competitive assay; b) Reducing the binding of human LAG-3 to human MHC class II on A375 cells by 35% to 85% compared to a negative control antibody at a concentration of 20 μg / mL, as determined by a flow cytometry competitive assay; c) blocking the binding between human LAG-3 expressed in Jurkat cells and human MHC class II expressed in Raji cells; d) binding to human LAG-3 with an EC50 of 0.1 nM or less when measured by flow cytometry; e) binding to cynomolgus monkey LAG-3 with an EC50 of 0.3 nM or less when measured by flow cytometry; f) binding to human LAG-3 with a K -8 of 3.0×10 D or less when measured by surface plasmon resonance; g) binding to cynomolgus monkey LAG-3 with a K -7 of 1.5×10 D or less when measured by surface plasmon resonance; h) binding to mouse LAG-3 with a K -8 of 3.5×10 D or less when measured by surface plasmon resonance; i) stimulating IL-2 production in human peripheral blood mononuclear cells (PBMCs) treated with Staphylococcus enterotoxin B (SEB); j) reducing the cellular level of LAG-3 in human T cells; k) reducing the soluble level of LAG-3 in human T cell culture; l) inducing regression of tumor growth in vivo; m) delaying tumor growth in vivo; and n) not binding to the same epitope of human LAG-3 as antibody 25F7-Lag-3.5 and having at least one of the above. In certain embodiments, the anti-LAG-3 antibody has at least properties a), c), d), e), f), g), i), j), k), m) and n).

[0029] In certain embodiments, the H-CDR1-3 and L-CDR1-3 of the anti-LAG-3 antibody are a) SEQ ID NOs: 318-323 respectively; b) Sequence numbers 308-313, respectively; c) Sequence numbers 328-333, respectively; d) Sequence numbers 338-343, respectively; e) Sequence numbers 348-353, respectively; f) Sequence numbers 358-363, respectively; or g) Sequence numbers 368-373, respectively It contains the amino acid sequence.

[0030] In some embodiments, the heavy and light chain variable domains of the anti-LAG-3 antibody are a) Sequence IDs 316 and 317, respectively; b) Sequence IDs 306 and 307, respectively; c) Sequence IDs 326 and 327, respectively; d) Sequence IDs 336 and 337, respectively; e) Sequence IDs 346 and 347, respectively; f) Sequence IDs 356 and 357, respectively; or g) Sequence IDs 366 and 367, respectively It contains the amino acid sequence.

[0031] In some embodiments, the anti-LAG-3 antibody is a) HC containing the amino acid sequence of SEQ ID NO: 316 and SEQ ID NO: 375, and LC containing the amino acid sequence of SEQ ID NO: 317 and SEQ ID NO: 378; b) HC containing the amino acid sequence of SEQ ID NO: 306 and SEQ ID NO: 375, and LC containing the amino acid sequence of SEQ ID NO: 307 and SEQ ID NO: 378; c) HC containing the amino acid sequence of SEQ ID NO: 326 and SEQ ID NO: 375, and LC containing the amino acid sequence of SEQ ID NO: 327 and SEQ ID NO: 378; d) HC containing the amino acid sequence of SEQ ID NO: 336 and SEQ ID NO: 375, and LC containing the amino acid sequence of SEQ ID NO: 337 and SEQ ID NO: 378; e) HC containing the amino acid sequences of SEQ ID NO: 346 and SEQ ID NO: 375, and LC containing the amino acid sequences of SEQ ID NO: 347 and SEQ ID NO: 378; f) HC containing the amino acid sequence of SEQ ID NO: 356 and SEQ ID NO: 375, and LC containing the amino acid sequence of SEQ ID NO: 357 and SEQ ID NO: 378; or g) HC containing the amino acid sequences of SEQ ID NO: 366 and SEQ ID NO: 375, and LC containing the amino acid sequences of SEQ ID NO: 367 and SEQ ID NO: 379 Includes.

[0032] In some embodiments, the method a) Anti-PD-1 antibodies in which H-CDR1-3 and L-CDR1-3 each contain the amino acid sequences of SEQ ID NOs. 228-233; and anti-TIM-3 antibodies in which H-CDR1-3 and L-CDR1-3 each contain the amino acid sequences of SEQ ID NOs. 8-13; b) Anti-PD-1 antibodies in which the VH and VL respectively contain the amino acid sequences of SEQ ID NOs. 226 and 227; and anti-TIM-3 antibodies in which the VH and VL respectively contain the amino acid sequences of SEQ ID NOs. 7 and 4; or c) Anti-PD-1 antibody comprising HC containing the amino acid sequences of SEQ ID NOs. 226 and 375, and LC containing the amino acid sequences of SEQ ID NOs. 227 and 379; and anti-TIM-3 antibody comprising HC containing the amino acid sequences of SEQ ID NOs. 7 and 377, and LC containing the amino acid sequences of SEQ ID NOs. 4 and 378. This includes administering the drug to the patient.

[0033] In some embodiments, the method a) Anti-PD-1 antibodies in which H-CDR1-3 and L-CDR1-3 each contain the amino acid sequences of SEQ ID NOs. 228-233; and anti-LAG-3 antibodies in which H-CDR1-3 and L-CDR1-3 each contain the amino acid sequences of SEQ ID NOs. 318-323; b) an anti-PD-1 antibody in which the VH and VL respectively contain the amino acid sequences of SEQ ID NOs. 226 and 227; and an anti-LAG-3 antibody in which the VH and VL respectively contain the amino acid sequences of SEQ ID NOs. 316 and 317; or c) Anti-PD-1 antibody comprising HC containing the amino acid sequences of SEQ ID NOs. 226 and 375, and LC containing the amino acid sequences of SEQ ID NOs. 227 and 379; and anti-LAG-3 antibody comprising HC containing the amino acid sequences of SEQ ID NOs. 316 and 375, and LC containing the amino acid sequences of SEQ ID NOs. 317 and 378. This includes administering the drug to the patient.

[0034] In some embodiments, the method a) Anti-PD-1 antibodies in which H-CDR1-3 and L-CDR1-3 each contain the amino acid sequences of SEQ ID NOs. 228-233; anti-TIM-3 antibodies in which H-CDR1-3 and L-CDR1-3 each contain the amino acid sequences of SEQ ID NOs. 8-13; and anti-LAG-3 antibodies in which H-CDR1-3 and L-CDR1-3 each contain the amino acid sequences of SEQ ID NOs. 318-323; b) an anti-PD-1 antibody in which the VH and VL respectively contain the amino acid sequences of SEQ ID NOs. 226 and 227; an anti-TIM-3 antibody in which the VH and VL respectively contain the amino acid sequences of SEQ ID NOs. 7 and 4; and an anti-LAG-3 antibody in which the VH and VL respectively contain the amino acid sequences of SEQ ID NOs. 316 and 317; or c) Anti-PD-1 antibody containing HC with amino acid sequences of SEQ ID NOs. 226 and 375, and LC with amino acid sequences of SEQ ID NOs. 227 and 379; anti-TIM-3 antibody containing HC with amino acid sequences of SEQ ID NOs. 7 and 377, and LC with amino acid sequences of SEQ ID NOs. 4 and 378; and anti-LAG-3 antibody containing HC with amino acid sequences of SEQ ID NOs. 316 and 375, and LC with amino acid sequences of SEQ ID NOs. 317 and 378 This includes administering the drug to the patient.

[0035] The antibody or antigen-binding moiety may be administered to the patient simultaneously (for example, in a single pharmaceutical composition) or sequentially.

[0036] The treatments of the present invention are useful for treating patients with cancer, such as hematological malignancies (e.g., leukemia, Hodgkin lymphoma, or non-Hodgkin lymphoma), or solid tumors. In some embodiments, patients may have melanoma, non-small cell lung cancer, bladder cancer, head and neck squamous cell carcinoma, ovarian cancer, colorectal cancer, renal cell carcinoma, Merkel cell carcinoma, fibrosarcoma, gliosarcoma, or glioblastoma. The treatments of the present invention may further comprise radiotherapy or at least one of chemotherapeutic agents, antineoplastic agents, and anti-angiogenic agents.

[0037] Similarly, the present invention provides a) human PD-1 and human TIM-3; b) human PD-1 and human LAG-3; or e) a polyspecific (e.g., bispecific or trispecific) antibody that specifically binds to human PD-1, human anti-TIM-3, and human LAG-3. In certain embodiments, the polyspecific antibody comprises an antigen-binding moiety of an anti-PD-1 antibody described herein, an antigen-binding moiety of an anti-TIM-3 antibody described herein, and / or an antigen-binding moiety of an anti-LAG-3 antibody described herein.

[0038] Similarly, the present invention also provides a pharmaceutical composition comprising (1) an anti-PD-1 antibody or its antigen-binding moiety as described herein, (2) an anti-TIM-3 antibody or its antigen-binding moiety, and / or an anti-LAG-3 antibody or its antigen-binding moiety, and (3) a pharmaceutically acceptable excipient. The anti-TIM-3 antibody and the anti-LAG-3 antibody can be selected from the antibodies described herein.

[0039] In some embodiments, the pharmaceutical composition is a) Anti-PD-1 antibodies in which H-CDR1-3 and L-CDR1-3 each contain the amino acid sequences of SEQ ID NOs. 228-233; anti-TIM-3 antibodies in which H-CDR1-3 and L-CDR1-3 each contain the amino acid sequences of SEQ ID NOs. 8-13; and anti-LAG-3 antibodies in which H-CDR1-3 and L-CDR1-3 each contain the amino acid sequences of SEQ ID NOs. 318-323; b) an anti-PD-1 antibody in which the VH and VL respectively contain the amino acid sequences of SEQ ID NOs. 226 and 227; an anti-TIM-3 antibody in which the VH and VL respectively contain the amino acid sequences of SEQ ID NOs. 7 and 4; and an anti-LAG-3 antibody in which the VH and VL respectively contain the amino acid sequences of SEQ ID NOs. 316 and 317; or c) Anti-PD-1 antibody containing HC with amino acid sequences of SEQ ID NOs. 226 and 375, and LC with amino acid sequences of SEQ ID NOs. 227 and 379; anti-TIM-3 antibody containing HC with amino acid sequences of SEQ ID NOs. 7 and 377, and LC with amino acid sequences of SEQ ID NOs. 4 and 378; and anti-LAG-3 antibody containing HC with amino acid sequences of SEQ ID NOs. 316 and 375, and LC with amino acid sequences of SEQ ID NOs. 317 and 378 Includes.

[0040] The antibodies in the composition may be present in equal amounts. In some embodiments, the pharmaceutical composition is for use in treating human patients in the methods described herein. In certain embodiments, the pharmaceutical composition is for use in enhancing immunity in human patients who require it, for example, to treat cancer.

[0041] The present invention also provides, for use in the treatment methods described herein, an anti-PD-1 antibody or its antigen-binding moiety, for use in combination with, for example, an anti-TIM-3 antibody or its antigen-binding moiety and / or an anti-LAG-3 antibody or its antigen-binding moiety, to enhance immunity and / or treat cancer in human patients requiring such enhancement. The anti-TIM-3 antibody and the anti-LAG-3 antibody can be selected from the antibodies described herein.

[0042] The present invention also provides an anti-PD-1 antibody or its antigen-binding moiety as described herein for use in the treatment of human patients in the methods described herein.

[0043] The present invention also provides the use of the anti-PD-1 antibody or its antigen-binding moiety described herein for producing an agent that enhances immunity and / or treats cancer in a patient (e.g., a human patient) who needs it, for example, in combination with an anti-TIM-3 antibody or its antigen-binding moiety and / or an anti-LAG-3 antibody or its antigen-binding moiety. In some embodiments, the present invention provides the use of the anti-PD-1 antibody or its antigen-binding moiety described herein, as well as an anti-TIM-3 antibody or its antigen-binding moiety, and / or an anti-LAG-3 antibody or its antigen-binding moiety, for producing an agent that enhances immunity in a human patient who needs it, for example, to treat cancer. The anti-TIM-3 antibody and the anti-LAG-3 antibody can be selected from the antibodies described herein.

[0044] The present invention also provides the use of the anti-PD-1 antibody or its antigen-binding moiety described herein for producing a drug for treating a human patient in the method described herein.

[0045] The present invention further provides a product comprising an anti-PD-1 antibody or its antigen-binding moiety, combined with an anti-TIM-3 antibody or its antigen-binding moiety, as described herein, suitable for enhancing immunity and / or treating cancer in a patient (e.g., a human patient) in a treatment method as described herein. The anti-TIM-3 antibody and the anti-LAG-3 antibody can be selected from the antibodies described herein. [Brief explanation of the drawing]

[0046] [Figure 1]Figure 1 is a set of graphs showing the percentage increase in IFN-γ levels in three donor pairs (Panels A-C) after 5 days of culture in a unidirectional MLR (mixed lymphocyte reaction) assay, following treatment with 10 μg / mL of control antibody (IgG1 LALA or IgG2), anti-PD-1 antibody 12819, anti-TIM-3 antibody 15086.17145, or a 1:1 combination of antibodies 12819 and 15086.17145. Each point in the graph represents a replicate experiment, and the mean value is shown by the horizontal line. [Figure 2] Figure 2 is a set of graphs showing the percentage increase in IFN-γ levels in three donor pairs (Panels A-C) after 5 days of culture in a bidirectional MLR assay, following treatment with 10 μg / mL of control antibody (IgG1 LALA or IgG2), anti-PD-1 antibody 12819, anti-TIM-3 antibody 15086.17145, or a 1:1 combination of antibodies 12819 and 15086.17145. Each point in the graph represents a replicate experiment, and the mean value is shown by the horizontal line. [Figure 3] Figure 3 shows a set of graphs illustrating the effect of anti-TIM-3 antibodies 15086 and 17145 on T cell proliferation in a unidirectional MLR assay compared to the positive control anti-PD-1 antibody 12819. The antibody was added at a final concentration of 25 μg / mL and incubated for 5 days with or without soluble anti-CD3 (Panel A) or with anti-CD3 (Panel B), followed by 1 μCi / well of 3H-thymidine for a further 18 hours. 3H-thymidine uptake in the recovered cells was determined by liquid scintillation counting. [Figure 4] Figure 4 is a graph showing the effect of anti-TIM-3 antibody 15086.17145 on IL-12p40 secretion from monocyte-derived dendritic cells incubated with 10 μg / mL antibody for 5 days. IL-12p40 levels in the cell supernatant were determined using ELISA. [Figure 5]Figure 5 is a set of graphs showing the expression levels of selected activating markers on monocyte-derived dendritic cells after 24 hours of treatment with 25 μg / mL anti-TIM-3 antibody 15086.17145. Panel A shows gene expression analysis of several activating markers and co-stimulatory molecules. Panel B shows verification of increased CD80 and Cd86 levels using FACS. The shown histogram overlay represents CD11c+ dendritic cells, and the numbers next to the histograms indicate the MFI (Mean Fluorescence Intensity) values. [Figure 6] Figure 6 is a graph showing the effect of TIM-3 targeting on tumor growth in a CD34+ humanized NSG-SGM3 mouse model transplanted with a human lung patient-derived xenograft (PDX) model LG1 306. Mice were treated with an initial dose of 10 mg / kg of anti-TIM-3 antibody 15086.17145, followed by 5 doses of 5 mg / kg every 5 days for 5 times. The gray area indicates the treatment period. Data are expressed as mean ± SEM. **p<0.01. [Figure 7] Figure 7 is a set of graphs showing the enhanced effect on IL-2 levels when anti-PD-1 antibody 12819 is combined with anti-LAG-3 antibody 15532 and / or anti-TIM-3 antibody 15086.17145 for two donor pairs (Panels A and B) in a SEB (Staphylococcal Enterotoxin B) + PBMC (Peripheral Blood Mononuclear Cell) assay. The bar graphs show IL-2 secretion from PBMCs treated with the antibody or mixture and SEB for 48 hours at a concentration of 10 μg / mL. The marks above the bar graphs indicate a single treatment (*) that is significantly different from the control antibody (p<0.05), or a mixture of two antibodies (#) that is significantly different from single antibody treatment of the constituent components. [Figure 8]Figure 8 is a set of graphs showing the effects of 10 mg / kg anti-PD-1 antibody 12819, anti-LAG-3 antibody C9B7W (reactive with mouse LAG-3), or a combination of anti-PD-1 and anti-LAG-3 antibodies, or media treatment on tumor growth in two syngeneic mouse tumor models: MC38 (colon cancer, panel A) and ASB-XIV (lung cancer, panel B). The gray area indicates the treatment period. Data represent mean ± SEM (standard error of the mean). **p<0.01, ***p<0.001, ****p<0.0001. [Figure 9] Figure 9 is a graph showing the effects of 10 mg / kg of anti-PD-1 antibody 12819, anti-LAG-3 antibody 15011, or a combination of anti-PD-1 and anti-LAG-3 antibodies, or media treatment, on tumor growth in an ASB-XIV syngeneic mouse tumor model. The gray area indicates the treatment period. Data are expressed as mean ± SEM. *p<0.05, ****p<0.0001. [Figure 10] Figure 10 is a set of graphs showing the effects of anti-PD-1 antibody 12819, anti-TIM-3 antibody 5D12, or a combination of anti-PD-1 and anti-TIM-3 antibodies, or media treatment, on tumor growth in two syngeneic mouse tumor models: ASB-XIV (lung cancer, panel A) and Sa1N (fibrosarcoma, panel B). Antibody treatment was administered at a dose of 10 mg / kg / target in mice with ASB-XIV tumors. Anti-PD-1 and anti-TIM-3 antibodies were administered at 1 mg / kg and 10 mg / kg, respectively, in mice with Sa1N tumors. The gray area indicates the treatment period. Data are expressed as mean ± SEM. **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 11]Figure 11 shows a set of graphs illustrating the effects of 10 mg / kg anti-PD-1 antibody 12819, anti-TIM-3 antibody 15086, or a combination of anti-PD-1 and anti-TIM-3 antibodies, or media treatment, on tumor growth in a human xenograft tumor model in which human melanoma cell line A375 was transplanted into mice reconstituted with human PBMCs. One human PBMC donor was used in each experiment, and the three graphs represent three different donors (Panels A-C). The gray area indicates the treatment period. Data represent mean ± SEM. *p<0.05 and **p<0.01. [Figure 12] Figure 12 is a set of graphs showing the effects of single and dual targeting of PD-1 and TIM-3 on percentage survival (Panel A) and tumor growth (Panels B-E) in NOD-scid mice transplanted with a mixture of human PBMCs and A375 melanoma cells. Mice were treated with anti-PD-1 antibody 12819, anti-TIM-3 antibody 15086, 17145, or a combination of anti-PD-1 and anti-TIM-3 antibodies. Antibody treatment was administered three times a week at a dose of 10 mg / kg for each antibody. The gray area indicates the treatment period. Data in Panel A are expressed as the percentage over time of mice with tumor size <400 mm3 in each treatment group. [Figure 13] Figure 13 is a set of graphs showing the effects of single, dual, and triple targeting of PD-1, LAG-3, and TIM-3 on percentage survival (Panel A) and tumor growth (Panels B-H) in an ASB-XIV syngeneic tumor model, using anti-PD-1 antibody 12819, anti-LAG-3 antibody C9B7W, anti-TIM-3 antibody 5D12, a combination of anti-PD1 and anti-LAG3 antibodies, a combination of anti-PD1 and anti-TIM-3 antibodies, or a "triple combo" (referring to a combination of anti-PD-1, anti-LAG-3, and anti-TIM-3 antibodies). Antibody treatment was administered at a dose of 10 mg / kg for each antibody. The gray area indicates the treatment period. [Modes for carrying out the invention]

[0047] The present invention provides novel combination therapies and compositions targeting human PD-1, human TIM-3, and / or human LAG-3 by using antibodies that bind to these targets. The therapies and compositions can be used to enhance the immune system in human patients, such as cancer patients. Unless otherwise specified, "PD-1" as used herein refers to human PD-1. The human PD-1 polypeptide sequence is available under Uniprot accession number Q15116 and is shown herein as SEQ ID NO: 388. Unless otherwise specified, "TIM-3" as used herein refers to human TIM-3. The human TIM-3 polypeptide sequence is available under Uniprot accession number Q8TDQ0 and is shown herein as SEQ ID NO: 389. Unless otherwise specified, "LAG-3" as used herein refers to human LAG-3. The human LAG-3 polypeptide sequence is available under Uniprot accession number P18627 and is shown herein as SEQ ID NO: 390.

[0048] As used herein, the terms “antibody” (Ab) or “immunoglobulin” (Ig) refer to a tetramer comprising two heavy (H) chains (approximately 50–70 kDa) and two light (L) chains (approximately 25 kDa) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable domain (VH) and a heavy chain constant region (CH). Each light chain consists of a light chain variable domain (VL) and a light chain constant region (CL). The VH and VL domains can be further subdivided into hypervariable regions called “complementarity-determining regions” (CDRs) that exist between more conserved regions called “framework regions” (FRs). Each VH and VL consists of three CDRs (wherein H-CDR refers to a CDR from the heavy chain and L-CDR refers to a CDR from the light chain) and four FRs, which are aligned from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The assignment of amino acid numbers in the heavy or light chain may be carried out according to the definition of IMGT® (Lefranc et al., Dev Comp Immunol 27(1):55-77 (2003)); or the definition of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991); Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); or Chothia et al., Nature 342:878-883 (1989). Unless otherwise specified, all antibody amino acid residue numbers referred to in this disclosure are numbers according to the IMGT® numbering scheme.

[0049] The term "recombinant antibody" refers to an antibody expressed from a cell or cell line containing a nucleotide sequence that codes for an antibody, wherein the nucleotide sequence does not normally associate with cells in nature.

[0050] The terms “isolated protein,” “isolated polypeptide,” or “isolated antibody” refer to a protein, polypeptide, or antibody that, for reasons of its origin or source of origin, (1) does not associate with the naturally associated component it is associated with in its native state, (2) does not contain other proteins of the same species, (3) is expressed by cells of a different species, and / or (4) does not exist in nature. For this reason, polypeptides that are chemically synthesized or synthesized in a cell system different from the cells of which they originate in nature are “isolated” from the naturally associated component. Proteins may also be made substantially free of the naturally associated component by isolation using protein purification techniques well known in the art.

[0051] The term "affinity" refers to a measure of the attractiveness between an antigen and an antibody. The intrinsic attractiveness of an antibody to an antigen is typically expressed by the binding affinity equilibrium constant (K) of a particular antibody-antigen interaction. D It is written as ). Antibodies are K D It is said that when the concentration is ≤1 mM, preferably ≤100 nM, it specifically binds to the antigen. D The binding affinity constant can be measured, for example, by surface plasmon resonance (SPR) (Biacore®) or biolayer interferometry, using, for example, IBIS Technologies' IBIS MX96 SPR system, Bio-Rad's ProteOn® XPR36 SPR system, or ForteBio's Octet® system.

[0052] The term "k" off " refers to the dissociation rate constant of a specific antibody-antigen interaction. off The dissociation rate constant can be measured, for example, by biolayer interferometry using one of the systems described above.

[0053] As used herein, the term “epitope” refers to a portion of an antigen (determinant) that specifically binds to an associated molecule, such as an antibody or a bispecific binding molecule. Epitope determinants generally consist of chemically active surface groups of molecules, such as amino acids, carbohydrates, or sugar side chains, and generally possess specific three-dimensional structural and charge characteristics. Epitopes can be “linear” or “conformation.” In the case of linear epitopes, all interaction points between a protein (e.g., an antigen) and an interacting molecule (such as an antibody) lie linearly along the primary amino acid sequence of the protein. In conformational epitopes, interaction points are located across amino acid residues on the protein that are separated from each other in the primary amino acid sequence. Once a desired epitope on an antigen is determined, it is possible to generate an antibody against that epitope using techniques known in the art. For example, an antibody against a linear epitope can be generated, for example, by immunizing an animal with a peptide containing the amino acid residues of the linear epitope. Antibodies against conformational epitopes can be produced, for example, by immunizing animals with a minidomain containing the relevant amino acid residues of the conformational epitope. Antibodies against specific epitopes can also be produced, for example, by immunizing animals with the target molecule or a related portion thereof, and then screening for binding to the epitope.

[0054] Whether an antibody binds to the same epitope as the antibody described herein, or competes for binding with the antibody described herein, can be determined by using methods known in the art, including but not limited to competition assays, epitope binning, and alanine scanning. In some embodiments, the test antibody and antibody described herein bind to at least one common residue (e.g., at least 2, 3, 4, 5, 6, 7, 8, or 9 residues) on the target protein (i.e., TIM-3, PD-1, or LAG-3). In further embodiments, the contact residues on the target protein are completely identical between the test antibody and antibody described herein. In one embodiment, the antibody described herein can be bound to the target protein under saturated conditions, and then the ability of the test antibody to bind to the target protein is measured. If the test antibody can bind to the target protein simultaneously with the reference antibody, the test antibody binds to a different epitope than the reference antibody. However, if the test antibody cannot bind to the target protein simultaneously, the test antibody binds to the same epitope, an overlapping epitope, or an epitope located proximal to the epitope to which the antibody described herein binds. This experiment can be carried out using, for example, ELISA, RIA, BIACORE®, SPR, biolayer interference, or flow cytometry. To test whether an antibody cross-competes with another antibody, the above competition methods may be used in two directions: namely, to determine whether a known antibody blocks the test antibody, and vice versa. Such cross-competition experiments can be carried out using, for example, an IBIS MX96 SPR instrument or an Octet® system.

[0055] The term "chimeric antibody," in its broadest sense, refers to an antibody that comprises one or more regions from one antibody and one or more regions from one or more other antibodies, and is typically partially of human origin and partially of non-human origin, i.e., partially derived from non-human animals, such as mice, rats, or other rodents, or birds such as chickens. Chimeric antibodies are preferred over non-human antibodies to reduce the risk of human anti-antibody responses, such as human anti-mouse antibody responses in the case of mouse antibodies. A typical example of a chimeric antibody is an antibody in which the variable domain sequence is mouse, but the constant region sequence is human. In the case of chimeric antibodies, further modifications may be made to the non-human portion to humanize the antibody. The chimeric antibody described herein has a chicken variable domain sequence and a human constant region sequence.

[0056] The term "humanization" refers to the fact that when an antibody is entirely or partially of non-human origin (e.g., mouse or chicken antibodies obtained from immunization of mice or chickens with the antigen of interest, respectively, or chimeric antibodies based on such mouse or chicken antibodies), it is possible to swap specific amino acids, particularly in the framework regions and constant regions of the heavy and light chains, in order to avoid or minimize the immune response in humans. While it is not possible to accurately predict immunogenicity and the resulting human anti-antibody response to a particular antibody, non-human antibodies tend to be more immunogenic in humans than human antibodies. Chimeric antibodies, in which the constant region of an exogenous source (e.g., rodent or bird) is swapped with a sequence of human origin, have generally been shown to be less immunogenic than antibodies of entirely exogenous origin, and the trend in therapeutic antibodies is towards humanized antibodies or fully human antibodies. For this reason, chimeric antibodies or other antibodies of non-human origin can be humanized to reduce the risk of anti-antibody response in humans.

[0057] With respect to chimeric antibodies, humanization typically involves modification of the framework region of the variable domain sequence. While amino acid residues that are part of the complementarity-determining region (CDR) most often remain unchanged in relation to humanization, in certain cases it may be desirable to alter individual CDR amino acid residues, for example, to remove glycosylation sites, deamide sites, aspartate isomerization sites, or undesirable cysteine ​​or methionine residues. N-linked glycosylation occurs by attaching an oligosaccharide chain to an asparagine residue in the tripeptide sequences Asn-X-Ser or Asn-X-Thr, where X can be any amino acid residue other than Pro. Removal of N-glycosylation sites can preferably be achieved by mutating either the Asn or Ser / Thr residue to a different residue by conservative substitution. Deamide of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation when they are present primarily in the Asn-Gly sequence, and to a lesser extent, in other dipeptide sequences such as Asn-Ala. Therefore, when such deamidation sites, especially Asn-Gly, are present in a CDR sequence, it is desirable to remove the site by a typically conservative substitution to eliminate one of the residues involved.

[0058] Numerous methods for humanizing antibody sequences are known in the art; see, for example, the review by Almagro & Fransson, Front Biosci. 13:1619-1633 (2008). One commonly used method is CDR grafting, which involves, for example, with respect to mouse-derived chimeric antibodies, the identification of human germline gene relatives to mouse variable domain genes and grafting mouse CDR sequences onto this framework. The specificity of an antibody's interaction with a target antigen lies primarily in the amino acid residues located in the six CDRs of the heavy and light chains. Therefore, the amino acid sequence within the CDR is considerably more variable among individual antibodies than the sequence outside the CDR. Since the CDR sequence is responsible for most antibody-antigen interactions, it is possible to express specific naturally occurring antibodies, or more generally recombinant antibodies that mimic the characteristics of any specific antibody having a given amino acid sequence, by constructing an expression vector that expresses a CDR sequence from a specific antibody transplanted onto a framework sequence from a different antibody, for example. As a result, it is possible to "humanize" non-human antibodies while still substantially maintaining the binding specificity and affinity of the original antibody. CDR grafting can be based on Kabat's definition of CDR, but more recent publications (Magdelaine-Beuzelin et al., Crit Rev. Oncol Hematol. 64:210-225 (2007))) suggest that the IMGT® definition (the international ImMunoGeneTics information system(R), www.imgt.org) can improve the results of humanization (see Lefranc et al., Dev. Comp Immunol. 27:55-77 (2003)).

[0059] In some cases, CDR grafting can reduce the binding specificity and affinity of the CDR-grafted non-human antibody compared to the parental antibody from which the CDR is derived, thus potentially reducing its biological activity. To re-establish the binding specificity and affinity of the parental antibody, a reverse mutation (sometimes called "framework repair") may be introduced, typically at a selected location on the CDR-grafted antibody within the framework region. Potential reverse mutation sites can be identified using information available in the literature and antibody databases. Candidate amino acid residues for reverse mutations are typically located on the surface of the antibody molecule; embedded residues or those with limited surface exposure usually remain unchanged.

[0060] A humanization technique that is an alternative to CDR grafting and reverse mutation is resurfacing, in which non-human, non-surface-exposed residues are retained, but surface residues are changed to human residues.

[0061] In certain cases, it may be desirable to alter one or more CDR amino acid residues to improve binding affinity to a target epitope. This is known as "affinity maturation" and may be performed in conjunction with humanization, for example, when humanization of an antibody leads to a reduction in binding specificity or affinity, and reverse mutation alone is insufficient to adequately improve binding specificity or affinity. Various affinity maturation methods are known in the art, such as the in vitro scanning saturation mutagenesis method described by Burks et al., Proc Natl Acad Sci USA, 94:412-417 (1997), and the stepwise in vitro affinity maturation method described by Wu et al., Proc Natl Acad Sci USA 95:6037-6042 (1998).

[0062] The term "human antibody" refers to antibodies whose variable domain and constant region sequences are derived from human sequences. The term encompasses antibodies derived from human genes but with sequences modified to, for example, reduce immunogenicity, increase affinity, and / or increase stability. Furthermore, the term includes antibodies produced by recombination using human-derived sequences in non-human cells, which may confer atypical glycosylation of human cells. The term also includes antibodies produced in transgenic non-human organisms possessing human antibody genes (e.g., OmniRat® rats).

[0063] As used herein, the term “antigen-binding moiety” (or simply “antibody moiety”) refers to one or more parts or fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human TIM-3, human PD-1, or human LAG-3 or a portion thereof). It has been shown that a particular fragment of a full-length antibody can perform the antigen-binding function of the antibody. Examples of binding fragments encompassed by the term “antigen-binding moiety” include (i) Fab fragments: VL, V H , C l , and C H (ii) A monovalent fragment consisting of one domain; (ii) F(ab')2 fragment; a divalent fragment containing two Fab fragments linked by disulfide bonds in the hinge region; (iii) V H (iv) Fd fragment consisting of the CH1 domain; (v) Fv fragment consisting of the VL and VH domains of the single arm of the antibody; (v) V H (vi) A dAb fragment consisting of domains; and an isolated complementarity-determining region (CDR) that can specifically bind to the antigen. Furthermore, the two domains of the Fv fragment, V L and V H These are encoded by different genes, but they can be linked together using recombination by a synthetic linker that can be made into a single protein chain, V L and V H Domains can pair up to form a monovalent molecule (known as single-stranded Fv (scFv)). Similarly, V Hand / or V L Antigen-binding molecules containing V are also included in the present invention. H In this case, the molecule may also contain one or more of the CH1, hinge, CH2, or CH3 regions. Such single-chain antibodies are also intended to be included in the term "antigen-binding portion" of the antibody. Other forms of single-chain antibodies, such as diabodies, are similarly included. Diabodies are V H and V L This is a bivalent, bispecific antibody in which the domain is expressed on a single polypeptide chain, but by using a linker that is too short to form a pair between two domains on the same chain, it forces a pair to form with a complementary domain on another chain, thereby creating two antigen-binding sites.

[0064] Antibody moieties, such as Fab and F(ab')2 fragments, can be prepared from the whole antibody using conventional techniques such as papain or pepsin digestion of the whole antibody. Furthermore, the antibody, antibody moieties, and immunoadhesins can be obtained, for example, using standard recombinant DNA techniques described herein.

[0065] The antibody classes (isotypes) and subclasses described herein can be determined by any method known in the art. Generally, antibody classes and subclasses can be determined using antibodies that are specific to a particular class and subclass of the antibody. Such antibodies are commercially available. Classes and subclasses can be determined by ELISA and Western blotting and other techniques. Alternatively, classes and subclasses can be determined by sequencing all or part of the constant regions of the heavy and / or light chains of the antibody, comparing its amino acid sequence with known amino acid sequences of various classes and subclasses of immunoglobulins, and determining the antibody class and subclass. The preferred isotype of the present invention is the IgG isotype.

[0066] When referring to a specific amino acid residue at a given position in an antibody sequence, for example, the designation "35S" refers to that position and residue, i.e., in this case, the serine residue (S) is located at position 35 of the sequence. Similarly, for example, the designation "13Q+35S" refers to two residues at each respective position. Unless otherwise specified, all antibody amino acid residue numbers referred to in this disclosure are numbers according to the IMGT® numbering scheme.

[0067] Anti-PD-1 antibody In some embodiments, the anti-PD-1 antibodies disclosed herein may be chimeric having a chicken-derived variable domain and a human constant region, or may be humanized.

[0068] The anti-PD-1 antibodies disclosed herein may be referred to by either a five-digit number, e.g., "12819", or a ten-digit number, e.g., "12819.15384". The five-digit number used herein refers to all antibodies having the heavy and light chain CDR1-3 sequences indicated by that number, while the ten-digit number refers to a specific humanized variant. For example, 12819.15384 is a specific humanized variant of the 12819 antibody having the CDR sequence. The five-digit number encompasses ten-digit variants that are identical except for some changes in the FR sequence (e.g., the absence of the residue SY at the N-terminus of the mature light chain, or the presence of the residue SS instead of SY). These modifications do not alter the functional (e.g., antigen-binding) properties of the antibody.

[0069] In some embodiments, the combination therapy or composition comprises an anti-PD-1 antibody or its antigen-binding moiety, the anti-PD-1 antibody being referred to herein as antibody 12819.15384, 12748.15381, 12748.16124, 12865.15377, 12892.15378, 12796.15376, 12777.15382, 12760.15375 or 13112.15380, or any variant thereof, the variant may have, for example, a certain minimum amino acid change compared to the antibody (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes, which may be present in the framework region) without losing the antigen-binding specificity of the antibody.

[0070] In some embodiments, the anti-PD-1 antibody competes with any one of the antibodies 12819.15384, 12748.15381, 12748.16124, 12865.15377, 12892.15378, 12796.15376, 12777.15382, 12760.15375, and 13112.15380 for binding to human PD-1, or binds to the same human PD-1 epitope as the antibody.

[0071] In some embodiments, any of the anti-PD-1 antibodies or antigen-binding moieties described herein may compete or cross-compete with antibodies 12865, 12892, and 12777 (e.g., antibodies 12865.15377, 12892.15378, and 12777.15382) for binding to PD-1. In some embodiments, any of the anti-PD-1 antibodies or antigen-binding moieties described herein may compete or cross-compete with antibody 12819 (e.g., antibody 12819.15384) for binding to PD-1. In some embodiments, any of the anti-PD-1 antibodies or antigen-binding moieties described herein may compete or cross-compete with antibodies 12760 and 13112 (e.g., antibodies 12760.15375 and 13112.15380) for binding to PD-1. In some embodiments, the antibodies have an IgG1 or IgG2 format. In a particular embodiment, the antibody has an IgG1 format.

[0072] In some embodiments, the anti-PD-1 antibody competes or cross-competes with antibodies whose heavy chain (H)CDR1-3 and light chain (L)CDR1-3 respectively contain SEQ ID NOs. 228-233, 238-243, 248-253, 258-263, 268-273, 278-283, 288-293, or 298-303 for binding to human PD-1, or binds to the same human PD-1 epitopes as the antibody.

[0073] In some embodiments, the anti-PD-1 antibody comprises an H-CDR3 containing the H-CDR3 amino acid sequence of SEQ ID NOs. 230, 240, 250, 260, 270, 280, 290, or 300.

[0074] In some embodiments, the anti-PD-1 antibody contains H-CDR1-3, each containing an H-CDR1-3 amino acid sequence of SEQ ID NOs. 228-230, 238-240, 248-250, 258-260, 268-270, 278-280, 288-290, or 298-300, respectively.

[0075] In some embodiments, the anti-PD-1 antibody has a VH whose amino acid sequence is at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NOs. 226, 236, 246, 256, 266, 276, 286, or 296.

[0076] In some embodiments, the anti-PD-1 antibody has a VH including SEQ ID NOs: 226, 236, 246, 256, 266, 276, 286, or 296.

[0077] In some embodiments, the anti-PD-1 antibody has a VH that is sequence-identical to at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) with SEQ ID NO: 226, 236, 246, 256, 266, 276, 286, or 296; and a CH that is sequence-identical to at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) with SEQ ID NO: 375.

[0078] In some embodiments, the anti-PD-1 antibody has an HC comprising the VH amino acid sequence of SEQ ID NO: 226, 236, 246, 256, 266, 276, 286, or 296 and the CH amino acid sequence of SEQ ID NO: 375.

[0079] In some embodiments, the anti-PD-1 antibody has an L-CDR3 containing the L-CDR3 amino acid sequence of SEQ ID NOs. 233, 243, 253, 263, 273, 283, 293, or 303.

[0080] In some embodiments, the anti-PD-1 antibody contains L-CDR1-3, each containing the L-CDR1-3 amino acid sequences of SEQ ID NOs. 231-233, 241-243, 251-253, 261-263, 271-273, 281-283, 291-293, or 301-303, respectively.

[0081] In some embodiments, the anti-PD-1 antibody has a VL whose sequence is at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the VL amino acid sequence of SEQ ID NOs. 227, 237, 247, 257, 267, 277, 287, 297, or 392.

[0082] In some embodiments, the anti-PD-1 antibody has a VL containing the VL amino acid sequence of SEQ ID NOs: 227, 237, 247, 257, 267, 277, 287, 297, or 392.

[0083] In some embodiments, the anti-PD-1 antibody has a VL whose sequence is at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the VL amino acid sequence of SEQ ID NO: 227, 237, 247, 257, 267, 277, 287, 297, or 392; and a CL whose sequence is at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the VL amino acid sequence of SEQ ID NO: 379.

[0084] In some embodiments, the anti-PD-1 antibody has an LC comprising the VL amino acid sequence of SEQ ID NO: 227, 237, 247, 257, 267, 277, 287, 297, or 392 and the CL amino acid sequence of SEQ ID NO: 379.

[0085] In some embodiments, the anti-PD-1 antibody comprises one of the above heavy chain sequences and one of the above light chain sequences.

[0086] In some embodiments, the anti-PD-1 antibody comprises H-CDR3 and L-CDR3, each containing the H-CDR3 and L-CDR3 amino acid sequences of SEQ ID NOs. 230 and 233, 240 and 243, 250 and 253, 260 and 263, 270 and 273, 280 and 283, 290 and 293, or 300 and 303, respectively.

[0087] In some embodiments, the anti-PD-1 antibody contains H-CDR1-3 and L-CDR1-3 sequences, each containing the H-CDR1-3 and L-CDR1-3 amino acid sequences of SEQ ID NOs. 228-233, 238-243, 248-253, 258-263, 268-273, 278-283, 288-293, or 298-303, respectively.

[0088] In some embodiments, the anti-PD-1 antibody includes VH, which has at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) the same amino acid sequence as SEQ ID NOs. 226, 236, 246, 256, 266, 276, 286, or 296, and VL, which has at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) the same amino acid sequence as SEQ ID NOs. 227, 237, 247, 257, 267, 277, 287, 297, or 392.

[0089] In some embodiments, the anti-PD-1 antibody has a VH containing the amino acid sequence of SEQ ID NOs. 226, 236, 246, 256, 266, 276, 286, or 296, and a VL containing the amino acid sequence of SEQ ID NOs. 227, 237, 247, 257, 267, 277, 287, 297, or 392.

[0090] In some embodiments, the anti-PD-1 antibody has an HC comprising the amino acid sequence of SEQ ID NOs. 226, 236, 246, 256, 266, 276, 286, or 296 and the amino acid sequence of SEQ ID NOs. 375; and an LC comprising the amino acid sequence of SEQ ID NOs. 227, 237, 247, 257, 267, 277, 287, 297, or 392 and the amino acid sequence of SEQ ID NOs. 379.

[0091] In some embodiments, the anti-PD-1 antibody is a) Sequence IDs 228-233, respectively; b) Sequence IDs 238-243, respectively; c) Sequence IDs 248-253, respectively; d) Sequence IDs 258-263, respectively; e) Sequence IDs 268-273, respectively; f) Sequence IDs 278-283, respectively; g) Sequence IDs 288-293, respectively; or h) Sequence IDs 298-303, respectively It contains the H-CDR1-3 and L-CDR1-3 amino acid sequences.

[0092] In some embodiments, the anti-PD-1 antibody is a) Sequence IDs 226 and 227, respectively; b) Sequence IDs 236 and 237, respectively; c) Sequence IDs 236 and 392, respectively; d) Sequence IDs 246 and 247, respectively; e) Sequence IDs 256 and 257, respectively; f) Sequence IDs 266 and 267, respectively; g) Sequence IDs 276 and 277, respectively; h) Sequence IDs 286 and 287, respectively; or i) Sequence numbers 296 and 297, respectively It contains VH and VL having the amino acid sequence.

[0093] In some embodiments, the anti-PD-1 antibody is a) HC containing the amino acid sequences of SEQ ID NO: 226 and SEQ ID NO: 375, and LC containing the amino acid sequences of SEQ ID NO: 227 and SEQ ID NO: 379; b) HC containing the amino acid sequence of SEQ ID NO: 236 and SEQ ID NO: 375, and LC containing the amino acid sequence of SEQ ID NO: 237 and SEQ ID NO: 379; c) HC containing the amino acid sequence of SEQ ID NO: 236 and SEQ ID NO: 375, and LC containing the amino acid sequence of SEQ ID NO: 392 and SEQ ID NO: 379; d) HC containing the amino acid sequence of SEQ ID NO: 246 and SEQ ID NO: 375, and LC containing the amino acid sequence of SEQ ID NO: 247 and SEQ ID NO: 379; e) HC containing the amino acid sequences of SEQ ID NO: 256 and SEQ ID NO: 375, and LC containing the amino acid sequences of SEQ ID NO: 257 and SEQ ID NO: 379; f) HC containing the amino acid sequences of SEQ ID NO: 266 and SEQ ID NO: 375, and LC containing the amino acid sequences of SEQ ID NO: 267 and SEQ ID NO: 379; g) HC containing the amino acid sequences of SEQ ID NO: 276 and SEQ ID NO: 375, and LC containing the amino acid sequences of SEQ ID NO: 277 and SEQ ID NO: 379; h) HC containing the amino acid sequence of SEQ ID NO: 286 and SEQ ID NO: 375, and LC containing the amino acid sequence of SEQ ID NO: 287 and SEQ ID NO: 379; or i) HC containing the amino acid sequence of SEQ ID NO: 296 and SEQ ID NO: 375, and LC containing the amino acid sequence of SEQ ID NO: 297 and SEQ ID NO: 379 Includes.

[0094] In some embodiments, the anti-PD-1 antibody is as follows: a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 228-230; b) An antibody in which the VH has at least 90% identical amino acid sequence to that of SEQ ID NO: 226; c) The VH antibody contains the amino acid sequence of SEQ ID NO: 226; d) The HC is an antibody containing the amino acid sequences of SEQ ID NOs. 226 and 375; e) Each of the L-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 231-233; f) The VL antibody has at least 90% identical amino acid sequence to that of SEQ ID NO: 227; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 227; h) The antibody in which the LC contains the amino acid sequences of SEQ ID NOs. 227 and 379; i) The H-CDR1-3 and L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 228-233; j) An antibody in which the VH is at least 90% identical in amino acid sequence to SEQ ID NO: 226, and the VL is at least 90% identical in amino acid sequence to SEQ ID NO: 227; k) An antibody in which VH contains the amino acid sequence of SEQ ID NO: 226 and VL contains the amino acid sequence of SEQ ID NO: 227; and l) An antibody in which the HC contains the amino acid sequence of SEQ ID NO: 226 and SEQ ID NO: 375, and the LC contains the amino acid sequences of SEQ ID NO: 227 and 379. It is selected from the group consisting of the following.

[0095] In some embodiments, the anti-PD-1 antibody is as follows: a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 238-240; b) An antibody in which the VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 236; c) The VH antibody contains the amino acid sequence of SEQ ID NO: 236; d) The HC is an antibody containing the amino acid sequences of SEQ ID NOs. 236 and 375; e) Each of the L-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 241-243; f) The VL antibody whose sequence is at least 90% identical to the amino acid sequence of SEQ ID NO: 237; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 237; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 237 and 379; i) The H-CDR1-3 and L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 238-243; j) An antibody in which the VH is at least 90% identical in amino acid sequence to SEQ ID NO: 236, and the VL is at least 90% identical in amino acid sequence to SEQ ID NO: 237; k) An antibody in which the VH contains the amino acid sequence of SEQ ID NO: 236 and the VL contains the amino acid sequence of SEQ ID NO: 237; and l) An antibody in which the HC contains the amino acid sequence of SEQ ID NO: 236 and SEQ ID NO: 375, and the LC contains the amino acid sequences of SEQ ID NO: 237 and 379. It is selected from the group consisting of the following.

[0096] In some embodiments, the anti-PD-1 antibody is as follows: a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 238-240; b) An antibody in which the VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 236; c) The VH antibody contains the amino acid sequence of SEQ ID NO: 236; d) The HC is an antibody containing the amino acid sequences of SEQ ID NOs. 236 and 375; e) Each of the L-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 241-243; f) The VL antibody has at least 90% identical amino acid sequence to that of SEQ ID NO: 392; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 392; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 392 and 379; i) The H-CDR1-3 and L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 238-243; j) An antibody in which the VH is at least 90% identical in amino acid sequence to SEQ ID NO: 236, and the VL is at least 90% identical in amino acid sequence to SEQ ID NO: 392; k) An antibody in which the VH contains the amino acid sequence of SEQ ID NO: 236 and the VL contains the amino acid sequence of SEQ ID NO: 392; and l) An antibody in which the HC contains the amino acid sequence of SEQ ID NO: 236 and SEQ ID NO: 375, and the LC contains the amino acid sequences of SEQ ID NOs: 392 and 379. It is selected from the group consisting of the following.

[0097] In some embodiments, the anti-PD-1 antibody is as follows: a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 248-250; b) An antibody in which the VH has at least 90% identical amino acid sequence to that of SEQ ID NO: 246; c) The VH antibody contains the amino acid sequence of SEQ ID NO: 246; d) The HC is an antibody containing the amino acid sequences of SEQ ID NOs. 246 and 375; e) Each of the L-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 251-253; f) The VL antibody has at least 90% identical amino acid sequence to that of SEQ ID NO: 247; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 247; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 247 and 379; i) The H-CDR1-3 and L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 248-253; j) An antibody in which the VH is at least 90% identical in amino acid sequence to SEQ ID NO: 246, and the VL is at least 90% identical in amino acid sequence to SEQ ID NO: 247; k) An antibody in which the VH contains the amino acid sequence of SEQ ID NO: 246 and the VL contains the amino acid sequence of SEQ ID NO: 247; and l) An antibody in which the HC contains the amino acid sequence of SEQ ID NO: 246 and SEQ ID NO: 375, and the LC contains the amino acid sequences of SEQ ID NO: 247 and 379. It is selected from the group consisting of the following.

[0098] In some embodiments, the anti-PD-1 antibody is as follows: a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 258-260; b) An antibody in which the VH has at least 90% identical amino acid sequence to that of SEQ ID NO: 256; c) The VH antibody contains the amino acid sequence of SEQ ID NO: 256; d) The HC is an antibody containing the amino acid sequences of SEQ ID NOs. 256 and 375; e) Each of the L-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 261-263; f) The VL antibody has at least 90% identical amino acid sequence to that of SEQ ID NO: 257; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 257; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 257 and 379; i) The H-CDR1-3 and L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 258-263; j) An antibody in which the VH is at least 90% identical in amino acid sequence to SEQ ID NO: 256, and the VL is at least 90% identical in amino acid sequence to SEQ ID NO: 257; k) An antibody in which the VH contains the amino acid sequence of SEQ ID NO: 256 and the VL contains the amino acid sequence of SEQ ID NO: 257; and l) An antibody in which the HC contains the amino acid sequence of SEQ ID NO: 256 and SEQ ID NO: 375, and the LC contains the amino acid sequences of SEQ ID NO: 257 and 379. It is selected from the group consisting of the following.

[0099] In some embodiments, the anti-PD-1 antibody is as follows: a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 268-270; b) An antibody in which the VH has at least 90% identical amino acid sequence to that of SEQ ID NO: 266; c) The VH antibody contains the amino acid sequence of SEQ ID NO: 266; d) The HC is an antibody containing the amino acid sequences of SEQ ID NOs. 266 and 375; e) Each of the L-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 271-273; f) The VL antibody has at least 90% identical amino acid sequence to that of SEQ ID NO: 267; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 267; h) The antibody in which the LC contains the amino acid sequences of SEQ ID NOs. 267 and 379; i) The H-CDR1-3 and L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 268-273; j) An antibody in which the VH is at least 90% identical in amino acid sequence to SEQ ID NO: 266, and the VL is at least 90% identical in amino acid sequence to SEQ ID NO: 267; k) An antibody in which the VH contains the amino acid sequence of SEQ ID NO: 266 and the VL contains the amino acid sequence of SEQ ID NO: 267; and l) An antibody in which the HC contains the amino acid sequence of SEQ ID NO: 266 and SEQ ID NO: 375, and the LC contains the amino acid sequences of SEQ ID NOs: 267 and 379. It is selected from the group consisting of the following.

[0100] In some embodiments, the anti-PD-1 antibody is as follows: a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 278-280; b) An antibody in which the VH has at least 90% identical amino acid sequence to that of SEQ ID NO: 276; c) The VH antibody contains the amino acid sequence of SEQ ID NO: 276; d) The HC is an antibody containing the amino acid sequences of SEQ ID NOs. 276 and 375; e) Each of the L-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 281-283; f) The VL antibody has at least 90% identical amino acid sequence to that of SEQ ID NO: 277; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 277; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 277 and 379; i) The H-CDR1-3 and L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 278-283; j) An antibody in which the VH is at least 90% identical in amino acid sequence to SEQ ID NO: 276, and the VL is at least 90% identical in amino acid sequence to SEQ ID NO: 277; k) An antibody in which the VH contains the amino acid sequence of SEQ ID NO: 276 and the VL contains the amino acid sequence of SEQ ID NO: 277; and l) An antibody in which the HC contains the amino acid sequence of SEQ ID NO: 276 and SEQ ID NO: 375, and the LC contains the amino acid sequences of SEQ ID NOs: 277 and 379. It is selected from the group consisting of the following.

[0101] In some embodiments, the anti-PD-1 antibody is as follows: a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 288-290; b) An antibody in which the VH has at least 90% identical amino acid sequence to that of SEQ ID NO: 286; c) The VH antibody contains the amino acid sequence of SEQ ID NO: 286; d) The HC is an antibody containing the amino acid sequences of SEQ ID NOs. 286 and 375; e) Each of the L-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 291-293; f) The VL antibody has at least 90% identical amino acid sequence to that of SEQ ID NO: 287; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 287; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 287 and 379; i) The H-CDR1-3 and L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 288-293; j) An antibody in which the VH is at least 90% identical in amino acid sequence to SEQ ID NO: 286, and the VL is at least 90% identical in amino acid sequence to SEQ ID NO: 287; k) An antibody in which the VH contains the amino acid sequence of SEQ ID NO: 286 and the VL contains the amino acid sequence of SEQ ID NO: 287; and l) An antibody in which the HC contains the amino acid sequence of SEQ ID NO: 286 and SEQ ID NO: 375, and the LC contains the amino acid sequences of SEQ ID NOs: 287 and 379. It is selected from the group consisting of the following.

[0102] In some embodiments, the anti-PD-1 antibody is a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 298-300; b) The VH antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 296; c) The VH is an antibody containing the amino acid sequence of SEQ ID NO: 296; d) The HC is an antibody containing the amino acid sequences of SEQ ID NOs. 296 and 375; e) The L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 301-303; f) The VL is an antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 297; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 297; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 297 and 379; i) The H-CDR1-3 and L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 298-303; j) The VH antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 296, and the VL antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 297; k) an antibody in which the VH contains the amino acid sequence of SEQ ID NO: 296, and the VL contains the amino acid sequence of SEQ ID NO: 297; and l) The HC contains the amino acid sequence of SEQ ID NO: 296 and the amino acid sequence of SEQ ID NO: 375; and the LC contains the amino acid sequences of SEQ ID NOs: 297 and 379. It is selected from the group consisting of the following.

[0103] In some embodiments, any anti-PD-1 antibody or antigen-binding moiety described herein contains at least 900, at least 850, at least 800, at least 750, at least 700, at least 650, at least 600, at least 550, at least 500, at least 450, at least 400, at least 350, at least 300, at least 250, at least 200, at least 150, at least 100, at least 50, at least 40, at least 30, or at least 20 pM of K D It can bind to human PD-1. In a particular embodiment, K D This is determined using surface plasmon resonance. In certain embodiments, the anti-PD-1 antibody or antigen-binding moiety binds to human PD-1 with higher affinity than nivolumab, pembrolizumab, or both.

[0104] In some embodiments, any anti-PD-1 antibody or antigen-binding moiety described herein contains at least 9000, at least 8000, at least 7000, at least 6000, at least 5000, at least 4000, at least 3000, at least 2500, at least 2000, at least 1500, at least 1000, at least 900, at least 800, at least 700, at least 600, at least 500, at least 400, at least 300, at least 200, at least 100, at least 75, at least 50, at least 25, at least 20, at least 15, at least 10, or at least 5 pM of K D It can bind to cynomolgus monkey PD-1. In a particular embodiment, K D This is determined using surface plasmon resonance.

[0105] In some embodiments, any anti-PD-1 antibody or antigen-binding moiety described herein contains at least 1000, at least 950, at least 900, or at least 850 pM of K D It can bind to mouse PD-1. In a particular embodiment, K D This is determined using surface plasmon resonance.

[0106] In some embodiments, any anti-PD-1 antibody or antigen-binding moiety described herein may inhibit the interaction between PD-1 and PD-L1 by at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% in a flow cytometry competitive assay at a concentration of 10 μg / ml. In certain embodiments, the anti-PD-1 antibody or its antigen-binding moiety may inhibit the interaction between PD-1 and PD-L1 by at least 83%.

[0107] In some embodiments, any anti-PD-1 antibody or antigen-binding moiety described herein may block the binding of PD-L1 and PD-L2 to PD-1 by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% at a concentration of 10 μg / ml, as determined by biolayer interferometry analysis. In certain embodiments, the anti-PD-1 antibody or antigen-binding moiety blocks the binding of PD-L1 and PD-L2 to PD-1 by at least 90%.

[0108] In some embodiments, the anti-PD-1 antibody or antigen-binding moiety described herein has the following characteristics: a) K₀ 750 pM or less D To bind to human PD-1; b) Kelvin of 7nM or less DIt binds to cynomolgus monkey PD-1; c) K(s) 1nM or less D To bind to mouse PD-1; d) Not bind to rat PD-1; e) Increasing IL-2 secretion in the SEB whole blood assay; f) To increase IFN-γ secretion in a unidirectional mixed lymphocyte reaction assay; g) Inhibit the interaction between PD-1 and PD-L1 by at least 60% at a concentration of 10 μg / ml in a flow cytometry assay; h) inhibiting at least 90% of the binding of PD-L1 and PD-L2 to PD-1 at a concentration of 10 μg / ml as determined by biolayer interferometry analysis; and i) Inhibiting tumor growth in vivo Having at least one of the above. Examples of such antibodies include, but are not limited to, the 12819 antibody (having properties a-i); the 12748, 12892, and 12777 antibodies (having at least properties a, b, and e-h); the 12865 and 12796 antibodies (having at least properties a, b, e, f, and h); and the 12760 and 13112 antibodies (having at least properties a, b, e, and f). In some embodiments, the anti-PD-1 antibody or antigen-binding moiety has all of the above properties. In some embodiments, the anti-PD-1 antibody or antigen-binding moiety has at least properties a, b, and e-h. In some embodiments, the anti-PD-1 antibody or antigen-binding moiety has at least properties a, b, e, f, and h. In some embodiments, the anti-PD-1 antibody or antigen-binding moiety has at least properties a, b, e, and f.

[0109] In some embodiments, the anti-PD-1 antibody or its antigen-binding moiety described herein binds to an epitope of PD-1 containing at least one of the following residues of SEQ ID NO: 388: V44, V64, L128, P130, K131, A132, E136, and T145 (e.g., at least one, at least two, at least three, at least four, or at least five). In certain embodiments, the antibody or antigen-binding moiety binds to an epitope of PD-1 containing residues V64, L128, P130, K131, and A132 of SEQ ID NO: 388 (e.g., antibody 12819, 15384). In certain embodiments, the antibody or antigen-binding moiety binds to an epitope of PD-1 containing residues K131 and E136 of SEQ ID NO: 388 (e.g., antibody 12865, 15377). In a particular embodiment, the antibody or antigen-binding moiety binds to the PD-1 epitope containing residues V44 and T145 of SEQ ID NO: 388 (13112 antibody, e.g., antibody 13112.15380).

[0110] In some embodiments, the combination therapy or composition comprises an anti-PD-1 antibody or its antigen-binding moiety that binds to the PD-1 epitope containing amino acid residue K131 of SEQ ID NO: 388 (e.g., 12819 or 12865 antibody). In some embodiments, the epitope may further comprise amino acid residues P130 and A132, and further comprise amino acid residues V64 and L128 (e.g., 12819 antibody). In some embodiments, the epitope further comprises amino acid residue E136 (e.g., 12865 antibody).

[0111] In some embodiments, the anti-PD-1 antibody or its antigen-binding moiety described herein binds to an epitope of PD-1 comprising residues 56-64, 69-90, and / or 122-140 of SEQ ID NO: 388. In certain embodiments, the antibody or antigen-binding moiety binds to an epitope of PD-1 comprising residues 69-90 and 122-140 of SEQ ID NO: 388 (12819 and 12865 antibodies, e.g., antibodies 12819, 15384, and 12865, 15377, etc.). In certain embodiments, the antibody or antigen-binding moiety binds to an epitope of PD-1 comprising residues 56-64, 69-90, and 122-140 of SEQ ID NO: 388 (e.g., antibody 12819). In certain embodiments, the antibody or antigen-binding moiety binds to an epitope of PD-1 comprising residues 69-90 and 122-140 of SEQ ID NO: 388 (e.g., antibody 12865). In some embodiments, the antibody or moiety binds to residues 69-75 (or fragments thereof, e.g., fragments of residues 1, 2, 3, 4, 5, or 6) of SEQ ID NO: 388 (e.g., antibodies 12819, 15384, and 12865, 15377). In some embodiments, the antibody or moiety binds to residues 136-140 (or fragments thereof, e.g., fragments of residues 1, 2, 3, or 4) of SEQ ID NO: 388 (e.g., antibodies 12819, 15384, and 12865, 15377). In some embodiments, the antibody or moiety binds to residues 69-75 (or fragments thereof) and residues 136-140 (or fragments thereof) of SEQ ID NO: 388 (e.g., antibodies 12819, 15384, and 12865, 15377). Epitopes having any combination of the above residues are similarly intended.

[0112] In some embodiments, the anti-PD-1 antibody or its antigen-binding moiety described herein is the anti-PD-1 antibody or its antigen-binding moiety described in whole by reference in PCT Patent Publication International Publication No. 2017 / 055547 or PCT Patent Application European Patent Application No. 2017 / 079615.

[0113] Anti-TIM-3 antibody In certain embodiments, the anti-TIM-3 antibody disclosed herein is a human antibody produced from a transgenic rat capable of producing an antibody having a human idiotype.

[0114] The anti-TIM-3 antibodies disclosed herein may be referred to by either a five-digit number, e.g., "20131", or a ten-digit number, e.g., "15086.16837". Ten-digit numbers with the same first five digits, such as antibodies 15086.15086, 15086.16837, 15086.17145, and 15086.17144, are derived from the same parent antibody. Such antibodies are expected to share the same six CDRs and have the same or substantially the same target binding properties. As is evident from the protein and DNA sequences provided herein, the 15086.16837, 15086.17145, and 15086.17144 variants differ from the parent 15086 antibody ("15086.15086") by only a single amino acid in the VH sequence, i.e., having E instead of Q at position 6, but the VL amino acid sequence is identical. Similarly, these variants differ primarily in their antibody format / subclass, i.e.: 15086.15086:IgG1 15086.16837:IgG1 LALA 15086.17145:IgG2 15086.17144:IgG4 It is clear that the VH sequences of IgG2 and IgG4 subclass antibodies are the same as those of the IgG1 LALA variant. The VL sequences are the same for all four antibody subclasses.

[0115] In some embodiments, the combination therapy or composition comprises an anti-TIM-3 antibody or its antigen-binding moiety, the anti-TIM-3 antibody as specified herein is antibody 15086.17145, 15086.15086, 15086.16837, 15086.17144, 20131, 20293, 15105, 15107, 15109, 15174, 15175, 15260, 15284, 15299, 15353, 15354, 1724 Antibodies referred to as 4, 17245, 19324, 19416, 19568, 20185, 20300, 20362, or 20621, or any of these variants, which may include a certain minimum amino acid change compared to the aforementioned antibody (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes, which may be present in the framework region) without losing the antigen-binding specificity of the antibody.

[0116] In some embodiments, the anti-TIM-3 antibody competes or cross-competes with antibody 15086.15086 having the IgG1 format, antibody 15086.16837 having the IgG LALA format, antibody 15086.17145 having the IgG2 format, or antibody 15086.17144 having the IgG4 format for binding to human TIM-3, or binds to the same human TIM-3 epitope as the antibody. In some embodiments, the antibody has the IgG1 or IgG2 format. In a particular embodiment, the antibody has the IgG2 format.

[0117] In some embodiments, the anti-TIM-3 antibody competes or cross-competes with antibodies 20131, 20293, 15105, 15107, 15109, 15174, 15175, 15260, 15284, 15299, 15353, 15354, 17244, 17245, 19324, 19416, 19568, 20185, 20300, 20362, or 20621 for binding to human TIM-3, or binds to the same human TIM-3 epitope as the antibody. In some embodiments, the antibody has an IgG1 or IgG2 format. In a particular embodiment, the antibody has an IgG2 format.

[0118] In some embodiments, an anti-TIM-3 antibody competes or cross-competes with antibodies whose heavy chain (H)CDR1-3 and light chain (L)CDR1-3 respectively contain SEQ ID NOs: 8-13, 18-23, 28-33, 38-43, 48-53, 58-63, 68-73, 78-83, 88-93, 98-103, 108-113, 118-123, 128-133, 138-143, 148-153, 158-163, 168-173, 178-183, 188-193, 198-203, 208-213, or 218-223 for binding to human TIM-3, or binds to the same human TIM-3 epitopes as the antibody.

[0119] In some embodiments, the anti-TIM-3 antibody contains H-CDR3 comprising the H-CDR3 amino acid sequence of SEQ ID NOs: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, or 220.

[0120] In some embodiments, the anti-TIM-3 antibody contains H-CDR1-3, each containing an H-CDR1-3 amino acid sequence of SEQ ID NOs: 8-10, 18-20, 28-30, 38-40, 48-50, 58-60, 68-70, 78-80, 88-90, 98-100, 108-110, 118-120, 128-130, 138-140, 148-150, 158-160, 168-170, 178-180, 188-190, 198-200, 208-210, or 218-220, respectively.

[0121] In some embodiments, the anti-TIM-3 antibody has a VH that is at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical in amino acid sequence to SEQ ID NOs. 3, 7, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, or 216.

[0122] In some embodiments, the anti-TIM-3 antibody has a VH including SEQ ID NOs: 3, 7, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, or 216.

[0123] In some embodiments, the anti-TIM-3 antibody has a VH that is sequence-identical by at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NOs. 3, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, or 216; and a CH that is sequence-identical by at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to SEQ ID NOs. 374.

[0124] In some embodiments, the anti-TIM-3 antibody has a HC comprising the VH amino acid sequence of SEQ ID NO: 3, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, or 216, and the CH amino acid sequence of SEQ ID NO: 374.

[0125] In some embodiments, the anti-TIM-3 antibody has a VH with a sequence that is at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 7, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, or 216; and a CH with a sequence that is at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 375, 376, or 377.

[0126] In some embodiments, the anti-TIM-3 antibody has a HC comprising the VH amino acid sequence of SEQ ID NO: 7, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, or 216, and the CH amino acid sequence of SEQ ID NO: 375, 376, or 377. In certain embodiments, the CH amino acid sequence is SEQ ID NO: 377.

[0127] In some embodiments, the anti-TIM-3 antibody comprises an L-CDR3 comprising the L-CDR3 amino acid sequence of SEQ ID NO: 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, 133, 143, 153, 163, 173, 183, 193, 203, 213, or 223.

[0128] In some embodiments, the anti-TIM-3 antibody comprises L-CDR1-3 comprising the L-CDR1-3 amino acid sequences of SEQ ID NOs: 11-13, 21-23, 31-33, 41-43, 51-53, 61-63, 71-73, 81-83, 91-93, 101-103, 111-113, 121-123, 131-133, 141-143, 151-153, 161-163, 171-173, 181-183, 191-193, 201-203, 211-213, or 221-223, respectively.

[0129] In some embodiments, the anti-TIM-3 antibody has a VL with an amino acid sequence of SEQ ID NOs: 4, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, 187, 197, 207, or 217 and is at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical in sequence.

[0130] In some embodiments, the anti-TIM-3 antibody has a VL comprising the VL amino acid sequence of SEQ ID NOs: 4, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, 187, 197, 207, or 217.

[0131] In some embodiments, the anti-TIM-3 antibody has a VL with an amino acid sequence of SEQ ID NOs: 4, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, or 187 and is at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical in sequence; and a CL with a sequence that is at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 378.

[0132] In some embodiments, the anti-TIM-3 antibody has an LC having the VL amino acid sequence of SEQ ID NOs. 4, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, 187, 197, 207, or 217, and the CL amino acid sequence of SEQ ID NOs. 378.

[0133] In some embodiments, the anti-TIM-3 antibody comprises one of the above heavy chain sequences and one of the above light chain sequences.

[0134] In some embodiments, the anti-TIM-3 antibody contains H-CDR3 and L-CDR3, each containing the H-CDR3 and L-CDR3 amino acid sequences of SEQ ID NOs: 10 and 13, 20 and 23, 30 and 33, 40 and 43, 50 and 53, 60 and 63, 70 and 73, 80 and 83, 90 and 93, 100 and 103, 110 and 113, 120 and 123, 130 and 133, 140 and 143, 150 and 153, 160 and 163, 170 and 173, 180 and 183, 190 and 193, 200 and 203, 210 and 213, or 220 and 223, respectively.

[0135] In some embodiments, the anti-TIM-3 antibody comprises H-CDR1-3 and L-CDR1-3, each containing H-CDR1-3 and L-CDR1-3 sequences of sequence numbers 8-13, 18-23, 28-33, 38-43, 48-53, 58-63, 68-73, 78-83, 88-93, 98-103, 108-113, 118-123, 128-133, 138-143, 148-153, 158-163, 168-173, 178-183, 188-193, 198-203, 208-213, or 218-223, respectively.

[0136] In some embodiments, the anti-TIM-3 antibody has an amino acid sequence of SEQ ID NOs. 3, 7, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, or 216, and the sequence has at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%). ) has a VH which is identical, and a VL which has a sequence that is at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence of SEQ ID NOs. 4, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, 187, 197, 207, or 217.

[0137] In some embodiments, the anti-TIM-3 antibody has a VH containing the amino acid sequence of SEQ ID NOs: 3, 7, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, or 216, and a VL containing the amino acid sequence of SEQ ID NOs: 4, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, 187, 197, 207, or 217.

[0138] In some embodiments, the anti-TIM-3 antibody has an HC containing the amino acid sequence of SEQ ID NOs: 3, 7, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, or 216, and the amino acid sequence of SEQ ID NOs: 378; and an LC containing the amino acid sequence of SEQ ID NOs: 4, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, 187, 197, 207, or 217, and the amino acid sequence of SEQ ID NOs: 374, 375, 376, or 377.

[0139] In some embodiments, the anti-TIM-3 antibody is a) respectively, SEQ ID NO: 8-13; b) respectively, SEQ ID NO: 18-23; c) respectively, SEQ ID NO: 28-33; d) respectively, SEQ ID NO: 38-43; e) respectively, SEQ ID NO: 48-53; f) respectively, SEQ ID NO: 58-63; g) respectively, SEQ ID NO: 68-73; h) respectively, SEQ ID NO: 78-83; i) respectively, SEQ ID NO: 88-93; j) respectively, SEQ ID NO: 98-103; k) respectively, SEQ ID NO: 108-113; l) respectively, SEQ ID NO: 118-123; m) respectively, SEQ ID NO: 128-133; n) respectively, SEQ ID NO: 138-143; o) respectively, SEQ ID NO: 148-153; p) respectively, SEQ ID NO: 158-163; q) respectively, SEQ ID NO: 168-173; r) respectively, SEQ ID NO: 178-183; s) respectively, SEQ ID NO: 188-193; t) respectively, SEQ ID NO: 198-203; u) respectively, SEQ ID NO: 208-213; or v) respectively, SEQ ID NO: 218-223 and comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences.

[0140] In some embodiments, the anti-TIM-3 antibody is a) respectively, SEQ ID NO: 7 and 4; b) respectively, SEQ ID NO: 3 and 4; c) respectively, SEQ ID NO: 16 and 17; d) respectively, SEQ ID NO: 26 and 27; e) respectively, SEQ ID NO: 36 and 37; f) respectively, SEQ ID NO: 46 and 47; g) Sequence IDs 56 and 57, respectively; h) Sequence IDs 66 and 67, respectively; i) Sequence numbers 76 and 77, respectively; j) Sequence IDs 86 and 87, respectively; k) Sequence IDs 96 and 97, respectively; l) Sequence IDs 106 and 107, respectively; m) Sequence numbers 116 and 117, respectively; n) Sequence IDs 126 and 127, respectively; o) Sequence IDs 136 and 137, respectively; p) Sequence IDs 146 and 147, respectively; q) Sequence IDs 156 and 157, respectively; r) Sequence IDs 166 and 167, respectively; s) Sequence IDs 176 and 177, respectively; t) Sequence IDs 186 and 187, respectively; u) Sequence IDs 196 and 197, respectively; v) Sequence IDs 206 and 207, respectively; or w) Sequence numbers 216 and 217, respectively It includes a heavy chain variable domain and a light chain variable domain having the following amino acid sequence.

[0141] In some embodiments, the anti-TIM-3 antibody is a) HC containing the amino acid sequence of SEQ ID NO: 7 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 4 and the amino acid sequence of SEQ ID NO: 378; b) HCs containing the amino acid sequence of SEQ ID NO: 3 and SEQ ID NOs: 374, 375, 376, or 377, and LCs containing the amino acid sequence of SEQ ID NO: 4 and SEQ ID NO: 378; c) HC containing the amino acid sequence of SEQ ID NO: 16 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 17 and the amino acid sequence of SEQ ID NO: 378; d) HC containing the amino acid sequence of SEQ ID NO: 26 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 27 and the amino acid sequence of SEQ ID NO: 378; e) HCs containing the amino acid sequence of SEQ ID NO: 36 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LCs containing the amino acid sequence of SEQ ID NO: 37 and the amino acid sequence of SEQ ID NO: 378; f) HCs containing the amino acid sequence of SEQ ID NO: 46 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, as well as HCs containing the amino acid sequence of SEQ ID NO: 47 and the amino acid sequence of SEQ ID NO: 378; g) HC containing the amino acid sequence of SEQ ID NO: 56 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 57 and the amino acid sequence of SEQ ID NO: 378; h) HC containing the amino acid sequence of SEQ ID NO: 66 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 67 and the amino acid sequence of SEQ ID NO: 378; i) HCs containing the amino acid sequence of SEQ ID NO: 76 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LCs containing the amino acid sequence of SEQ ID NO: 77 and the amino acid sequence of SEQ ID NO: 378; j) HC containing the amino acid sequence of SEQ ID NO: 86 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 87 and the amino acid sequence of SEQ ID NO: 378; k) HC containing the amino acid sequence of SEQ ID NO: 96 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 97 and the amino acid sequence of SEQ ID NO: 378; l) HC containing the amino acid sequence of SEQ ID NO: 106 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 107 and the amino acid sequence of SEQ ID NO: 378; m) HC containing the amino acid sequence of SEQ ID NO: 116 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 117 and the amino acid sequence of SEQ ID NO: 378; n) HC containing the amino acid sequence of SEQ ID NO: 126 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 127 and the amino acid sequence of SEQ ID NO: 378; o) HC containing the amino acid sequence of SEQ ID NO: 136 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 137 and the amino acid sequence of SEQ ID NO: 378; p) HC containing the amino acid sequence of SEQ ID NO: 146 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 147 and the amino acid sequence of SEQ ID NO: 378; q) HC containing the amino acid sequence of SEQ ID NO: 156 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 157 and the amino acid sequence of SEQ ID NO: 378; r) HC containing the amino acid sequence of SEQ ID NO: 166 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 167 and the amino acid sequence of SEQ ID NO: 378; s) HC containing the amino acid sequence of SEQ ID NO: 176 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 177 and the amino acid sequence of SEQ ID NO: 378; t) HC containing the amino acid sequence of SEQ ID NO: 186 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 187 and the amino acid sequence of SEQ ID NO: 378; u) HC containing the amino acid sequence of SEQ ID NO: 196 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 197 and the amino acid sequence of SEQ ID NO: 378; v) HC containing the amino acid sequence of SEQ ID NO: 206 and the amino acid sequences of SEQ ID NO: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 378; or w) HC containing the amino acid sequence of SEQ ID NO: 216 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377, and LC containing the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 378 Includes.

[0142] In some embodiments, the anti-TIM-3 antibody is a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 8-10; b) The VH antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 3 or 7; c) The VH is an antibody containing the amino acid sequence of SEQ ID NO: 3 or 7; d) The HC is an antibody containing the amino acid sequences of SEQ ID NOs: 3 and 374, or SEQ ID NO: 7, and SEQ ID NOs: 375, 376, or 377; e) Each of the L-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 11-13; f) The VL antibody has at least 90% identical amino acid sequence to SEQ ID NO: 4; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 4; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs: 4 and 378; i) Antibodies in which H-CDR1-3 and L-CDR1-3 each contain the amino acid sequences of SEQ ID NOs: 8-13; j) The VH antibody is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 3 or 7, and the VL antibody is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 4; k) an antibody in which the VH contains the amino acid sequence of SEQ ID NO: 3 or 15, and the VL contains the amino acid sequence of SEQ ID NO: 4; and l) The antibody whose HC contains the amino acid sequences of SEQ ID NOs. 3 and 374, or SEQ ID NOs. 7, and SEQ ID NOs. 375, 376, or 377; and whose LC contains the amino acid sequences of SEQ ID NOs. 4 and 378. It is selected from the group consisting of the following.

[0143] In some embodiments, the anti-TIM-3 antibody is a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 18-20; b) The VH antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 16; c) The VH is an antibody containing the amino acid sequence of SEQ ID NO: 16; d) The HC is an antibody containing the amino acid sequence of SEQ ID NO: 16 and SEQ ID NOs: 374, 375, 376, or 377; e) The L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 21-23; f) The VL is an antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 17; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 17; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 17 and 378; i) The H-CDR1-3 and L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 18-23; j) The VH antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 16, and the VL antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 17; k) The antibody in which the VH contains the amino acid sequence of SEQ ID NO: 16, and the antibody in which the VL contains the amino acid sequence of SEQ ID NO: 17; and l) The HC contains the amino acid sequence of SEQ ID NO: 16 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377; and the LC contains the amino acid sequences of SEQ ID NOs: 17 and 378. It is selected from the group consisting of the following.

[0144] In some embodiments, the anti-TIM-3 antibody is a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 28-30; b) The VH antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 26; c) The VH is an antibody containing the amino acid sequence of SEQ ID NO: 26; d) The HC is an antibody containing the amino acid sequence of SEQ ID NO: 26 and SEQ ID NOs: 374, 375, 376, or 377; e) The L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 31-33; f) The VL antibody has at least 90% identical amino acid sequence to SEQ ID NO: 27; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 27; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 27 and 378; i) Antibodies in which H-CDR1-3 and L-CDR1-3 each contain the amino acid sequences of SEQ ID NOs. 28-33; j) The VH antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 26, and the VL antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 27; k) an antibody in which the VH contains the amino acid sequence of SEQ ID NO: 26, and the VL contains the amino acid sequence of SEQ ID NO: 27; and l) The HC contains the amino acid sequence of SEQ ID NO: 26 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377; and the LC contains the amino acid sequences of SEQ ID NOs: 27 and 378. It is selected from the group consisting of the following.

[0145] In some embodiments, the anti-TIM-3 antibody is a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 38-40; b) The VH antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 36; c) The VH is an antibody containing the amino acid sequence of SEQ ID NO: 36; d) The HC is an antibody containing the amino acid sequence of SEQ ID NO: 36 and SEQ ID NOs: 374, 375, 376, or 377; e) The L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 41-43; f) The VL antibody has at least 90% identical amino acid sequence to SEQ ID NO: 37; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 37; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 37 and 378; i) Antibodies in which H-CDR1-3 and L-CDR1-3 each contain the amino acid sequences of SEQ ID NOs. 38-43; j) The VH antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 36, and the VL antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 37; k) an antibody in which the VH contains the amino acid sequence of SEQ ID NO: 36, and the VL contains the amino acid sequence of SEQ ID NO: 37; and l) The antibody whose HC contains the amino acid sequence of SEQ ID NO: 36, and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377; and whose LC contains the amino acid sequences of SEQ ID NOs: 37 and 378. It is selected from the group consisting of the following.

[0146] In some embodiments, the anti-TIM-3 antibody is a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 48-50; b) The VH antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 46; c) The VH is an antibody containing the amino acid sequence of SEQ ID NO: 46; d) The HC is an antibody containing the amino acid sequence of SEQ ID NO: 46 and SEQ ID NOs: 374, 375, 376, or 377; e) The L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 51-53; f) The VL antibody has at least 90% identical amino acid sequence to SEQ ID NO: 47; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 47; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 47 and 378; i) Antibodies in which H-CDR1-3 and L-CDR1-3 each contain the amino acid sequences of SEQ ID NOs. 48-53; j) The VH antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 46, and the VL antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 47; k) an antibody in which the VH contains the amino acid sequence of SEQ ID NO: 46, and the VL contains the amino acid sequence of SEQ ID NO: 47; and l) The HC contains the amino acid sequence of SEQ ID NO: 46, and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377; and the LC contains the amino acid sequences of SEQ ID NOs: 47 and 378. It is selected from the group consisting of the following.

[0147] In some embodiments, the anti-TIM-3 antibody is a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 58-60; b) The VH antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 56; c) The VH is an antibody containing the amino acid sequence of SEQ ID NO: 56; d) The HC is an antibody containing the amino acid sequence of SEQ ID NO: 56 and SEQ ID NOs: 374, 375, 376, or 377; e) The L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 61-63; f) The VL antibody has at least 90% identical amino acid sequence to SEQ ID NO: 57; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 57; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 57 and 378; i) Antibodies in which H-CDR1-3 and L-CDR1-3 each contain the amino acid sequences of SEQ ID NOs. 58-63; j) The VH antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 56, and the VL antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 57; k) The antibody in which the VH contains the amino acid sequence of SEQ ID NO: 56, and the antibody in which the VL contains the amino acid sequence of SEQ ID NO: 57; and l) The HC contains the amino acid sequence of SEQ ID NO: 56 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377; and the LC contains the amino acid sequences of SEQ ID NOs: 57 and 378. It is selected from the group consisting of the following.

[0148] In some embodiments, the anti-TIM-3 antibody is a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 68-70; b) The VH antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 66; c) The VH is an antibody containing the amino acid sequence of SEQ ID NO: 66; d) The HC is an antibody containing the amino acid sequence of SEQ ID NO: 66 and SEQ ID NOs: 374, 375, 376, or 377; e) The L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 71-73; f) The VL antibody has at least 90% identical amino acid sequence to SEQ ID NO: 67; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 67; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 67 and 378; i) Antibodies in which H-CDR1-3 and L-CDR1-3 each contain the amino acid sequences of SEQ ID NOs. 68-73; j) The VH antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 66, and the VL antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 67; k) an antibody in which the VH contains the amino acid sequence of SEQ ID NO: 66, and the VL contains the amino acid sequence of SEQ ID NO: 67; and l) The HC contains the amino acid sequence of SEQ ID NO: 66 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377; and the LC contains the amino acid sequences of SEQ ID NOs: 67 and 378. It is selected from the group consisting of the following.

[0149] In some embodiments, the anti-TIM-3 antibody is a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 78-80; b) The VH antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 76; c) The VH is an antibody containing the amino acid sequence of SEQ ID NO: 76; d) The HC is an antibody containing the amino acid sequence of SEQ ID NO: 76 and SEQ ID NOs: 374, 375, 376, or 377; e) The L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 81-83; f) The VL antibody has at least 90% identical amino acid sequence to SEQ ID NO: 77; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 77; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 77 and 378; i) Antibodies in which H-CDR1-3 and L-CDR1-3 each contain the amino acid sequences of SEQ ID NOs. 78-83; j) The VH antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 76, and the VL antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 77; k) an antibody in which the VH contains the amino acid sequence of SEQ ID NO: 76, and the VL contains the amino acid sequence of SEQ ID NO: 77; and l) The HC contains the amino acid sequence of SEQ ID NO: 76 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377; and the LC contains the amino acid sequences of SEQ ID NOs: 77 and 378. It is selected from the group consisting of the following.

[0150] In some embodiments, the anti-TIM-3 antibody is a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 88-90; b) The VH antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 86; c) The VH is an antibody containing the amino acid sequence of SEQ ID NO: 86; d) The HC is an antibody containing the amino acid sequence of SEQ ID NO: 86 and SEQ ID NOs: 374, 375, 376, or 377; e) The L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 91-93; f) The VL antibody has at least 90% identical amino acid sequence to that of SEQ ID NO: 87; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 87; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 87 and 378; i) Antibodies in which H-CDR1-3 and L-CDR1-3 each contain the amino acid sequences of SEQ ID NOs. 88-93; j) The VH antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 86, and the VL antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 87; k) an antibody in which the VH contains the amino acid sequence of SEQ ID NO: 86, and the VL contains the amino acid sequence of SEQ ID NO: 87; and l) The HC contains the amino acid sequence of SEQ ID NO: 86, and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377; and the LC contains the amino acid sequences of SEQ ID NOs: 87 and 378. It is selected from the group consisting of the following.

[0151] In some embodiments, the anti-TIM-3 antibody is a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 98-100; b) The VH antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 96; c) The VH is an antibody containing the amino acid sequence of SEQ ID NO: 96; d) The HC is an antibody containing the amino acid sequence of SEQ ID NO: 96 and SEQ ID NOs: 374, 375, 376, or 377; e) The L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 101-103; f) The VL antibody has at least 90% identical amino acid sequence to that of SEQ ID NO: 97; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 97; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 97 and 378; i) Antibodies in which H-CDR1-3 and L-CDR1-3 each contain the amino acid sequences of SEQ ID NOs. 98-103; j) The VH antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 96, and the VL antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 97; k) an antibody in which the VH contains the amino acid sequence of SEQ ID NO: 96, and the VL contains the amino acid sequence of SEQ ID NO: 97; and l) The HC contains the amino acid sequence of SEQ ID NO: 96 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377; and the LC contains the amino acid sequences of SEQ ID NOs: 97 and 378. It is selected from the group consisting of the following.

[0152] In some embodiments, the anti-TIM-3 antibody is a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 108-110; b) The VH antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 106; c) The VH is an antibody containing the amino acid sequence of SEQ ID NO: 106; d) The HC is an antibody containing the amino acid sequence of SEQ ID NO: 106 and SEQ ID NOs: 374, 375, 376, or 377; e) The L-CDR1-3 antibodies each contain the amino acid sequences of sequence numbers 111-113; f) The VL is an antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 107; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 107; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 107 and 378; i) The H-CDR1-3 and L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 108-113; j) The VH antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 106, and the VL antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 107; k) an antibody in which the VH contains the amino acid sequence of SEQ ID NO: 106, and the VL contains the amino acid sequence of SEQ ID NO: 107; and l) The HC contains the amino acid sequence of SEQ ID NO: 106 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377; and the LC contains the amino acid sequences of SEQ ID NOs: 107 and 378. It is selected from the group consisting of the following.

[0153] In some embodiments, the anti-TIM-3 antibody is a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 118-120; b) The VH antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 116; c) The VH is an antibody containing the amino acid sequence of SEQ ID NO: 116; d) The HC is an antibody containing the amino acid sequence of SEQ ID NO: 116 and SEQ ID NOs: 374, 375, 376, or 377; e) The L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 121-123; f) The VL is an antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 117; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 117; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 117 and 378; i) The H-CDR1-3 and L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 118-123; j) The VH antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 116, and the VL antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 117; k) an antibody in which the VH contains the amino acid sequence of SEQ ID NO: 116, and the VL contains the amino acid sequence of SEQ ID NO: 117; and l) The HC comprises the amino acid sequence of SEQ ID NO: 116 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377; and the LC comprises the amino acid sequences of SEQ ID NOs: 117 and 378. It is selected from the group consisting of the following.

[0154] In some embodiments, the anti-TIM-3 antibody is a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 128-130; b) The VH antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 126; c) The VH is an antibody containing the amino acid sequence of SEQ ID NO: 126; d) The HC is an antibody containing the amino acid sequence of SEQ ID NO: 126 and SEQ ID NOs: 374, 375, 376, or 377; e) The L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 131-133; f) The VL is an antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 127; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 127; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 127 and 378; i) The H-CDR1-3 and L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 128-133; j) The VH antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 126, and the VL antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 127; k) an antibody in which the VH contains the amino acid sequence of SEQ ID NO: 126, and the VL contains the amino acid sequence of SEQ ID NO: 127; and l) The HC comprises the amino acid sequence of SEQ ID NO: 126 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377; and the LC comprises the amino acid sequences of SEQ ID NOs: 127 and 378. It is selected from the group consisting of the following.

[0155] In some embodiments, the anti-TIM-3 antibody is a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 138-140; b) The VH antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 136; c) The VH is an antibody containing the amino acid sequence of SEQ ID NO: 136; d) The HC is an antibody containing the amino acid sequence of SEQ ID NO: 136 and SEQ ID NOs: 374, 375, 376, or 377; e) The L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 141-143; f) The VL is an antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 137; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 137; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 137 and 378; i) The H-CDR1-3 and L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 138-143; j) The VH antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 136, and the VL antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 137; k) The VH of the antibody contains the amino acid sequence of SEQ ID NO: 136, and the VL of the antibody contains the amino acid sequence of SEQ ID NO: 137; and l) The HC contains the amino acid sequence of SEQ ID NO: 136 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377; and the LC contains the amino acid sequences of SEQ ID NOs: 137 and 378. It is selected from the group consisting of the following.

[0156] In some embodiments, the anti-TIM-3 antibody is a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 148-150; b) The VH antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 146; c) The VH is an antibody containing the amino acid sequence of SEQ ID NO: 146; d) The HC is an antibody containing the amino acid sequence of SEQ ID NO: 146 and SEQ ID NOs: 374, 375, 376, or 377; e) The L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 151-153; f) The VL is an antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 147; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 147; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 147 and 378; i) The H-CDR1-3 and L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 148-153; j) The VH antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 146, and the VL antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 147; k) The VH of the antibody contains the amino acid sequence of SEQ ID NO: 146, and the VL of the antibody contains the amino acid sequence of SEQ ID NO: 147; and l) The HC contains the amino acid sequence of SEQ ID NO: 146 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377; and the LC contains the amino acid sequences of SEQ ID NOs: 147 and 378. It is selected from the group consisting of the following.

[0157] In some embodiments, the anti-TIM-3 antibody is a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 158-160; b) The VH antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 156; c) The VH is an antibody containing the amino acid sequence of SEQ ID NO: 156; d) The HC is an antibody containing the amino acid sequence of SEQ ID NO: 156 and SEQ ID NOs: 374, 375, 376, or 377; e) The L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 161-163; f) The VL is an antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 157; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 157; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 157 and 378; i) The H-CDR1-3 and L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 158-163; j) The VH antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 156, and the VL antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 157; k) The VH of the antibody contains the amino acid sequence of SEQ ID NO: 156, and the VL of the antibody contains the amino acid sequence of SEQ ID NO: 157; and l) The HC contains the amino acid sequence of SEQ ID NO: 156 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377; and the LC contains the amino acid sequences of SEQ ID NOs: 157 and 378. It is selected from the group consisting of the following.

[0158] In some embodiments, the anti-TIM-3 antibody is a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 168-170; b) The VH antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 166; c) The VH is an antibody containing the amino acid sequence of SEQ ID NO: 166; d) The HC is an antibody containing the amino acid sequence of SEQ ID NO: 166 and SEQ ID NOs: 374, 375, 376, or 377; e) The L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 171-173; f) The VL is an antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 167; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 167; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 167 and 378; i) The H-CDR1-3 and L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 168-173; j) The VH antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 166, and the VL antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 167; k) an antibody in which the VH contains the amino acid sequence of SEQ ID NO: 166, and the VL contains the amino acid sequence of SEQ ID NO: 167; and l) The HC contains the amino acid sequence of SEQ ID NO: 166 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377; and the LC contains the amino acid sequences of SEQ ID NOs: 167 and 378. It is selected from the group consisting of the following.

[0159] In some embodiments, the anti-TIM-3 antibody is a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 178-180; b) The VH antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 176; c) The VH is an antibody containing the amino acid sequence of SEQ ID NO: 176; d) The HC is an antibody containing the amino acid sequence of SEQ ID NO: 176 and SEQ ID NOs: 374, 375, 376, or 377; e) The L-CDR1-3 antibodies each contain the amino acid sequences of sequence numbers 181-183; f) The VL is an antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 177; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 177; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 177 and 378; i) The H-CDR1-3 and L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 178-183; j) The VH antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 176, and the VL antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 177; k) an antibody in which the VH contains the amino acid sequence of SEQ ID NO: 176, and the VL contains the amino acid sequence of SEQ ID NO: 177; and l) The HC contains the amino acid sequence of SEQ ID NO: 176 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377; and the LC contains the amino acid sequences of SEQ ID NOs: 177 and 378. It is selected from the group consisting of the following.

[0160] In some embodiments, the anti-TIM-3 antibody is a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 188-190; b) The VH antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 186; c) The VH is an antibody containing the amino acid sequence of SEQ ID NO: 186; d) The HC is an antibody containing the amino acid sequence of SEQ ID NO: 186 and SEQ ID NOs: 374, 375, 376, or 377; e) The L-CDR1-3 antibodies each contain the amino acid sequences of sequence numbers 191-193; f) The VL is an antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 187; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 187; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 187 and 378; i) The H-CDR1-3 and L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 188-193; j) The VH antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 186, and the VL antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 187; k) The antibody in which the VH contains the amino acid sequence of SEQ ID NO: 186, and the antibody in which the VL contains the amino acid sequence of SEQ ID NO: 187; and l) The HC contains the amino acid sequence of SEQ ID NO: 186 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377; and the LC contains the amino acid sequences of SEQ ID NOs: 187 and 378. It is selected from the group consisting of the following.

[0161] In some embodiments, the anti-TIM-3 antibody is a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 198-200; b) The VH antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 196; c) The VH is an antibody containing the amino acid sequence of SEQ ID NO: 196; d) The HC is an antibody containing the amino acid sequence of SEQ ID NO: 196 and SEQ ID NOs: 374, 375, 376, or 377; e) The L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 201-203; f) The VL is an antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 197; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 197; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 197 and 378; i) The H-CDR1-3 and L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 198-203; j) The VH antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 196, and the VL antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 197; k) The antibody in which VH contains the amino acid sequence of SEQ ID NO: 196, and the antibody in which VL contains the amino acid sequence of SEQ ID NO: 197; and l) The HC contains the amino acid sequence of SEQ ID NO: 196 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377; and the LC contains the amino acid sequences of SEQ ID NOs: 197 and 378. It is selected from the group consisting of the following.

[0162] In some embodiments, the anti-TIM-3 antibody is a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 208-210; b) The VH antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 206; c) The VH is an antibody containing the amino acid sequence of SEQ ID NO: 206; d) The HC is an antibody containing the amino acid sequence of SEQ ID NO: 206 and SEQ ID NOs: 374, 375, 376, or 377; e) The L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 211-213; f) The VL is an antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 207; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 207; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 207 and 378; i) The H-CDR1-3 and L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 208-213; j) The VH antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 206, and the VL antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 207; k) an antibody in which the VH contains the amino acid sequence of SEQ ID NO: 206, and the VL contains the amino acid sequence of SEQ ID NO: 207; and l) The HC contains the amino acid sequence of SEQ ID NO: 206 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377; and the LC contains the amino acid sequences of SEQ ID NOs: 207 and 378. It is selected from the group consisting of the following.

[0163] In some embodiments, the anti-TIM-3 antibody is a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 218-220; b) The VH antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 216; c) The VH is an antibody containing the amino acid sequence of SEQ ID NO: 216; d) The HC is an antibody containing the amino acid sequence of SEQ ID NO: 216 and SEQ ID NOs: 374, 375, 376, or 377; e) The L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 221-223; f) The VL is an antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 217; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 217; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 217 and 378; i) The H-CDR1-3 and L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 218-223; j) The VH antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 216, and the VL antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 217; k) The antibody in which the VH contains the amino acid sequence of SEQ ID NO: 216, and the antibody in which the VL contains the amino acid sequence of SEQ ID NO: 217; and l) The HC contains the amino acid sequence of SEQ ID NO: 216 and the amino acid sequences of SEQ ID NOs: 374, 375, 376, or 377; and the LC contains the amino acid sequences of SEQ ID NOs: 217 and 378. It is selected from the group consisting of the following.

[0164] In some embodiments, any of the anti-TIM-3 antibodies or antigen-binding moieties described herein may inhibit the binding of ligands such as galectin-9, CEACAM1, HMGB-1, and phosphatidylserine to TIM-3.

[0165] In some embodiments, any of the anti-TIM-3 antibodies or antigen-binding moieties described herein may increase NK cell activity. In some embodiments, this activity may be mediated by ADCC.

[0166] In some embodiments, administration of the anti-TIM-3 antibody or its antigen-binding moiety described herein can activate dendritic cells, induce their maturation, and thereby induce their T-cell stimulating ability. While we do not wish to be bound by any particular theory, the anti-TIM-3 antibody described herein is thought to function as a TIM-3 dendritic cell activator, thereby its effect on dendritic cells playing a role in stimulating T cells. For this reason, in tumor-related contexts, the anti-TIM-3 antibody induces maturation and activation of tumor-associated dendritic cells, leading to the activation of tumor-specific T cells.

[0167] In some embodiments, administration of the anti-TIM-3 antibody or its antigen-binding moiety described herein may directly activate T cells.

[0168] In some embodiments, the anti-TIM-3 antibody or antigen-binding moiety described herein has the following characteristics: a) K(T) 23 nM or less when measured by surface plasmon resonance D To bind to human TIM-3; b) K(T) 22 nM or less when measured by surface plasmon resonance D To bind to TIM-3 in cynomolgus monkeys; c) Binding to human TIM-3 at an EC50 of 1.2 nM or less as measured by ELISA; d) Binding to cynomolgus monkey TIM-3 at an EC50 of 46 nM or less when measured by ELISA; e) To increase IFN-γ secretion in a unidirectional mixed lymphocyte reaction assay; f) To increase IFN-γ secretion in a bidirectional mixed lymphocyte reaction assay; g) Increasing TNF-α secretion in a unidirectional mixed lymphocyte reaction assay; h) Increasing TNF-α secretion from dendritic cells; and i) Inhibiting the interaction between TIM-3 and phosphatidylserine. It has at least one of the following. Examples of such antibodies include, but are not limited to, antibody 15086.15086 (having at least properties a, c, d, e, g, and h), antibody 15086.17145 (having at least properties a, c, d, e, g, h, and i), antibody 15086.16837 or 15086.17144 (having at least properties a, c, and d), antibody 20293 or 20131 (having at least properties a, b, c, d, e, f, and h), antibody 20362 (having at least properties c, e, f, and h), and antibodies 19324, 19416, 19568, 20185, 20300, or 20621 (having at least properties c, d, e, f, and h). In some embodiments, the anti-TIM-3 antibody or antigen-binding moiety has all of the aforementioned properties. In some embodiments, the anti-TIM-3 antibody or antigen-binding moiety has at least characteristics a, c, d, e, g, and h. In some embodiments, the anti-TIM-3 antibody or antigen-binding moiety has at least characteristics a, c, d, e, g, h, and i. In some embodiments, the anti-TIM-3 antibody or antigen-binding moiety has at least characteristics a, c, and d. In some embodiments, the anti-TIM-3 antibody or antigen-binding moiety has at least characteristics a, b, c, d, e, f, and h. In some embodiments, the anti-TIM-3 antibody or antigen-binding moiety has at least characteristics c, e, f, and h. In some embodiments, the anti-TIM-3 antibody or antigen-binding moiety has at least characteristics c, d, e, f, and h.

[0169] In some embodiments, the anti-TIM-3 antibody or antigen-binding moiety described herein binds to an epitope of TIM-3 containing at least one of the following residues of SEQ ID NO: 389: P50, V60, F61, E62, G64, R69, I117, M118, and D120 (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine). In certain embodiments, the antibody or antigen-binding moiety binds to an epitope of TIM-3 containing the residues P50, V60, F61, E62, G64, R69, I117, M118, and D120 of SEQ ID NO: 389 (e.g., antibodies 15086.15086, 15086.16837, 15086.17145, or 15086.17144). In certain embodiments, the antibody or antigen-binding moiety binds to an epitope of TIM-3 containing residues F61, R69, and I117 of SEQ ID NO: 389 (e.g., antibody 20293). In certain embodiments, the antibody or antigen-binding moiety binds to an epitope of TIM-3 containing residues P50, F61, E62, I117, M118, and D120 of SEQ ID NO: 389 (e.g., antibody 20131).

[0170] In some embodiments, the anti-TIM-3 antibody or its antigen-binding moiety binds to the TIM-3 epitope containing amino acid residues F61 and I117 of SEQ ID NO: 389 (e.g., antibodies 15086.15086, 15086.16837, 15086.17145, 15086.17144, 20293, or 20131). In some embodiments, the epitope further contains amino acid residue R69 (e.g., antibodies 15086.15086, 15086.16837, 15086.17145, 15086.17144, or 20293). In some embodiments, the epitope further comprises P50, E62, M118, and D120 (e.g., antibodies 15086.15086, 15086.16837, 15086.17145, 15086.17144, or 20131), and may further comprise amino acid residues V60 and G64 (e.g., antibodies 15086.15086, 15086.16837, 15086.17145, or 15086.17144).

[0171] In some embodiments, the anti-TIM-3 antibody or its antigen-binding moiety described herein binds to an epitope of TIM-3 containing at least one of the following residues of SEQ ID NO: 236: P50, V60, F61, E62, G64, R69, I117, M118, and D120 (e.g., at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine). An epitope having any combination of the above residues is intended.

[0172] In some embodiments, the anti-TIM-3 antibody or its antigen-binding moiety described herein binds to an epitope of TIM-3 comprising residues 62-67 and / or 114-117 of SEQ ID NO: 389. In some embodiments, the antibody or moiety binds to residues 62-67 (or fragments thereof, e.g., 1, 2, 3, 4, or 5 residue fragments) of SEQ ID NO: 389 (e.g., antibodies 15086, 15086, 16837, 15086, 17145, 15086, 17144, and 20293). In some embodiments, the antibody or moiety binds to residues 114-117 (or fragments thereof, e.g., 1, 2, or 3 residue fragments) of SEQ ID NO: 389 (e.g., antibody 20131). Epitopes having any combination of the above residues are also intended.

[0173] In some embodiments, the anti-TIM-3 antibody or its antigen-binding moiety does not compete with ABTIM3 (PCT Patent Publication International Publication No. 2015 / 117002) and / or mAb15 (PCT Patent Publication International Publication No. 2016 / 111947) for binding to TIM-3. In some embodiments, the anti-TIM-3 antibody or antigen-binding moiety does not bind to the same epitopes as ABTIM3 and / or mAB15; for example, the antibody or moiety binds to one or more residues on TIM-3 to which ABTIM3 and / or mAb15 do not bind.

[0174] In some embodiments, the anti-TIM-3 antibody or antigen-binding moiety described herein is the anti-TIM-3 antibody or antigen-binding moiety described herein, which is incorporated herein by reference in whole, in PCT Patent Publication International Publication No. 2017 / 178493.

[0175] Anti-LAG-3 antibody In some embodiments, the anti-LAG-3 antibody disclosed herein is a human antibody produced from a transgenic rat capable of producing an antibody having a human idiotype. In another embodiment, the antibody is a chicken-derived chimeric antibody comprising a chicken CDR sequence and a human framework region, the framework region of which is subjected to humanization.

[0176] One advantage of the novel anti-LAG-3 antibodies described herein is their ability to enhance T cell activity, as measured by increased IL-2 production. While we do not wish to be bound by any particular theory, it is thought that anti-LAG-3 antibodies can block the interaction of LAG-3 with its putative ligands, such as MHCII and LSECtin. The antibody may achieve this directly, either through blockade of the ligand-binding region or through induction of LAG-3 internalization. Another potential advantage of the anti-LAG-3 antibodies described herein is the low level of secondary effector function in antibodies with the "LALA" mutation (L234A / L235A), which interferes with significant antibody binding to human FcgR (Fc gamma receptor) and therefore leads to the depletion of effector T cells.

[0177] In some embodiments, the combination therapy or composition comprises an anti-LAG-3 antibody or its antigen-binding moiety, the anti-LAG-3 antibody being referred herein to as antibody 15646, 15532, 15723, 15595, 15431, 15572, or 15011, or any variant thereof, the variants may contain a certain minimum amino acid change compared to the antibody (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes that may be present in the framework region) without losing the antigen-binding specificity of the antibody.

[0178] In some embodiments, the anti-LAG-3 antibody or its antigen-binding moiety competes or cross-competes with antibodies 15646, 15532, 15723, 15595, 15431, 15572, or 15011 for binding to human LAG-3, or binds to the same human LAG-3 epitope as the antibody. In some embodiments, the antibody has an IgG1 or IgG2 format. In a particular embodiment, the antibody has an IgG2 format.

[0179] In some embodiments, the anti-LAG-3 antibody competes or cross-competes with antibodies containing SEQ ID NOs. 308-313, 318-323, 328-333, 338-343, 348-353, 358-363, or 368-373 for binding to human LAG-3, or binds to the same human LAG-3 epitopes as the antibody.

[0180] In some embodiments, the anti-LAG-3 antibody contains H-CDR3 comprising the H-CDR3 amino acid sequence of SEQ ID NOs. 310, 320, 330, 340, 350, 360, or 370.

[0181] In some embodiments, the anti-LAG-3 antibody contains H-CDR1-3, each containing the H-CDR1-3 amino acid sequence of SEQ ID NOs. 308-310, 318-320, 328-330, 338-340, 348-350, 358-360, or 368-370, respectively.

[0182] In some embodiments, the anti-LAG-3 antibody has a VH whose amino acid sequence is at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NOs. 306, 316, 326, 336, 346, 356, or 366.

[0183] In some embodiments, the anti-LAG-3 antibody has a VH including SEQ ID NOs: 306, 316, 326, 336, 346, 356, or 366.

[0184] In some embodiments, the anti-LAG-3 antibody has a VH that is sequence-identical to at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) with SEQ ID NO: 306, 316, 326, 336, 346, 356, or 366; and a CH that is sequence-identical to at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) with SEQ ID NO: 375.

[0185] In some embodiments, the anti-LAG-3 antibody has an HC comprising the VH amino acid sequence of SEQ ID NO: 306, 316, 326, 336, 346, 356, or 366, and the CH amino acid sequence of SEQ ID NO: 375.

[0186] In some embodiments, the anti-LAG-3 antibody contains L-CDR3 comprising the L-CDR3 amino acid sequence of SEQ ID NOs. 313, 323, 333, 343, 353, 363, or 373.

[0187] In some embodiments, the anti-LAG-3 antibody contains L-CDR1-3, each containing the L-CDR1-3 amino acid sequences of SEQ ID NOs. 311-313, 321-323, 331-333, 341-343, 351-353, 361-363, or 371-373, respectively.

[0188] In some embodiments, the anti-LAG-3 antibody has a VL whose sequence is at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the VL amino acid sequence of SEQ ID NO: 307, 317, 327, 337, 347, 357, or 367.

[0189] In some embodiments, the anti-LAG-3 antibody has a VL containing the VL amino acid sequence of SEQ ID NOs. 307, 317, 327, 337, 347, 357, or 367.

[0190] In some embodiments, the anti-LAG-3 antibody has a VL whose sequence is at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the VL amino acid sequence of SEQ ID NO: 307, 317, 327, 337, 347, or 357; and a CL whose sequence is at least 90% (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the VL amino acid sequence of SEQ ID NO: 378.

[0191] In some embodiments, the anti-LAG-3 antibody has an LC comprising the VL amino acid sequence of SEQ ID NO: 307, 317, 327, 337, 347, or 357, and the CL amino acid sequence of SEQ ID NO: 378. In some embodiments, the anti-LAG-3 antibody has an LC comprising the VL amino acid sequence of SEQ ID NO: 367, and the CL amino acid sequence of SEQ ID NO: 379.

[0192] In some embodiments, the anti-LAG-3 antibody comprises one of the above heavy chain sequences and one of the above light chain sequences.

[0193] In some embodiments, the anti-LAG-3 antibody comprises H-CDR3 and L-CDR3, each containing the H-CDR3 and L-CDR3 amino acid sequences of SEQ ID NOs. 310 and 313, 320 and 323, 330 and 333, 340 and 343, 350 and 353, 360 and 363, or 370 and 373, respectively.

[0194] In some embodiments, the anti-LAG-3 antibody comprises H-CDR1-3 and L-CDR1-3, each containing H-CDR1-3 and L-CDR1-3 sequences of sequence numbers 308-313, 318-323, 328-333, 338-343, 348-353, 358-363, or 368-373, respectively.

[0195] In some embodiments, the anti-LAG-3 antibody has a VH variant whose sequence is at least 90% (e.g., at least 92%, at least 95%, at least 98%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 306, 316, 326, 336, 346, 356, or 366, and a VL variant whose sequence is at least 90% (e.g., at least 92%, at least 95%, at least 98%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 307, 317, 327, 337, 347, 357, or 367.

[0196] In some embodiments, the anti-LAG-3 antibody has a VH containing the amino acid sequence of SEQ ID NOs. 306, 316, 326, 336, 346, 356, or 366, and a VL containing the amino acid sequence of SEQ ID NOs. 307, 317, 327, 337, 347, 357, or 367.

[0197] In some embodiments, the anti-LAG-3 antibody has an HC comprising the amino acid sequence of SEQ ID NO: 306, 316, 326, 336, 346, 356, or 366 and the amino acid sequence of SEQ ID NO: 375; and an LC comprising the amino acid sequence of SEQ ID NO: 307, 317, 327, 337, 347, or 357 and the amino acid sequence of SEQ ID NO: 378.

[0198] In some embodiments, the anti-LAG-3 antibody has an HC comprising the amino acid sequence of SEQ ID NO: 306, 316, 326, 336, 346, 356, or 366, and the amino acid sequence of SEQ ID NO: 375; and an LC comprising the amino acid sequence of SEQ ID NO: 367 and the amino acid sequence of SEQ ID NO: 379.

[0199] In some embodiments, the anti-LAG-3 antibody is a) Sequence numbers 308-313, respectively; b) Sequence numbers 318-323, respectively; c) Sequence numbers 328-333, respectively; d) Sequence numbers 338-343, respectively; e) Sequence numbers 348-353, respectively; f) Sequence numbers 358-363, respectively; or g) Sequence numbers 368-373, respectively It contains the H-CDR1-3 and L-CDR1-3 amino acid sequences.

[0200] In some embodiments, the anti-LAG-3 antibody is a) Sequence IDs 306 and 307, respectively; b) Sequence IDs 316 and 317, respectively; c) Sequence IDs 326 and 327, respectively; d) Sequence IDs 336 and 337, respectively; e) Sequence IDs 346 and 347, respectively; f) Sequence IDs 356 and 357, respectively; or g) Sequence IDs 366 and 367, respectively It includes a heavy chain variable domain and a light chain variable domain having the following amino acid sequence.

[0201] In some embodiments, the anti-LAG-3 antibody is a) HC containing the amino acid sequence of SEQ ID NO: 306 and SEQ ID NO: 375, and LC containing the amino acid sequence of SEQ ID NO: 307 and SEQ ID NO: 378; b) HC containing the amino acid sequence of SEQ ID NO: 316 and SEQ ID NO: 375, and LC containing the amino acid sequence of SEQ ID NO: 317 and SEQ ID NO: 378; c) HC containing the amino acid sequence of SEQ ID NO: 326 and SEQ ID NO: 375, and LC containing the amino acid sequence of SEQ ID NO: 327 and SEQ ID NO: 378; d) HC containing the amino acid sequence of SEQ ID NO: 336 and SEQ ID NO: 375, and LC containing the amino acid sequence of SEQ ID NO: 337 and SEQ ID NO: 378; e) HC containing the amino acid sequences of SEQ ID NO: 346 and SEQ ID NO: 375, and LC containing the amino acid sequences of SEQ ID NO: 347 and SEQ ID NO: 378; f) HC containing the amino acid sequence of SEQ ID NO: 356 and SEQ ID NO: 375, and LC containing the amino acid sequence of SEQ ID NO: 357 and SEQ ID NO: 378; or g) HC containing the amino acid sequences of SEQ ID NO: 366 and SEQ ID NO: 375, and LC containing the amino acid sequences of SEQ ID NO: 367 and SEQ ID NO: 379 Includes.

[0202] In some embodiments, the anti-LAG-3 antibody is a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 308-310; b) The VH antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 306; c) The VH antibody contains the amino acid sequence of SEQ ID NO: 306; d) The HC is an antibody containing the amino acid sequences of SEQ ID NOs. 306 and 375; e) Each of the L-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 311-313; f) The VL is an antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 307; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 307; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 307 and 378; i) The H-CDR1-3 and L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 308-313; j) The VH antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 306, and the VL antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 307; k) an antibody in which the VH contains the amino acid sequence of SEQ ID NO: 306, and the VL contains the amino acid sequence of SEQ ID NO: 307; and l) The antibody whose HC contains the amino acid sequences of SEQ ID NOs. 306 and 375, and whose LC contains the amino acid sequences of SEQ ID NOs. 307 and 378. It is selected from the group consisting of the following.

[0203] In some embodiments, the anti-LAG-3 antibody is a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 318-320; b) The VH antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 316; c) The VH antibody contains the amino acid sequence of SEQ ID NO: 316; d) The HC is an antibody containing the amino acid sequences of SEQ ID NOs. 316 and 375; e) Each of the L-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 321-323; f) The VL is an antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 317; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 317; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 317 and 378; i) The H-CDR1-3 and L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 318-323; j) The VH antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 316, and the VL antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 317; k) The antibody in which the VH contains the amino acid sequence of SEQ ID NO: 316, and the antibody in which the VL contains the amino acid sequence of SEQ ID NO: 317; and l) The antibody whose HC contains the amino acid sequences of SEQ ID NOs. 316 and 375, and whose LC contains the amino acid sequences of SEQ ID NOs. 317 and 378. It is selected from the group consisting of the following.

[0204] In some embodiments, the anti-LAG-3 antibody is a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 328-330; b) The VH antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 326; c) The VH antibody contains the amino acid sequence of SEQ ID NO: 326; d) The HC is an antibody containing the amino acid sequences of SEQ ID NOs. 326 and 375; e) Each of the L-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 331-333; f) The VL is an antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 327; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 327; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 327 and 378; i) The H-CDR1-3 and L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 328-333; j) The VH antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 326, and the VL antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 327; k) The antibody in which VH contains the amino acid sequence of SEQ ID NO: 326, and the antibody in which VL contains the amino acid sequence of SEQ ID NO: 327; and l) The antibody whose HC contains the amino acid sequences of SEQ ID NOs. 326 and 375, and whose LC contains the amino acid sequences of SEQ ID NOs. 327 and 378. It is selected from the group consisting of the following.

[0205] In some embodiments, the anti-LAG-3 antibody is a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 338-340; b) The VH antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 336; c) The VH antibody contains the amino acid sequence of SEQ ID NO: 336; d) The HC is an antibody containing the amino acid sequences of SEQ ID NOs. 336 and 375; e) Each of the L-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 341-343; f) The VL is an antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 337; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 337; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 337 and 378; i) The H-CDR1-3 and L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 338-343; j) The VH antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 336, and the VL antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 337; k) The VH of the antibody contains the amino acid sequence of SEQ ID NO: 336, and the VL of the antibody contains the amino acid sequence of SEQ ID NO: 337; and l) The antibody whose HC contains the amino acid sequences of SEQ ID NOs. 336 and 375, and whose LC contains the amino acid sequences of SEQ ID NOs. 337 and 378. It is selected from the group consisting of the following.

[0206] In some embodiments, the anti-LAG-3 antibody is a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 348-350; b) The VH antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 346; c) The VH antibody contains the amino acid sequence of SEQ ID NO: 346; d) The HC is an antibody containing the amino acid sequences of SEQ ID NOs. 346 and 375; e) Each of the L-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 351-353; f) The VL is an antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 347; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 347; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 347 and 378; i) The H-CDR1-3 and L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 348-353; j) The VH antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 346, and the VL antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 347; k) The VH of the antibody contains the amino acid sequence of SEQ ID NO: 346, and the VL of the antibody contains the amino acid sequence of SEQ ID NO: 347; and l) The antibody whose HC contains the amino acid sequences of SEQ ID NOs. 346 and 375, and whose LC contains the amino acid sequences of SEQ ID NOs. 347 and 378. It is selected from the group consisting of the following.

[0207] In some embodiments, the anti-LAG-3 antibody is a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 358-360; b) The VH antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 356; c) The VH antibody contains the amino acid sequence of SEQ ID NO: 356; d) The HC is an antibody containing the amino acid sequences of SEQ ID NOs. 356 and 375; e) Each of the L-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 361-363; f) The VL is an antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 357; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 357; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 357 and 378; i) The H-CDR1-3 and L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 358-363; j) The VH antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 356, and the VL antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 357; k) The antibody in which the VH contains the amino acid sequence of SEQ ID NO: 356, and the antibody in which the VL contains the amino acid sequence of SEQ ID NO: 357; and l) The antibody whose HC contains the amino acid sequences of SEQ ID NOs. 356 and 375, and whose LC contains the amino acid sequences of SEQ ID NOs. 357 and 378. It is selected from the group consisting of the following.

[0208] In some embodiments, the anti-LAG-3 antibody is a) Each of the H-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 368-370; b) The VH antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 366; c) The VH antibody contains the amino acid sequence of SEQ ID NO: 366; d) The HC is an antibody containing the amino acid sequences of SEQ ID NOs. 366 and 375; e) Each of the L-CDR1-3 antibodies contains the amino acid sequence of SEQ ID NOs. 371-373; f) The VL is an antibody whose amino acid sequence is at least 90% identical to that of SEQ ID NO: 367; g) The VL is an antibody containing the amino acid sequence of SEQ ID NO: 367; h) The LC is an antibody containing the amino acid sequences of SEQ ID NOs. 367 and 379; i) The H-CDR1-3 and L-CDR1-3 antibodies each contain the amino acid sequences of SEQ ID NOs. 368-373; j) The VH antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 366, and the VL antibody is at least 90% identical in amino acid sequence to SEQ ID NO: 367; k) The VH of the antibody contains the amino acid sequence of SEQ ID NO: 366, and the VL of the antibody contains the amino acid sequence of SEQ ID NO: 367; and l) The antibody whose HC contains the amino acid sequences of SEQ ID NOs. 366 and 375, and whose LC contains the amino acid sequences of SEQ ID NOs. 367 and 379. It is selected from the group consisting of the following.

[0209] In some embodiments, any of the anti-LAG-3 antibodies or antigen-binding moieties described herein may bind to human LAG-3 at an EC50 of, for example, 0.2 nM or less, 0.15 nM or less, 0.1 nM or less, 0.09 nM or less, 0.08 nM or less, 0.07 nM or less, 0.06 nM or less, 0.05 nM or less, or 0.04 nM or less. In some embodiments, any of the anti-LAG-3 antibodies or antigen-binding moieties described herein may bind to cynomolgus monkey LAG-3 at EC50 levels of, for example, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, 0.1 nM or less, 0.09 nM or less, 0.08 nM or less, 0.07 nM or less, 0.06 nM or less, 0.05 nM or less, 0.04 nM or less, or 0.03 nM or less. In certain embodiments, any of the anti-LAG-3 antibodies or antigen-binding moieties described herein may bind to human LAG-3 at EC50 levels of, for example, 0.1 nM or less, and to cynomolgus monkey LAG-3 at EC50 levels of, for example, 0.3 nM or less.

[0210] In some embodiments, any of the anti-LAG-3 antibodies or antigen-binding moieties described herein may bind to human LAG-3 at, for example, 0.1 nM or less EC50. In some embodiments, any of the anti-LAG-3 antibodies or antigen-binding moieties described herein may bind to cynomolgus monkey LAG-3 at, for example, 0.3 nM or less EC50. In specific embodiments, any of the anti-LAG-3 antibodies or antigen-binding moieties described herein may bind to human LAG-3 at, for example, 0.1 nM or less EC50, and to cynomolgus monkey LAG-3 at, for example, 0.3 nM or less EC50.

[0211] In some embodiments, any of the anti-LAG-3 antibodies or antigen-binding moieties described herein may inhibit the binding of ligands such as MHC class II (MHCII) or LSECtin to LAG-3. For example, a 20 μg / mL anti-LAG-3 antibody or antigen-binding moiety may reduce the binding of LAG-3 to MHCII by at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% compared to binding in the presence of a negative control antibody. In one embodiment, an anti-LAG-3 antibody or antigen-binding protein may reduce the binding of LAG-3 to MHCII by more than 85% compared to a negative control. In one embodiment, an anti-LAG-3 antibody or antigen-binding protein can reduce the binding of LAG-3 to MHCII by approximately 25% to 95%, 30% to 90%, or 35% to 85% compared to a negative control.

[0212] In some embodiments, either the anti-LAG-3 antibody or antigen-binding moiety described herein may block the binding between LAG-3 and MHC class II, for example, between human LAG-3 expressed in Jurkat cells and human MHC class II expressed in Raji cells (for example, at concentrations of 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, or 50 μg / mL).

[0213] In some embodiments, either the anti-LAG-3 antibody or antigen-binding moiety described herein yields a reading of 5.0 × 10⁻¹⁴ when measured by surface plasmon resonance. -8 Or less, 4.0 × 10 -8 Or less, 3.0 × 10 -8 Or less, 2.0 × 10-8 Or less, 1.0 × 10 -8 Or less, 9.0 × 10 -9 Or less, 8.0 × 10 -9 Or less, 7.0 × 10 -9 Or less, 6.0 × 10 -9 Or less, 5.0 × 10 -9 Or less, 4.0 × 10 -9 Or less, 3.0 × 10 -9 Or less, 2.0 × 10 -9 Or less than that, or 1.0 × 10 -9 Or a K less than that. D It can bind to human LAG-3.

[0214] In some embodiments, either the anti-LAG-3 antibody or antigen-binding moiety described herein yields a reading of 1.5 × 10⁻¹⁶ when measured by surface plasmon resonance. -7 Or less, 1.0 × 10 -7 Or less, 9.0 × 10 -8 Or less, 8.0 × 10 -8 Or less, 7.0 × 10 -8 Or less, 6.0 × 10 -8 Or less, 5.0 × 10 -8 Or less, 4.0 × 10 -8 Or less, 3.0 × 10 -8 Or less, 2.0 × 10 -8 Or less than that, or 1.0 × 10 -8 Or a K less than that. D It can bind to LAG-3 in cynomolgus monkeys.

[0215] In some embodiments, either the anti-LAG-3 antibody or antigen-binding moiety described herein yields a reading of 5.0 × 10⁻¹⁴ when measured by surface plasmon resonance. -8 Or less, 4.5 × 10 -8 Or less, 4.0 × 10 -8 Or less, 3.5 × 10 -8Or less, or 3.0 × 10 -8 Or a K less than that. D It can bind to mouse LAG-3.

[0216] In some embodiments, either the anti-LAG-3 antibody or antigen-binding moiety described herein may stimulate IL-2 production in, for example, SEB-stimulated PBMCs.

[0217] In some embodiments, either the anti-LAG-3 antibody or antigen-binding moiety described herein may reduce the cellular and / or soluble levels of LAG-3 in, for example, human T cell lines (e.g., human T cell lines overexpressing LAG-3).

[0218] In some embodiments, either the anti-LAG-3 antibody or antigen-binding moiety described herein may induce regression of tumor growth in vivo and / or slow tumor growth.

[0219] In some embodiments, either the anti-LAG-3 antibody or antigen-binding moiety described herein may bind to a human LAG-3 epitope different from antibody 25F7-Lag3.5.

[0220] In some embodiments, either the anti-LAG-3 antibody or antigen-binding moiety described herein may activate T cells and lead to enhanced antitumor activity.

[0221] In some embodiments, the anti-LAG-3 antibody or antigen-binding moiety described herein has the following characteristics: a) At a concentration of 20 μg / mL, reduce the binding of human LAG-3 to human MHC class II on A375 cells by more than 85% compared to a negative control antibody, as determined by a flow cytometry competitive assay; b) Reducing the binding of human LAG-3 to human MHC class II on A375 cells by 35% to 85% compared to a negative control antibody at a concentration of 20 μg / mL, as determined by a flow cytometry competitive assay; c) Blocking the binding between human LAG-3 expressed in Jurkat cells and human MHC class II expressed in Raji cells; d) Binding to human LAG-3 at an EC50 of 0.1 nM or less as measured by flow cytometry; e) Binding to cynomolgus monkey LAG-3 at an EC50 of 0.3 nM or less as measured by flow cytometry; f) 3.0 × 10⁻¹⁶ when measured by surface plasmon resonance. -8 or less than K D Binding to human LAG-3; g) When measured by surface plasmon resonance, 1.5 × 10 -7 or less than K D It binds to LAG-3 in cynomolgus monkeys; h) When measured by surface plasmon resonance, 3.5 × 10⁻⁶ -8 or less than K D To bind to mouse LAG-3; i) Stimulating IL-2 production in human peripheral blood mononuclear cells (PBMCs) treated with Staphylococcus enterotoxin B (SEB); j) To reduce the cellular level of LAG-3 in human T cells; k) To reduce the solubility level of LAG-3 in human T cell cultures; l) Inducing regression of tumor growth in vivo; m) Slowing tumor growth in vivo; and n) The antibody 25F7-Lag3.5 should not bind to the same human LAG-3 epitope. Having at least one of the following characteristics: Examples of such antibodies include, but are not limited to, antibody 15646 (having at least characteristics b, c, d, e, i, and n), antibody 15532 (having at least characteristics a, c, d, e, f, g, i, j, k, m, and n), antibody 15723 (having at least characteristics b, c, d, e, i, and n), antibody 15595 (having at least characteristics a, c, d, e, i, and n), antibody 15431 (having at least characteristics a, c, d, e, f, g, i, and n), antibody 15572 (having at least characteristics b, c, d, e, f, g, i, and n), and antibody 15011 (having at least characteristics a, c, d, e, f, g, h, i, j, k, l, m, and n). In some embodiments, the anti-TIM-3 antibody or antigen-binding moiety of the present invention has at least 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 of the aforementioned properties. In some embodiments, the anti-TIM-3 antibody or antigen-binding moiety of the present invention has at least properties b, c, d, e, i, and n; at least properties a, c, d, e, f, g, i, j, k, m, and n; at least properties a, c, d, e, i, and n; at least properties a, c, d, e, f, g, i, and n; or at least properties a, c, d, e, f, g, h, i, j, k, l, m, and n.

[0222] In some embodiments, the anti-LAG-3 antibody or antigen-binding moiety of the present invention competes with antibodies 15011, 15572, and / or 15431 for binding to human LAG-3.

[0223] In some embodiments, the anti-LAG-3 antibody or antigen-binding moiety of the present invention is a) Amino acid residues H85, P86, A87, P89, S91, W92, and G93 of SEQ ID NO: 68; b) Amino acid residues A40, Q41, P43, P46, P49, D52, T62, Q64, H65, Q66, P67, D68, G93, P94, P96, R98, Y99, T100, V101, P106, G107, R119, E124, R129, G130, D131, S133, R137, P138, D143, R148, and R163 of SEQ ID NO: 68; c) Amino acid residues A40, Q41, P43, P46, P49, D52, T62, Q64, H65, Q66, P67, D68, P96, Y99, T100, V101, P106, G107, R119, E124, R129, G130, D131, S133, R137, P138, D143, R148, and R163 of SEQ ID NO: 68; or d) Amino acid residues G107, L109, R110, and S111 of SEQ ID NO: 68 It binds to the human LAG-3 epitope that has [specific characteristic].

[0224] In some embodiments, the anti-LAG-3 antibody or antigen-binding moiety of the present invention binds to an epitope having amino acid residues 98-105 of SEQ ID NO: 68. Examples of such antibodies include, but are not limited to, antibodies 15532, 15431, 15572, and 15011.

[0225] In some embodiments, the anti-LAG-3 antibody or antigen-binding moiety of the present invention is a) Amino acid residues 78-105 and 123-131 of Sequence ID No. 68; b) Amino acid residues 23-30, 40-66, 88-105, 123-137, and 148-152 of SEQ ID NO: 68; or c) Amino acid residues 23-30, 40-66, 98-105, 118-137, and 148-161 of SEQ ID NO: 68 It binds to an epitope that has [a certain characteristic].

[0226] In some embodiments, the anti-LAG-3 antibody or antigen-binding moiety described herein is the anti-LAG-3 antibody or antigen-binding moiety described herein in whole by reference in PCT Patent Publication European Patent Application No. 2017 / 076188.

[0227] The class of antibodies described herein may be changed or switched to another class or subclass. In one embodiment, a nucleic acid molecule encoding VL or VH is isolated using methods known in the art so as not to contain a nucleic acid sequence encoding CL or CH. The nucleic acid molecule encoding VL or VH is then operably ligated to a nucleic acid sequence encoding CL or CH from a different class of immunoglobulin molecule. This can be done using a vector or nucleic acid molecule containing a CL or CH chain as described above. For example, an antibody that was originally IgM may be class-switched to IgG. Furthermore, class switching may be used to convert one IgG subclass to another, for example, from IgG1 to IgG2. The κ light chain constant region can be changed to, for example, the λ light chain constant region. A preferred method for producing the antibody described herein having a desired Ig isotype includes the steps of: isolating nucleic acid molecules encoding the heavy chain and light chain of the antibody; obtaining a variable domain of the heavy chain; ligating the variable domain of the heavy chain to the constant region of the heavy chain of the desired isotype; expressing the light chain and the ligated heavy chain in cells; and collecting the antibody having the desired isotype.

[0228] The antibodies described herein may be IgG, IgM, IgE, IgA, or IgD molecules, but are typically IgG isotypes, such as IgG subclass IgG1, IgG2a, or IgG2b, IgG3, or IgG4 molecules. In one embodiment, the antibody is IgG1. In another embodiment, the antibody is IgG2.

[0229] In one embodiment, an antibody may contain at least one mutation in its Fc region. Numerous different Fc mutations are known that provide altered effector function. For example, in many cases, such as when ligand / receptor interaction is undesirable, or in the case of antibody-drug conjugates, it is desirable to reduce or eliminate effector function.

[0230] In one embodiment, the antibody contains at least one mutation in the Fc region that reduces effector function. The amino acid positions in the Fc region that are advantageous to mutate to reduce effector function include one or more of positions 228, 233, 234, and 235, where the amino acid positions are numbered according to the IMGT® numbering scheme.

[0231] In some embodiments, one or both of the amino acid residues at positions 234 and 235 may be mutated, for example, from Leu to Ala (L234A / L235A). These mutations reduce the effector function of the Fc region of the IgG1 antibody. Furthermore, or alternatively, the amino acid residue at position 228 may be mutated, for example, to Pro. In some embodiments, the amino acid residue at position 233 may be mutated, for example, to Pro, the amino acid residue at position 234 may be mutated, for example, to Val, and / or the amino acid residue at position 235 may be mutated, for example, to Ala. The amino acid positions are numbered according to the IMGT® numbering scheme.

[0232] In some embodiments, when the antibody is an IgG4 subclass antibody, the antibody may contain the S228P mutation, where the amino acid position is numbered according to the Eu IMGT® numbering scheme, i.e., it may have a proline at position 228. This mutation is known to reduce undesirable Fab arm exchange (Angal et al., Mol Immunol. 30:105-8 (1993)).

[0233] In certain embodiments, the antibody or its antigen-binding moiety described herein may be part of a larger immunoadhesin formed by covalent or noncovalent bonding of the antibody or antibody moiety to one or more other proteins or peptides. Examples of such immunoadhesins include the use of a streptavidin core region to construct a tetrameric scFv molecule (Kipriyanov et al., Human Antibodies and Hybridomas 6:93-101 (1995)), and the use of cysteine ​​residues, a marker peptide, and a C-terminal polyhistidine tag to construct a divalent biotinylated scFv molecule (Kipriyanov et al., Mol. Immunol. 31:1047-1058 (1994)). Other examples include incorporating one or more CDRs from an antibody into a molecule by covalent or noncovalent bonding to form an immunoadhesin that specifically binds to the antigen of interest. In such embodiments, the CDR may be incorporated as part of a larger polypeptide chain, covalently bonded to another polypeptide chain, or incorporated non-covalently.

[0234] In another embodiment, a fusion antibody or immunoadhesin may be prepared containing all or some of the antibodies described herein linked to another polypeptide. In a particular embodiment, only the variable domain of the antibody is linked to the polypeptide. In a particular embodiment, the VH domain of the antibody is linked to a first polypeptide, and the VL domain of the antibody is linked to a second polypeptide that associates with the first polypeptide, so that the VH and VL domains can interact with each other to form an antigen-binding site. In another preferred embodiment, the VH domain is separated from the VL domain by a linker so that the VH and VL domains can interact with each other (e.g., a single-chain antibody). The VH-linker-VL antibody is then linked to the polypeptide of choice. Furthermore, a fusion antibody can be prepared in which two (or more) single-chain antibodies are linked to each other. This is useful when it is desired to prepare a bivalent or polyvalent antibody on a single polypeptide chain, or when it is desired to prepare a bispecific antibody.

[0235] To produce single-chain antibodies (scFv), DNA fragments encoding VH and VL can be operably linked to another fragment encoding a flexible linker, for example, the amino acid sequence (Gly4-Ser)3 (SEQ ID NO: 396), allowing the VH and VL sequences to be expressed as a continuous single-chain protein with the VL and VH domains linked by the flexible linker. See, for example, Bird et al., Science 242:423 426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879 5883 (1988); and McCafferty et al., Nature 348:552 554 (1990). Single-chain antibodies can be monovalent when using only one VH and VL; bivalent when using two VH and VL; or polyvalent when using more than two VH and VL. Bispecific or polyvalent antibodies may be prepared that specifically bind to human PD-1, TIM-3, or LAG-3, and, for example, another molecule. In some embodiments, the bispecific or polyvalent antibodies may bind to PD-1 and TIM-3, PD-1 and LAG-3, TIM-3 and LAG-3, or PD-1, TIM-3, and LAG-3.

[0236] In other embodiments, other modified antibodies may be prepared using antibody-coding nucleic acid molecules. For example, "kappa bodies" (Ill et al., Protein Eng. 10:949-57 (1997)), "mini bodies" (Martin et al., EMBO J. 13:5303-9 (1994)), "dia bodies" (Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)), or "janusin" (Traunecker et al., EMBO J. 10:3655-3659 (1991) and Traunecker et al., Int. J. Cancer (Suppl.) 7:51-52 (1992)) may be prepared using standard molecular biology techniques according to the teachings herein.

[0237] The antibodies or antigen-binding moieties described herein can be derivatized or linked to another molecule (e.g., another peptide or protein). Generally, the antibody or its moiety is derivatized in such a way that the antigen binding is not adversely affected by the derivatization or labeling. Accordingly, the antibodies and antibody moieties that can be used in the combination therapies and compositions of the present invention are intended to include both intact and modified forms of the antibodies described herein. For example, the antibodies or antibody moieties described herein can be functionally linked to one or more other molecules, such as another antibody (e.g., a bispecific antibody or diabody), a detection agent, a drug, and / or a protein or peptide that can mediate the association of the antibody or antibody moiety with another molecule (e.g., a streptavidin core region or polyhistidine tag) (e.g., by chemical coupling, gene fusion, non-covalent bonding, or other means).

[0238] One type of derivatized antibody is produced by crosslinking two or more antibodies (of the same type or different types, for example, to produce bispecific antibodies). Suitable crosslinkers include heterobifunctional (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate) crosslinkers having two distinct reactive groups separated by a suitable spacer. Such linkers can be obtained, for example, from Pierce Chemical Company, Rockford, 1.

[0239] Antibodies can also be derivatized with chemical groups such as polyethylene glycol (PEG), methyl or ethyl groups, or carbohydrate groups. These groups may be useful to improve the biological characteristics of the antibody, for example, by increasing its serum half-life.

[0240] The antibodies described herein may also be labeled. As used herein, “labeling” or “labeled” refers to the incorporation of another molecule into an antibody. In one embodiment, labeling is the incorporation of a detectable marker, such as a radiolabeled amino acid, or the conjugation of a biotinylated moiety to a polypeptide that can be detected by a labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity detectable by optical or colorimetric methods). In another embodiment, the label or marker may be therapeutic, such as a drug conjugate or toxin. Various methods for labeling polypeptides and glycoproteins are known in the art and can be used. Examples of polypeptide labeling include radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 1111n, 125I, 1311), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predefined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, secondary antibody binding sites, metal-binding domains, epitope tags), and gadolinium chelates. Examples of magnetic agents such as t, toxins such as pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as their analogues or homologues, are, but are not limited to, these. In some embodiments, the labels are coupled by spacer arms of varying lengths to reduce possible steric interference.

[0241] In certain embodiments, the antibodies described herein may exist as neutral (including amphoteric) or positively charged or negatively charged species. In some embodiments, antibodies may be complexed with counterions to form pharmaceutically acceptable salts.

[0242] The term "pharmaceutically acceptable salt" refers to a complex comprising one or more antibodies and one or more counterions derived from pharmaceutically acceptable inorganic and organic acids and bases.

[0243] Combination therapy The present invention provides a combination therapy (e.g., a composition) comprising any (e.g., one) of the anti-PD-1 antibodies or their antigen-binding moieties described herein and any (e.g., one) of the anti-TIM-3 antibodies or their antigen-binding moieties described herein. In some embodiments, the combination therapy comprises any (e.g., one) of the anti-PD-1 antibodies or their antigen-binding moieties described herein and any (e.g., one) of the anti-TIM-3 antibodies or their antigen-binding moieties described herein. In some embodiments, the combination therapy comprises any (e.g., one) of the anti-TIM-3 antibodies or their antigen-binding moieties described herein and any (e.g., one) of the anti-LAG-3 antibodies or their antigen-binding moieties described herein. In certain embodiments, the combination therapy comprises any (e.g., one) of the anti-PD-1 antibodies or their antigen-binding moieties described herein and any (e.g., one) of the anti-TIM-3 antibodies or their antigen-binding moieties described herein and any (e.g., one) of the anti-LAG-3 antibodies or their antigen-binding moieties described herein. The combination therapy may take the form of a treatment method using the antibody or antigen-binding portion, or a pharmaceutical composition containing the antibody or antigen-binding portion.

[0244] In certain embodiments, the combination therapy or composition of the present invention comprises anti-PD-1 antibody 12819 and anti-TIM-3 antibody 15086.17145. In certain embodiments, the combination therapy or composition of the present invention comprises anti-PD-1 antibody 12819 and anti-LAG-3 antibody 15532. In certain embodiments, the combination therapy or composition of the present invention comprises anti-PD-1 antibody 12819, anti-TIM-3 antibody 15086.17145, and anti-LAG-3 antibody 15532.

[0245] In a particular embodiment, the combination therapy or composition of the present invention is - Anti-PD-1 antibodies or their antigen-binding moieties containing the H-CDR1-3 and L-CDR1-3 amino acid sequences of SEQ ID NOs. 228-233, respectively; and anti-TIM-3 antibodies or their antigen-binding moieties containing the H-CDR1-3 and L-CDR1-3 amino acid sequences of SEQ ID NOs. 8-13, respectively; - Anti-PD-1 antibodies or their antigen-binding moieties containing the VH and VL amino acid sequences of SEQ ID NOs. 226 and 227, respectively; and anti-TIM-3 antibodies or their antigen-binding moieties containing the VH and VL amino acid sequences of SEQ ID NOs. 7 and 4, respectively; or - Anti-PD-1 antibody containing HC with amino acid sequences of SEQ ID NOs. 226 and 375, and LC with amino acid sequences of SEQ ID NOs. 227 and 379; and anti-TIM-3 antibody containing HC with amino acid sequences of SEQ ID NOs. 7 and 377, and LC with amino acid sequences of SEQ ID NOs. 4 and 378. Includes.

[0246] In a particular embodiment, the combination therapy or composition of the present invention is - Anti-PD-1 antibodies or their antigen-binding moieties containing the H-CDR1-3 and L-CDR1-3 amino acid sequences of SEQ ID NOs. 228-233, respectively; and anti-LAG-3 antibodies or their antigen-binding moieties containing the H-CDR1-3 and L-CDR1-3 amino acid sequences of SEQ ID NOs. 318-323, respectively; - Anti-PD-1 antibodies or their antigen-binding moieties containing the VH and VL amino acid sequences of SEQ ID NOs. 226 and 227, respectively; and anti-LAG-3 antibodies or their antigen-binding moieties containing the VH and VL amino acid sequences of SEQ ID NOs. 316 and 317, respectively; or - Anti-PD-1 antibodies containing HC with amino acid sequences of SEQ ID NOs. 226 and 375, and LC with amino acid sequences of SEQ ID NOs. 227 and 379; and anti-LAG-3 antibodies containing HC with amino acid sequences of SEQ ID NOs. 316 and 375, and LC with amino acid sequences of SEQ ID NOs. 317 and 378. Includes.

[0247] In a particular embodiment, the combination therapy or composition of the present invention is - Anti-TIM-3 antibodies or their antigen-binding moieties containing the H-CDR1-3 and L-CDR1-3 amino acid sequences of SEQ ID NOs. 8-13, respectively; and anti-LAG-3 antibodies or their antigen-binding moieties containing the H-CDR1-3 and L-CDR1-3 amino acid sequences of SEQ ID NOs. 318-323, respectively; - Anti-TIM-3 antibodies or their antigen-binding moieties containing the VH and VL amino acid sequences of SEQ ID NOs. 7 and 4, respectively; and anti-LAG-3 antibodies or their antigen-binding moieties containing the VH and VL amino acid sequences of SEQ ID NOs. 316 and 317, respectively; or - Anti-TIM-3 antibody containing HC with amino acid sequences of SEQ ID NOs. 7 and 377, and LC with amino acid sequences of SEQ ID NOs. 4 and 378; and anti-LAG-3 antibody containing HC with amino acid sequences of SEQ ID NOs. 316 and 375, and LC with amino acid sequences of SEQ ID NOs. 317 and 378. Includes.

[0248] In a particular embodiment, the combination therapy or composition of the present invention is - Anti-PD-1 antibodies or their antigen-binding moieties containing the H-CDR1-3 and L-CDR1-3 amino acid sequences of SEQ ID NOs. 228-233, respectively; anti-TIM-3 antibodies or their antigen-binding moieties containing the H-CDR1-3 and L-CDR1-3 amino acid sequences of SEQ ID NOs. 8-13, respectively; and anti-TIM-3 antibodies or their antigen-binding moieties containing the H-CDR1-3 and L-CDR1-3 amino acid sequences of SEQ ID NOs. 318-323; - Anti-PD-1 antibodies or their antigen-binding moieties containing the VH and VL amino acid sequences of SEQ ID NOs. 226 and 227, respectively; anti-TIM-3 antibodies or their antigen-binding moieties containing the VH and VL amino acid sequences of SEQ ID NOs. 7 and 4, respectively; and anti-LAG-3 antibodies or their antigen-binding moieties containing the VH and VL amino acid sequences of SEQ ID NOs. 316 and 317, respectively; or - Anti-PD-1 antibody containing HC with amino acid sequences of SEQ ID NOs. 226 and 375, and LC with amino acid sequences of SEQ ID NOs. 227 and 379; anti-TIM-3 antibody containing HC with amino acid sequences of SEQ ID NOs. 7 and 377, and LC with amino acid sequences of SEQ ID NOs. 4 and 378; and anti-LAG-3 antibody containing HC with amino acid sequences of SEQ ID NOs. 316 and 375, and LC with amino acid sequences of SEQ ID NOs. 317 and 378 Includes.

[0249] In some embodiments, one or all of the antibodies in the combination therapy or composition may be IgG, for example, IgG1 or IgG2.

[0250] Multispecific binding molecules In a further embodiment, the present invention is - The anti-PD-1 antibody and the anti-TIM-3 antibody described herein; - The anti-PD-1 antibody and the anti-LAG-3 antibody described herein; or - Anti-TIM-3 antibody and anti-LAG-3 antibody as described herein The present invention provides a polyspecific conjugating molecule having binding specificity (e.g., including an antigen-binding moiety, e.g., an antigen-binding moiety containing six CDRs). In some embodiments, the anti-PD-1 antibody, anti-TIM-3 antibody, and / or anti-LAG-3 antibody are selected from the antibodies described herein. In certain embodiments, the polyspecific conjugating molecule has the binding specificity of the anti-PD-1 antibody, anti-TIM-3 antibody, and anti-LAG-3 antibody described herein. Polyspecific conjugating molecules are known in the art, and examples of different types of polyspecific conjugating molecules are shown elsewhere herein. Such polyspecific (e.g., bispecific or trispecific) conjugating molecules are included in the combination therapy of the present invention.

[0251] In a further embodiment, the present invention provides combination therapy with two or more of the following: a bispecific conjugate molecule targeting PD-1, a bispecific conjugate molecule targeting TIM-3, and a bispecific conjugate molecule targeting LAG-3. The bispecific conjugate molecules targeting PD-1, TIM-3, or LAG-3 may have the binding specificity of an antibody targeting the aforementioned antigen as described herein, and the binding specificity of another antibody targeting the same antigen (e.g., another antibody as described herein) or an antibody targeting a different protein, such as another immune checkpoint protein, a cancer antigen, or another cell surface molecule whose activity mediates a disease condition such as cancer. Such bispecific conjugate molecules are known in the art, and examples of different types of bispecific conjugate molecules are shown elsewhere herein.

[0252] nucleic acid molecules and vectors Similarly, nucleic acid molecules and sequences encoding the anti-PD-1, anti-TIM-3, and / or anti-LAG-3 antibodies or their antigen-binding moieties are also described herein. In some embodiments, different nucleic acid molecules encode the heavy and light chain amino acid sequences of the anti-PD-1 antibody or its antigen-binding moiety, the anti-TIM-3 antibody or its antigen-binding moiety, or the anti-LAG-3 antibody or its antigen-binding moiety. In other embodiments, the same nucleic acid molecule encodes the heavy and light chain amino acid sequences of the anti-PD-1 antibody or its antigen-binding moiety, the anti-TIM-3 antibody or its antigen-binding moiety, or the anti-LAG-3 antibody or its antigen-binding moiety.

[0253] References to nucleotide sequences, unless otherwise specified, include their complements. Therefore, references to nucleic acids with a particular sequence should be understood to include their complementary strands with complementary sequences. As used herein, the term "polynucleotide" means a polymer of nucleotides of at least 10 bases in length, which may be ribonucleotides, deoxynucleotides, or modified forms of either type of nucleotide. This term includes single-stranded and double-stranded forms.

[0254] In some embodiments, the nucleotide sequence is one or more nucleotide sequences listed herein, for example, SEQ ID NOs: 1, 2, 5, 6, 14, 15, 24, 25, 34, 35, 44, 45, 54, 55, 64, 65, 74, 75, 84, 85, 94, 95, 104, 105, 114, 115, 124, 125, 134, 135, 144, 145, 154, 155, 164, 165, 174, 175, 184, 185, 194, 195, 204, 205, 214, The nucleotide sequence encoding an amino acid sequence selected from the group consisting of 215, 224, 225, 234, 235, 244, 245, 254, 255, 264, 265, 274, 275, 284, 285, 294, 295, 304, 305, 314, 315, 324, 325, 334, 335, 344, 345, 354, 355, 364, 365, or 391 is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical. In the context of nucleic acid sequences, the term "percent sequence identity" refers to the residues that are identical in two sequences when aligned to maximize matching. The length of a sequence identity comparison can be at least about 9 nucleotides, typically at least about 18 nucleotides, more typically at least about 24 nucleotides, usually at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36, 48, or more nucleotides. There are many different algorithms known in the art that can be used to measure nucleotide sequence identity. For example, polynucleotide sequences can be compared using FASTA, Gap, or Bestfit, programs in Wisconsin Package version 10.0, Genetics Computer Group (GCG), Madison, Wisconsin.For example, FASTA, including programs FASTA2 and FASTA3, provides the best overlap region alignment and percent sequence identity between the query sequence and the search sequence (see, for example, Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219 (2000); Pearson, Methods Enzymol. 266:227-258 (1996); and Pearson, J. Mol. Biol. 276:71-84 (1998), which are incorporated herein by reference in their entirety). Unless otherwise specified, the default parameters of a particular program or algorithm are used. For example, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (word size 6 and NOPAM factor with respect to the score matrix), or using Gap with its default parameters provided in GCG version 6.1, which is incorporated herein by reference in its entirety.

[0255] In some embodiments, nucleic acid molecules are SEQ ID NOs: 1, 2, 5, 6, 14, 15, 24, 25, 34, 35, 44, 45, 54, 55, 64, 65, 74, 75, 84, 85, 94, 95, 104, 105, 114, 115, 124, 125, 134, 135, 144, 145, 154, 155, 164, 165, 174, 175, 184, 185, 194, 195, 204, It contains one or more nucleotide sequences selected from the group consisting of 205, 214, 215, 224, 225, 234, 235, 244, 245, 254, 255, 264, 265, 274, 275, 284, 285, 294, 295, 304, 305, 314, 315, 324, 325, 334, 335, 344, 345, 354, 355, 364, 365, or 391.

[0256] In any of the embodiments described above, the nucleic acid molecule may be isolated. Nucleic acid molecules encoding the heavy and / or light chains or their antigen-binding moieties of the antibodies described herein can be isolated from any source that produces such antibodies or moieties. In various embodiments, the nucleic acid molecule is isolated from antibody-expressing B cells isolated from animals immunized with PD-1, TIM-3, or LAG-3 antigens, or from immortalized cells produced from such B cells. Methods for isolating nucleic acids encoding antibodies are well known in the art. mRNA may be isolated and used to produce cDNA for use in polymerase chain reaction (PCR) or cDNA cloning of antibody genes. In certain embodiments, the nucleic acid molecules described herein can be synthesized rather than isolated.

[0257] In some embodiments, the nucleic acid molecules described herein may include a nucleotide sequence encoding a VH domain or antigen-binding moiety from an antibody described herein, in-frame conjugated to a nucleotide sequence encoding a heavy chain constant region from any origin. Similarly, the nucleic acid molecules described herein may include a nucleotide sequence encoding a VL domain or antigen-binding moiety from an antibody described herein, in-frame conjugated to a nucleotide sequence encoding a light chain constant region from any origin.

[0258] In a further embodiment, nucleic acid molecules encoding variable domains of the heavy (VH) and / or light (VL) chains may be "converted" into a full-length antibody gene. In one embodiment, a nucleic acid molecule encoding the VH or VL domain is converted into a full-length antibody gene by inserting it into an expression vector already encoding a heavy-chain constant (CH) or light-chain constant (CL) region, respectively, such that the VH segment is operably ligated to a CH segment in the vector and / or the VL segment is operably ligated to a CL segment in the vector. In another embodiment, a nucleic acid molecule encoding the VH and / or VL domains is converted into a full-length antibody gene by ligating, for example, the nucleic acid molecule encoding the VH and / or VL domains to a nucleic acid molecule encoding the CH and / or CL region using standard molecular biology techniques. The full-length nucleic acid molecules encoding the heavy and / or light chains may then be expressed from the cells into which they are introduced to isolate the antibody.

[0259] Nucleic acid molecules may be used to recombinantly express large quantities of antibodies. Nucleic acid molecules may also be used to produce the chimeric antibodies, bispecific antibodies, single-chain antibodies, immunoadhesins, diabodies, mutant antibodies, and antibody derivatives described herein.

[0260] Vectors suitable for expressing one or both of the anti-PD-1 antibody or its antigen-binding moiety, the anti-TIM-3 antibody or its antigen-binding moiety, and / or the anti-LAG-3 antibody or its antigen-binding moiety are also described herein. As used herein, the term “vector” means a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. In some embodiments, the vector is a plasmid, i.e., a circular double-stranded piece of DNA into which an additional DNA segment may be ligated. In some embodiments, the vector is a viral vector into which an additional DNA segment may be ligated within a viral genome. In some embodiments, the vector may be capable of autonomous replication in the host cell into which it is introduced (e.g., bacterial vectors with bacterial replication origins and episomal mammalian vectors). In other embodiments, the vector (e.g., non-episomal mammalian vectors), once introduced into a host cell, may be integrated into the host cell’s genome and thereby replicate with the host genome. Furthermore, certain vectors may be capable of directing the expression of a gene to which it is operably ligated. Such vectors are referred herein as “recombinant expression vectors” (or simply “expression vectors”).

[0261] In some embodiments, the vector comprises a nucleic acid molecule or its antigen-binding moiety encoding the heavy chain, light chain, or both of the heavy and light chains of an antibody described herein. In some embodiments, the vector comprises a nucleic acid molecule encoding a fusion protein, a modified antibody, an antibody fragment, and its probe.

[0262] In some embodiments, anti-PD-1, anti-TIM-3, or anti-LAG-3 antibodies or antigen-binding moieties are expressed by inserting the partially or full-length light and heavy chain encoding DNA obtained above into an expression vector so that the gene is operably linked to the required expression regulatory elements, such as transcription and translation regulatory elements. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAVs), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, cosmids, YACs, and EBV-derived episomes. The antibody-coding sequence may be ligated into the vector so that the transcription and translation regulatory elements within the vector perform their intended function of regulating the transcription and translation of the antibody-coding sequence. The expression vector and expression regulatory elements may be selected to be compatible with the expression host cell used. The antibody light chain encoding sequence and antibody heavy chain encoding sequence may be inserted into separate vectors and operably linked to the same or different expression regulatory elements (e.g., promoters). In one embodiment, both coding sequences may be inserted into the same expression vector and operably ligated to the same expression regulatory sequence (e.g., a common promoter) or to different expression regulatory sequences (e.g., promoters) to separate the same expression regulatory sequence (e.g., promoter). The antibody coding sequence may be inserted into the expression vector by a standard method (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt-end ligation if no restriction sites exist).

[0263] A convenient vector is a vector encoding a functionally complete human CH or CL immunoglobulin sequence, having appropriate restriction sites that have been engineered to allow for the easy insertion and expression of any VH or VL sequence as described above. The HC and LC coding genes in such vectors may contain intronic sequences that increase the overall antibody protein yield by stabilizing the associated mRNA. The intronic sequences are adjacent to splice donor and splice acceptor sites that determine where RNA splicing occurs. The location of the intronic sequences may be either a variable or constant region of the antibody chain, or both if a large number of introns are used. Polyadenylation and transcription termination may occur at a native chromosomal site downstream of the coding region. Recombinant expression vectors may also encode a signal peptide that facilitates the secretion of the antibody chain from host cells. The antibody chain gene may be cloned in the vector such that the signal peptide is in-frame ligated to the amino terminus of the immunoglobulin chain. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0264] In addition to antibody chain genes, recombinant expression vectors may have regulatory sequences that control the expression of antibody chain genes in host cells. Those skilled in the art will recognize that the design of expression vectors, including the selection of regulatory sequences, may depend on factors such as the selection of host cells to be transformed and the desired protein expression level. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as retroviral LTRs, cytomegalovirus (CMV) (CMV promoter / enhancer, etc.), Simian virus 40 (SV40) (SV40 promoter / enhancer), adenovirus (e.g., adenovirus major late promoter (AdMLP)), polyoma, and promoters and / or enhancers derived from strong mammalian promoters such as native immunoglobulin and actin promoters. For a more detailed description of viral regulatory elements and their sequences, see, for example, U.S. Patents 5,168,062, 4,510,245, and 4,968,615. Methods for expressing antibodies in plants, including descriptions of promoters and vectors, and plant transformations, are well known in the art. See, for example, U.S. Patent No. 6,517,529. Methods for expressing polypeptides in bacterial or fungal cells, such as yeast cells, are also well known in the art.

[0265] In addition to antibody chain genes and regulatory sequences, recombinant expression vectors may have additional sequences such as sequences that regulate vector replication in host cells (e.g., replication origins) and selectable marker genes. Selectable marker genes facilitate the selection of host cells into which the vector has been introduced (see, for example, U.S. Patents 4,399,216, 4,634,665, and 5,179,017). For example, selectable marker genes typically confer resistance to drugs such as G418, hygromycin, or methotrexate to host cells into which the vector has been introduced. Examples of selectable marker genes include the dihydrofolate reductase (DHFR) gene (for methotrexate selection / amplification in dhfr-host cells), the neo gene (for G418 selection), and the glutamate synthetase gene.

[0266] As used herein, the term “regulatory sequence” means a polynucleotide sequence that is necessary for the expression and processing of the coding sequence it ligates. Regulatory sequences include appropriate transcription start, stop, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that increase protein stability; and sequences that, if desired, increase protein secretion. The nature of such regulatory sequences varies depending on the host organism; in prokaryotes, such regulatory sequences generally include promoters, ribosome binding sites, and transcription stop sequences, while in eukaryotes, such regulatory sequences generally include promoters and transcription stop sequences. The term “regulatory sequence” is intended to include at least all components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0267] Host cells and methods for antibody and antibody composition production Similarly, methods for producing the combination therapy (e.g., composition) of the present invention are also described. One embodiment relates to a method for producing an antibody as described herein, comprising providing a recombinant host cell capable of expressing an antibody, culturing the host cell under conditions suitable for antibody expression, and isolating the obtained antibody. The antibody produced by such expression in such recombinant host cells is referred to herein as a “recombinant antibody.” Progeny cells of such host cells and antibodies produced by such progeny cells are also described.

[0268] As used herein, the term “recombinant host cell” (or simply “host cell”) means a cell into which a recombinant expression vector has been introduced. A host cell may include, for example, one or more vectors described herein. A host cell may include the nucleotide sequence encoding the heavy chain or its antigen-binding portion, the nucleotide sequence encoding the light chain or its antigen-binding portion, or both, of the anti-PD-1, anti-TIM-3, and / or anti-LAG-3 antibodies or their antigen-binding portions described herein. “Recombinant host cell” and “host cell” should be understood to mean not only a specific target cell but also the offspring of such a cell. Since certain modifications may occur in subsequent generations due to either mutation or environmental influences, such offspring are not actually identical to the parent cell, but are still included in the scope of the term “host cell” as used herein.

[0269] Nucleic acid molecules encoding anti-PD-1, anti-TIM-3, and / or anti-LAG-3 antibodies or their antigen-binding moieties, and vectors containing these nucleic acid molecules, can be used for transfection of suitable mammalian, plant, bacterial, or yeast host cells. Transformation can be carried out by any known method for introducing polynucleotides into host cells. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran transfection, calcium phosphate precipitation, polyblen transfection, protoplast fusion, electroporation, liposome encapsulation of polynucleotides, and direct microinjection of DNA into the nucleus. Furthermore, nucleic acid molecules may be introduced into mammalian cells by viral vectors. Methods for transforming cells are well known in the art. See, for example, U.S. Patents 4,399,216, 4,912,040, 4,740,461, and 4,959,455. Methods for transforming plant cells, including transformation by Agrobacterium, bioristic transformation, direct injection, electroporation, and viral transformation, are well known in the art. Similarly, methods for transforming bacterial and yeast cells are also well known in the art.

[0270] Mammalian cell lines available as hosts for expression are well known in the art, including many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, among others, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, 293 Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and many other cell lines. Particularly preferred cell lines are selected by determining which cell lines have high expression levels. Other cell lines that can be used are insect cell lines, e.g., Sf9 or Sf21 cells. When a recombinant expression vector encoding an antibody gene is introduced into mammalian host cells, the antibody is produced by culturing the host cells for a sufficient period to allow antibody expression in the host cells, or more preferably to allow secretion of the antibody into the culture medium in which the host cells grow. Antibodies can be recovered from the culture medium using standard protein purification methods. Plant cells include, for example, Nicotiana, Arabidopsis, duckweed, maize, wheat, and potato. Bacterial host cells include E. coli and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Pichia pastoris.

[0271] Furthermore, the expression of the antibodies described herein or their antigen-binding moieties from producing cell lines can be enhanced using a number of known techniques. For example, glutamine synthetase gene expression systems (GS systems) are a common approach for enhancing expression under certain conditions. GS systems have been discussed in whole or in part in connection with European Patent Nos. 0216846, 0256055, 0323997, and 0338841.

[0272] Antibodies expressed by different cell lines or in transgenic animals may have different glycosylation patterns. However, all antibodies encoded by the nucleic acid molecules provided herein, or containing the amino acid sequences provided herein, are part of the antibodies of the present invention, regardless of the glycosylation state of the antibody, and more generally, regardless of the presence or absence of post-translational modifications.

[0273] In some embodiments, the present invention relates to a method for producing an antibody composition comprising an anti-PD-1 antibody and an anti-TIM-3 antibody, - To provide first and second host cells, in which a first host cell can express the anti-PD-1 antibody described herein, and a second host cell can express the anti-TIM-3 antibody described herein. - Culture the first and second host cells under conditions suitable for the expression of anti-PD-1 antibody and anti-TIM-3 antibody, and - Isolate the obtained antibodies. Regarding methods including

[0274] In some embodiments, the present invention relates to a method for producing an antibody composition comprising an anti-PD-1 antibody and an anti-LAG-3 antibody, - To provide first and second host cells, in which a first host cell can express the anti-PD-1 antibody described herein, and a second host cell can express the anti-LAG-3 antibody described herein. - Culture the first and second host cells under conditions suitable for the expression of anti-PD-1 antibody and anti-LAG-3 antibody, and - Isolate the obtained antibodies. Regarding methods including

[0275] In some embodiments, the present invention relates to a method for producing an antibody composition comprising an anti-TIM-3 antibody and an anti-LAG-3 antibody, - To provide first and second host cells, in which a first host cell can express the anti-TIM-3 antibody described herein, and a second host cell can express the anti-LAG-3 antibody described herein. - Culture the first and second host cells under conditions suitable for the expression of anti-TIM-3 antibody and anti-LAG-3 antibody, and - Isolate the obtained antibodies. Regarding methods including

[0276] In some embodiments, the present invention relates to a method for producing an antibody composition comprising an anti-PD-1 antibody, an anti-TIM-3 antibody, and an anti-LAG-3 antibody, - To provide first, second, and third host cells, the first host cell capable of expressing the anti-PD-1 antibody described herein, the second host cell capable of expressing the anti-TIM-3 antibody described herein, and the third host cell capable of expressing the anti-LAG-3 antibody described herein. - Culturing the first, second, and third host cells under conditions suitable for the expression of anti-PD-1 antibody, anti-TIM-3 antibody, and anti-LAG-3 antibody, and - Isolate the obtained antibodies. Regarding methods including

[0277] Regarding the production of the antibody composition of the present invention, antibodies against different targets may be produced individually, that is, each antibody may be produced in a separate bioreactor, or the individual antibodies may be produced together in a single bioreactor. When the antibody composition is produced in more than one bioreactor, a purified antibody composition can be obtained by pooling antibodies obtained from individually purified supernatants from each bioreactor. Various approaches for the production of polyclonal antibody compositions in multiple bioreactors, such as combining cell lines or antibody preparations at a later point in the upstream process or before or during downstream processing, are described in PCT International Publication No. 2009 / 129814.

[0278] When producing individual antibodies in a single bioreactor, this may be carried out as described, for example, in PCT International Publication No. 2004 / 061104 or International Publication No. 2008 / 145133. The method described in International Publication No. 2004 / 061104 is based on site-specific incorporation of antibody coding sequences into the genomes of individual host cells, while the method described in International Publication No. 2008 / 145133 involves an alternative approach that uses random incorporation to produce antibodies in a single bioreactor.

[0279] Further information regarding suitable methods for preparing the antibody composition of the present invention can be found in PCT International Publication No. 2012 / 059857 and International Publication No. 2013 / 164689.

[0280] This invention also: - At least one anti-PD-1 antibody or its antigen-binding moiety as described herein, and at least one anti-TIM-3 antibody or its antigen-binding moiety as described herein; - At least one anti-PD-1 antibody or its antigen-binding moiety as described herein, and at least one anti-LAG-3 antibody or its antigen-binding moiety as described herein; - At least one anti-TIM-3 antibody or its antigen-binding moiety as described herein, and at least one anti-LAG-3 antibody or its antigen-binding moiety as described herein; or - At least one anti-PD-1 antibody or its antigen-binding moiety as described herein, at least one anti-TIM-3 antibody or its antigen-binding moiety as described herein, and at least one anti-LAG-3 antibody or its antigen-binding moiety as described herein We also provide polyclonal cell lines that produce [the substance].

[0281] The present invention also provides a method for producing the above-mentioned polyclonal cell line, comprising providing a host cell each containing a nucleotide sequence encoding a heavy chain or its antigen-binding portion, and a nucleotide sequence encoding a light chain or its antigen-binding portion, of at least one antibody or portion produced by the polyclonal cell line.

[0282] This invention also: - A nucleotide sequence encoding the heavy chain or its antigen-binding portion of the anti-PD-1 antibody described herein, a nucleotide sequence encoding the light chain or its antigen-binding portion, or both; and a nucleotide sequence encoding the heavy chain or its antigen-binding portion of the anti-TIM-3 antibody described herein, a nucleotide sequence encoding the light chain or its antigen-binding portion, or both; - A nucleotide sequence encoding the heavy chain or its antigen-binding portion of an anti-PD-1 antibody as described herein, a nucleotide sequence encoding the light chain or its antigen-binding portion, or both; and a nucleotide sequence encoding the heavy chain or its antigen-binding portion of an anti-LAG-3 antibody as described herein, a nucleotide sequence encoding the light chain or its antigen-binding portion, or both; - A nucleotide sequence encoding the heavy chain or its antigen-binding portion of the anti-TIM-3 antibody described herein, a nucleotide sequence encoding the light chain or its antigen-binding portion, or both; and a nucleotide sequence encoding the heavy chain or its antigen-binding portion of the anti-LAG-3 antibody described herein, a nucleotide sequence encoding the light chain or its antigen-binding portion, or both; or - A nucleotide sequence encoding the heavy chain or its antigen-binding portion of the anti-PD-1 antibody described herein, a nucleotide sequence encoding the light chain or its antigen-binding portion, or both; a nucleotide sequence encoding the heavy chain or its antigen-binding portion of the anti-TIM-3 antibody described herein, a nucleotide sequence encoding the light chain or its antigen-binding portion, or both; and a nucleotide sequence encoding the heavy chain or its antigen-binding portion of the anti-LAG-3 antibody described herein, a nucleotide sequence encoding the light chain or its antigen-binding portion, or both. It also provides host cells containing these cells.

[0283] Pharmaceutical composition Another aspect of the present invention is the active ingredient (for example, as the sole active ingredient): - At least one anti-PD-1 antibody or its antigen-binding moiety as described herein and at least one anti-TIM-3 antibody or its antigen-binding moiety as described herein; - At least one anti-PD-1 antibody or its antigen-binding moiety as described herein and at least one anti-LAG-3 antibody or its antigen-binding moiety as described herein; - At least one anti-TIM-3 antibody or its antigen-binding moiety as described herein and at least one anti-LAG-3 antibody or its antigen-binding moiety as described herein; or - At least one anti-PD-1 antibody or its antigen-binding moiety as described herein, at least one anti-TIM-3 antibody or its antigen-binding moiety as described herein, and at least one anti-LAG-3 antibody or its antigen-binding moiety as described herein The pharmaceutical composition includes the following: In some embodiments, the pharmaceutical composition includes a polyspecific binding molecule (for example, the anti-PD-1 antibody described herein and the anti-TIM-3 or anti-LAG-3 antibody described herein; or a polyspecific binding molecule having binding specificity for the anti-PD1 antibody, anti-TIM-3 antibody, and anti-LAG-3 antibody described herein).

[0284] In some embodiments, the pharmaceutical composition may further comprise one or more additional antibodies that target one or more relevant cell surface receptors, such as one or more cancer-related receptors.

[0285] In some embodiments, the pharmaceutical composition is intended for the improvement, prevention, and / or treatment of disorders, diseases, or conditions that are improved or whose progression is slowed by the modulation of PD-1, TIM-3, and / or LAG-3. In some embodiments, the pharmaceutical composition is intended for the improvement, prevention, and / or treatment of cancer. In some embodiments, the pharmaceutical composition is intended for the activation of the immune system.

[0286] The ratio of antibodies or their antigen-binding moieties in the pharmaceutical compositions of the present invention (or the individual antibodies or portions described herein administered simultaneously, sequentially, or individually) is often a ratio in which the antibodies are administered in equal amounts, but this is not always necessary. Thus, compositions of the present invention comprising anti-PD-1 antibody and anti-TIM-3 antibody, anti-PD-1 antibody and anti-LAG-3 antibody, or anti-TIM-3 antibody and anti-LAG-3 antibody may contain the antibodies in approximately a 1:1 ratio. Compositions of the present invention comprising anti-PD-1 antibody, anti-TIM-3 antibody, and anti-LAG-3 antibody may contain the antibodies in approximately a 1:1:1 ratio (i.e., in equal amounts). However, it may be desirable to use unequal amounts of different antibodies based on the characteristics of each antibody. The suitable ratio of different antibodies in the composition of the present invention may be determined as described in PCT International Publication No. 2010 / 040356, which describes a method for identifying and selecting the optimal stoichiometric ratio between compounds in a combinatorial drug product, such as a polyclonal antibody composition, in order to obtain a combinatorial drug with optimal potency and efficacy.

[0287] Generally, the pharmaceutical compositions described herein are suitable for administration as formulations in association with one or more pharmaceutically acceptable excipients, such as those described below.

[0288] The term “excipient” is used herein to describe any component other than the compounds of the present invention. The choice of excipient depends to a great extent on factors such as the specific mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. As used herein, “pharmaceutically acceptable excipient” includes any physiologically compatible solvent, dispersion medium, coating, antimicrobial and antifungal agents, isotonic and absorption retardants, etc. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, etc., and combinations thereof. Often, it is preferable to include isotonic agents, such as sugars, polyhydric alcohols, such as mannitol, sorbitol, or sodium chloride in the composition. Further examples of pharmaceutically acceptable substances are wetting agents or trace amounts of auxiliary substances, such as wetting agents or emulsifiers, preservatives or buffers that enhance the shelf life or efficacy of antibodies.

[0289] The pharmaceutical composition and method for preparing the present invention will be readily apparent to those skilled in the art. Such compositions and methods for preparing them can be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995). The pharmaceutical composition is preferably manufactured in accordance with GMP (Good Manufacturing Practice).

[0290] The pharmaceutical compositions of the present invention may be prepared, packaged, or sold in bulk, as single unit doses, or as multiple single unit doses. As used herein, “unit dose” refers to a distinct amount of the pharmaceutical composition containing a predetermined amount of the active ingredient. The amount of the active ingredient may be equal to the dose of the active ingredient generally administered to a subject, or equal to a convenient fraction of such a dose, such as half or one-third of such a dose.

[0291] Any method of administering peptides, proteins, or antibodies that is accepted in the art can be appropriately used for the antibody and antigen-binding moieties described herein.

[0292] The pharmaceutical compositions of the present invention are typically suitable for parenteral administration. As used herein, “parenteral administration” of a pharmaceutical composition includes administration of the pharmaceutical composition through tissue intrusion, any route of administration characterized by physical intrusion of the tissue of interest, and thereby generally resulting in direct administration into the bloodstream, muscle, or internal organs. For this reason, parenteral administration includes, but is not limited to, administration of the pharmaceutical composition by injection, application of the composition through surgical incision, application of the composition through non-surgical wounds penetrating tissue, etc. In particular, parenteral administration is intended to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, ​​intracranial, intratumoral and synovial injection or infusion, as well as renal dialysis infusion techniques. Local perfusion is also intended. Certain embodiments include intravenous and subcutaneous routes.

[0293] Formulations of pharmaceutical compositions suitable for parenteral administration typically include an active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in dosage forms suitable for bolus or continuous administration. Injectable formulations may be prepared, packaged, or sold in unit-dosage forms, such as ampoules or multi-dose containers containing preservatives. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous media, pastes, etc. Such formulations may further include one or more additional components, including, but not limited to, suspensions, stabilizers, or dispersants. In one embodiment of a formulation for parenteral administration, the active ingredient is reconstituted with a suitable medium (e.g., sterile pyrogen-free water), and the reconstituted composition is then provided in a dry (i.e., powder or granule) dosage form for parenteral administration. Parenteral formulations also include aqueous solutions that may contain excipients such as salts, carbohydrates, and buffers (preferably pH 3-9), but for some applications, they may more appropriately be formulated as sterile non-aqueous solutions or as desiccants used with a suitable medium such as sterile pyrotherm-free water. Exemplary parenteral formulations include solutions or suspensions in sterile aqueous solutions, such as aqueous solutions of propylene glycol or dextrose. Such dosage forms may be appropriately buffered as needed. Other useful parenterally administered formulations include formulations containing active ingredients in microcrystalline or liposomal preparations. Formulations for parenteral administration may be formulated to have immediate and / or controlled release. Controlled release formulations include delayed, sustained, pulsed, controlled, targeted, and programmed release.

[0294] For example, in one embodiment, a sterile injectable solution can be prepared by incorporating the required amount of the composition with one or a combination of the components listed above into a suitable solvent, and then sterilizing by filtration as necessary. Generally, dispersants are prepared by incorporating the active compound into a sterile medium containing a basic dispersion medium and other necessary components from the components listed above. In the case of sterile powders for preparing sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying, which produce a powder of the active ingredient plus any additional desired components from a pre-filtered, sterilized solution. Appropriate fluidity of the solution can be maintained by using a coating such as lecithin, by maintaining the required particle size in the case of dispersants, and by using a surfactant. Sustained absorption of the injectable composition can be achieved by including absorption-delaying agents, such as monostearate and gelatin, in the composition, and / or by using a release-controlled coating (e.g., a sustained-release coating).

[0295] Therapeutic use of combination therapy and composition of the present invention In one embodiment, the combination therapies and compositions of the present invention are used to enhance or activate the immune system in a patient (e.g., a human) who needs them. In some embodiments, the patient is in an immunosuppressed state. In some embodiments, a physician can enhance the anti-cancer activity of the patient's own immune system by administering the combination therapies or compositions of the present invention alone or in combination with other therapeutic agents (sequentially or simultaneously). The combination therapies or compositions modulate the activity of PD-1, TIM-3, and / or LAG-3 in immune cells, thereby resulting in enhanced anti-cancer immunity. In certain embodiments, the combination therapies and compositions of the present invention are intended for use in the treatment of cancers originating from tissues such as the skin, lung, intestine, colon, ovaries, brain, prostate, kidney, soft tissue, hematopoietic system, head and neck, liver, bladder, breast, stomach, uterus, and pancreas, as well as any cancer or other condition that depends on PD-1, TIM-3, and / or LAG-3 activity, and / or in which the patient expresses or overexpresses any of these ligands.

[0296] In some embodiments, cancers treated by the combination therapy and compositions of the present invention include, for example, melanoma (e.g., advanced melanoma or metastatic melanoma), non-small cell lung cancer, head and neck squamous cell carcinoma, renal cell carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, glioblastoma, glioma, pulmonary squamous cell carcinoma, small cell lung cancer, hepatocellular carcinoma, bladder cancer, upper urinary tract urothelial carcinoma, esophageal cancer, gastroesophageal junction cancer, stomach cancer, liver cancer, colon cancer, colorectal cancer, multiple myeloma, sarcoma, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, nasopharyngeal cancer, chronic lymphocytic leukemia, acute lymphoblastic leukemia, small lymphocytic lymphoma, ovarian cancer, gastrointestinal cancer, and Examples of cancers that may be included are primary abdominal cancer, ductal fallopian carcinoma, urothelial carcinoma, HTLV-associated T-cell leukemia / lymphoma, prostate cancer, genitourinary cancer, meningioma, adrenocortical carcinoma, gliosarcoma, fibrosarcoma, kidney cancer, breast cancer, pancreatic cancer, endometrial cancer, basal cell carcinoma, appendiceal cancer, bile duct cancer, salivary gland cancer, advanced Merkel cell carcinoma, diffuse large B lymphoma, follicular lymphoma, mesothelioma, neuroendocrine tumors, urological cancers, bone cancer, thoracic cancer, respiratory cancer, adenoid cystic carcinoma, cervical cancer, astrocytoma, chordoma, neuroblastoma, oral cancer, cutaneous squamous cell carcinoma, thyroid cancer, Kaposi's sarcoma, anal cancer, gallbladder cancer, thymic cancer, uterine cancer, and solid tumors. Cancers may be at, for example, early, intermediate, advanced, or metastatic stages.

[0297] In certain embodiments, cancers treated by the combination therapies and compositions of the present invention may include, for example, melanoma (e.g., advanced melanoma or unresectable or metastatic melanoma), non-small cell lung cancer (e.g., advanced non-small cell lung cancer), lung cancer, head and neck squamous cell carcinoma, glioblastoma (e.g., recurrent glioblastoma), gliosarcoma, Merkel cell carcinoma, fibrosarcoma, ovarian cancer, bladder cancer, renal cell carcinoma, colorectal cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, leukemia (e.g., acute myeloid leukemia), hematological malignancies, solid tumors (e.g., advanced or metastatic solid tumors), high-frequency MSI tumors, HPV and HIV-related malignancies, and tumors having BRAC1 and BRAC2 mutations.

[0298] In certain embodiments, the pharmaceutical compositions of the present invention are intended for the treatment of immune-mediated disorders such as psoriasis, systemic lupus erythematosus, MLS (sclerosis), Crohn's disease, diabetes mellitus, and / or ulcerative colitis.

[0299] In some embodiments, the combination therapy or composition is intended for use, for example, in treating viral and / or parasitic infections in which the pathogen inhibits the host immune response. For example, pathogens may include, for instance, HIV, hepatitis (types A, B, or C), human papillomavirus (HPV), lymphocytic choriomeningitis virus (LCMV), adenovirus, flavivirus, echovirus, rhinovirus, coxsackievirus, comovirus, respiratory polynuclear virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, human T-cell lymphotropic virus (HTLV), human cytomegalovirus (HCMV), dengue virus, molluscum contagiosum virus, poliovirus, rabies virus, John Cunningham (JC) virus, arbovirus encephalitis virus, simian immunodeficiency virus (SIV), influenza, herpes, giardia, malaria, leishmania, Staphylococcus aureus, or Pseudomonas aeruginosa.

[0300] In some embodiments, the combination therapies and compositions of the present invention may be used to treat patients who are immunocompromised (e.g., due to chemotherapy or radiation therapy) or at risk of immunocompromise.

[0301] In some embodiments, the combination therapies and compositions of the present invention may be used for ex vivo activation and enlargement of antigen-specific T cells.

[0302] In some embodiments, the patient may have been previously treated for a condition characterized by overexpression or overactivity of PD-1, TIM-3, and / or LAG-3, or any of their ligands (e.g., cancer, or immunodeficiency). For example, the patient may have been treated with one or more drugs targeting PD-1, TIM-3, and / or LAG-3, and may have acquired resistance to said drugs.

[0303] "To treat," "treating," and "treatment" refer to a method of reducing or eliminating at least one of a biological disorder and / or its associated symptoms. As used herein, "to reduce" a disease, disorder, or condition means to reduce the severity and / or frequency of the symptoms of the disease, disorder, or condition. Furthermore, references to "treatment" herein include references to curative, mitigating, and preventive treatments.

[0304] The "therapeutic dose" refers to the amount of a therapeutic agent administered that alleviates, to some extent, one or more symptoms of the disorder being treated. A therapeutic dose of an anticancer drug may result in, for example, tumor reduction, increased survival, disappearance of cancer cells, reduced disease progression, reversal of metastasis, or other clinical endpoints desirable to the medical professional.

[0305] In some embodiments, the antibody, antigen-binding moiety, or polyspecific binding molecule in the combination therapy of the present invention is administered in a single composition. In other embodiments, the antibody, antigen-binding moiety, or polyspecific molecule is administered in one or more compositions. For example, a combination therapy comprising an anti-PD-1 antibody, an anti-TIM-3 antibody, and an anti-LAG-3 antibody may involve the administration of a single composition containing all three antibodies, a composition containing two of the antibodies, a composition containing one of the antibodies, or individual compositions of each antibody. In the case of one or more compositions, the compositions may be administered simultaneously, sequentially, individually, or in any combination.

[0306] The combination therapies and compositions of the present invention may be administered alone or in combination with one or more other drugs or antibodies (as any combination thereof). The pharmaceutical compositions, methods, and uses of the present invention also encompass embodiments of combination (concurrent administration) with other activators, as detailed below.

[0307] In this specification, the terms “concurrent administration,” “concurrently administered,” and “combined use” with one or more other therapeutic agents used with reference to the combination therapy or composition of the present invention are defined as follows: - If such components are formulated together in a single dosage form that releases the components substantially simultaneously to the patient, such combination therapy (or its components) to a patient requiring treatment, or simultaneous administration of the composition and the therapeutic agent, - If such components are formulated separately into individual dosage forms that are taken substantially simultaneously by the patient, such combination therapy (or its components) to a patient requiring treatment, or substantially simultaneous administration of the composition and the therapeutic agent, thereby the components are released substantially simultaneously to the patient. - If such components are separately formulated into individual dosage forms to be taken by the patient at consecutive times with a significant time interval between each administration, such combination therapy (or its components) or composition to a patient requiring treatment and consecutive administration of the therapeutic agent, thereby releasing the components to the patient at substantially different times. - Such combination therapy (or its components) or composition to a patient in need of treatment, or a continuous administration of a therapeutic agent with such components, where the components are formulated together into a single dosage form that releases the components in a controlled manner, thereby releasing them to the patient simultaneously and / or at different times, in a continuous manner and / or overlapping manner, where each portion may be administered via the same route or different routes.

[0308] The combination therapies and compositions of the present invention may be administered without additional therapeutic intervention, i.e., as a single therapy (i.e., monotherapy). Alternatively, treatment with the combination therapy or composition of the present invention may include at least one additional therapeutic intervention, e.g., another immunostimulant, anticancer agent, antiviral agent, or vaccine (e.g., oncology vaccine). In some embodiments, the combination therapy or composition may be administered concurrently with or formulated together with another agent / drug for the treatment of cancer. Additional therapeutic interventions may include, for example, chemotherapeutic agents, antineoplastic agents, anti-angiogenic agents, different anti-cancer antibodies, and / or radiotherapy.

[0309] The efficacy of the combination therapies and compositions of the present invention can be further improved by combining them with agents known to induce terminal differentiation of cancer cells. Such compounds may be selected from the group consisting of, for example, retinoic acid, trans retinoic acid, cis retinoic acid, phenyl butyrate, nerve growth factor, dimethyl sulfoxide, active vitamin D3, peroxisome proliferator-activated receptor gamma, 12-O-tetradecanoylphorbol-13-acetate, hexamethylene-bis-acetamide, transforming proliferator-beta, butyrate, cyclic AMP, and vesnarinone. In some embodiments, the compound is selected from the group consisting of retinoic acid, phenyl butyrate, all-trans retinoic acid, and active vitamin D.

[0310] A pharmaceutical product comprising the combination therapy or composition of the present invention and at least one other agent (e.g., a chemotherapeutic agent, an antineoplastic agent, or an anti-angiogenic agent) can be used as a combination treatment for simultaneous, individual, or sequential administration in cancer treatment. The other agent may be any agent suitable for the treatment of the particular cancer in question, e.g., alkylating agents, e.g., platinum derivatives such as cisplatin, carboplatin, and / or oxaliplatin; plant alkaloids, e.g., paclitaxel, docetaxel, and / or irinotecan; antitumor antibiotics, e.g., doxorubicin (adriamycin), daunorubicin, epirubicin, idarubicin, mitoxantrone, dactinomycin, bleomycin, actinomycin, luteomycin, and / or mitomycin; topoisomerase inhibitors such as topotecan; and / or antimetabolites, e.g., fluorouracil and / or other fluoropyrimidines. In some embodiments, the other agent is dacarbazine or gemcitabine.

[0311] The combination therapy or composition of the present invention may also be used in combination with other anticancer therapies such as vaccines, cytokines, enzyme inhibitors, immunostimulatory compounds, and T-cell therapy. In the case of vaccines, this may be, for example, a protein, peptide, or DNA vaccine containing one or more antigens related to the cancer being treated, or a vaccine containing dendritic cells together with the antigens. Suitable cytokines include, for example, IL-2, IFN-gamma, and GM-CSF. An example of an enzyme inhibitor of the type having anticancer activity is an indoleamine-2,3-dioxygenase (IDO) inhibitor, such as 1-methyl-D-tryptophan (1-D-MT). Adoptive T-cell therapy refers to a variety of immunotherapy techniques that involve increasing or manipulating the patient's own T cells that recognize and attack their own tumors.

[0312] Similarly, the combination therapies or compositions of the present invention are intended to be used in adjunctive therapy in conjunction with tyrosine kinase inhibitors. These are synthetic, primarily quinazolone-derived, low molecular weight molecules that inhibit ligand-induced receptor phosphorylation by interacting with the intracellular tyrosine kinase domain of the receptor and competing for the intracellular Mg-ATP binding site.

[0313] In some embodiments, the combination therapy or composition can be used in combination with another agent / drug that mediates the activation of the immune system, including but not limited to agents that modulate the expression or activity of A2AR, BTLA, B7-H3, B7-H4, CTLA-4, CD27, CD28, CD39, CD40, CD47, CD55, CD73, CD122, CD137, CD160, CGEN-15049, LY108, CHK1, CHK2, CTLA-3, CEACAM (e.g., CEACAM-1 and / or CEACAM-5), GAL9, GITR, HVEM, ICOS, IDO, KIR, LAIR1, NKG2A, OX40, PD-L1 / PD-L2, LILRB2, CMTM6, TIGIT, TGFR-beta, TNFR2, VISTA and / or 2B4. In certain embodiments, the agent is an antibody or its antigen-binding fragment that binds to one of the above molecules. In certain embodiments, the antibody or its antigen-binding moiety, composition, or bispecific conjugation molecule of the present invention may be administered in combination with a CTLA-4 inhibitor (e.g., an anti-CTLA-4 antibody, e.g., tremelimumab or ipilimumab). In one embodiment, the antibody or its antigen-binding moiety, composition, or bispecific conjugation molecule of the present invention may be administered in combination with ipilimumab. Similarly, the combination therapy or composition of the present invention is intended to be used in combination with cytokines (e.g., IL-1, IL-2, IL-12, IL-15, or IL-21), EGFR inhibitors, VEGF inhibitors, etc.

[0314] In certain embodiments, the combination therapies and compositions of the present invention may be administered in combination with other inhibitors of the PD-1, TIM-3, or LAG-3 pathway that may target PD-1, TIM-3, or LAG-3, or one or more ligands of these targets. Examples of such inhibitors include: - Other anti-PD-1 antibodies, as well as antibodies targeting PD-1 ligands and / or co-receptors, such as PD-L1 or PD-L2 (e.g., pembrolizumab and / or nivolumab); - Other anti-TIM-3 antibodies, as well as antibodies targeting TIM-3 ligands and / or coreceptors, e.g., galectin-9, HMGB-1, phosphatidylserolelipids, CEACAM1, LILRA1-6, or LILRB1-5 (e.g., MGB453, TSR-022, and / or LY3321367); and - Other anti-LAG-3 antibodies, as well as antibodies targeting LAG-3 ligands and / or co-receptors, such as MHCII, galectin-3, and LSECtin (e.g., BMS-986016, GSK2831781, REGN3767, BAP050, or BAP050-chi, or LAG525) These are some examples.

[0315] The combination therapies and compositions of the present invention are understood to be for use in the treatment methods described herein, for use in the treatments described herein, and / or for use in the manufacture of agents for the treatments described herein. The present invention also provides kits and products comprising the combination therapies or compositions of the present invention as described herein.

[0316] Dosage and route of administration The combination therapies and compositions of the present invention are administered in an effective dose for treating the condition in question, i.e., in the dose and duration necessary to achieve the desired outcome. The therapeutically effective dose may vary depending on factors such as the specific condition being treated, the patient's age, sex, and weight, as well as whether the antibody is administered as a monotherapy or in combination with one or more additional anticancer therapies.

[0317] The administration regimen can be adjusted to provide the optimal desired response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased depending on the urgency of the treatment situation. Formulating parenteral compositions into unit dosage forms is particularly advantageous for ease of administration and dose uniformity. As used herein, unit dosage forms refer to physically distinct units suitable as unit doses for the patient / subject being treated, each containing a predetermined amount of the active compound calculated to associate with the required pharmaceutically acceptable carrier to produce the desired therapeutic effect. The specifications of the unit dosage forms of the present invention are generally subject to and directly depend on (a) the unique characteristics of the chemotherapeutic agent and the specific therapeutic or prophylactic effect to be achieved, and (b) the inherent limitations in the art of synthesizing such active compounds for the treatment of susceptibility in an individual.

[0318] Therefore, those skilled in the art will recognize, based on the disclosures provided herein, that doses and administration regimens are adjusted according to methods well known in the art of therapy. That is, the maximum tolerable dose can be easily established, and the effective dose that provides a detectable therapeutic benefit to the patient can be determined as well as the time requirements for administering each agent to provide a detectable therapeutic benefit to the patient. Accordingly, while certain doses and administration regimens are illustrated herein, these examples do not in any way limit the doses and administration regimens that may be provided to a patient in practice of the invention.

[0319] It should be noted that dose values ​​may vary depending on the type and severity of the condition being alleviated, and may include single or multiple doses. Furthermore, it should be understood that for any particular subject, a specific dose regimen should be adjusted over time according to the individual needs and the professional judgment of the person administering or managing the administration of the composition, and that the dose ranges described herein are merely examples and are not intended to limit the scope or practice of concretized combination therapy. Moreover, the administration regimens of the combination therapy and compositions of the present invention may be based on a variety of factors, including the type of disease, the patient's age, weight, sex, medical condition, severity of the condition, route of administration, and the specific antibodies used. For this reason, administration regimens may vary widely, but can be routinely determined using standard methods. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include toxic effects and / or clinical effects such as clinical laboratory values. For this reason, the present invention includes intra-patient dose increases as determined by those skilled in the art. Determining appropriate doses and regimens is well known in the relevant art and will be understood to be encompassed by those skilled in the art if the teachings disclosed herein are provided.

[0320] The appropriate dose of the antibody or composition in the combination therapy of the present invention is intended to be in the range of 0.1 to 100 mg / kg, such as about 0.5 to 50 mg / kg, for example, about 1 to 20 mg / kg. The antibody may be administered in doses of at least 0.25 mg / kg, at least 0.5 mg / kg, such as at least 1 mg / kg, for example, at least 1.5 mg / kg, such as at least 2 mg / kg, for example, at least 3 mg / kg, for example, at least 4 mg / kg, for example, at least 5 mg / kg; and at most 50 mg / kg, such as at most 30 mg / kg, for example, at most 20 mg / kg, for example, at most 15 mg / kg. Administration is usually repeated at appropriate intervals, for example, once a week, once every two weeks, once every three weeks, or once every four weeks, and for a period of time that the responsible physician deems appropriate, with the dose being increased or decreased as appropriate as needed.

[0321] The effective dose of tumor therapy can be measured by its ability to stabilize disease progression and / or alleviate symptoms in the patient, and preferably by its ability to reverse disease progression, for example, by reducing tumor size. The ability of the combination therapy of the present invention to inhibit cancer can be evaluated, for example, by in vitro assays described in the Examples, and in suitable animal models that predict efficacy in human tumors (see, for example, the Examples). A suitable dosing regimen can be selected to provide the optimal therapeutic response in each specific situation, for example, when administered as a single bolus or as a series of infusions, and the dose can be adjusted according to the urgency of each case.

[0322] Products and Kits The present invention also provides products comprising an antibody selected from the group consisting of 12819.15384, 12748.15381, 12748.16124, 12865.15377, 12892.15378, 12796.15376, 12777.15382, 12760.15375, and 13112.15380, and an anti-PD-1 antibody that competes for binding to human PD-1 or binds to the same human PD-1 epitope as said antibody; as well as an anti-TIM-3 antibody or an anti-LAG-3 antibody.

[0323] In some embodiments, the product - At least one anti-PD-1 antibody or its antigen-binding moiety as described herein and at least one anti-TIM-3 antibody or its antigen-binding moiety as described herein; - At least one anti-PD-1 antibody or its antigen-binding moiety as described herein and at least one anti-LAG-3 antibody or its antigen-binding moiety as described herein; - At least one anti-TIM-3 antibody or its antigen-binding moiety as described herein and at least one anti-LAG-3 antibody or its antigen-binding moiety as described herein; or - At least one anti-PD-1 antibody or its antigen-binding moiety as described herein, at least one anti-TIM-3 antibody or its antigen-binding moiety as described herein, and at least one anti-LAG-3 antibody or its antigen-binding moiety as described herein This includes, and also includes a method for manufacturing the said product.

[0324] The present invention also provides a kit comprising an antibody selected from the group consisting of 12819.15384, 12748.15381, 12748.16124, 12865.15377, 12892.15378, 12796.15376, 12777.15382, 12760.15375 and 13112.15380, an anti-PD-1 antibody that competes for binding to human PD-1 or binds to the same human PD-1 epitope as the antibody; and an anti-TIM-3 antibody or an anti-LAG-3 antibody.

[0325] In some embodiments, the kit is - At least one anti-PD-1 antibody or its antigen-binding moiety as described herein and at least one anti-TIM-3 antibody or its antigen-binding moiety as described herein; - At least one anti-PD-1 antibody or its antigen-binding moiety as described herein and at least one anti-LAG-3 antibody or its antigen-binding moiety as described herein; - At least one anti-TIM-3 antibody or its antigen-binding moiety as described herein and at least one anti-LAG-3 antibody or its antigen-binding moiety as described herein; or - At least one anti-PD-1 antibody or its antigen-binding moiety as described herein, at least one anti-TIM-3 antibody or its antigen-binding moiety as described herein, and at least one anti-LAG-3 antibody or its antigen-binding moiety as described herein Includes.

[0326] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention have meanings generally understood by those skilled in the art. Exemplary methods and materials are described below, but similar or equivalent methods and materials can also be used in the practice or testing of the present invention. In case of any conflict, this specification, including its definitions, shall prevail.

[0327] In general, the nomenclature and techniques used in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medical chemistry and medicinal chemistry, as well as protein and nucleic acid chemistry and hybridization, as described herein, are well known and commonly used in the art. Enzyme reactions and purification techniques are carried out as is commonly done in the art or in accordance with the manufacturer's specifications as described herein.

[0328] Furthermore, unless otherwise explicitly stated in the text, singular forms include plural forms, and plural forms include singular forms. Throughout this specification and its embodiments, the terms “have” and “comprise,” or their variations such as “has,” “having,” “comprises,” or “comprising,” are understood to imply that they include the integer or group of integers described, but do not exclude any other integer or group of integers.

[0329] All publications and other references mentioned herein are incorporated herein by reference in their entirety. Numerous documents are cited herein, but this citation does not imply that any of these documents constitute part of the general knowledge in the art.

[0330] To better understand the present invention, the following examples are provided. These examples are for illustrative purposes only and should be construed as not limiting the scope of the present invention in any way. [Examples]

[0331] Example 1 Enhancement of in vitro activity in unidirectional MLRs through a combination of PD-1 / TIM-3 targeting. This example demonstrates that a combination of PD-1 and TIM-3 targeting using anti-PD-1 antibody 12819 and anti-TIM-3 antibody 15086.17145 enhances IFN-γ secretion in a unidirectional mixed lymphocyte response (NLR) assay.

[0332] In a unidirectional MLR assay, dendritic cells (DCs) and CD4 isolated from two different healthy human donors were used. + T cells were co-cultured to induce an alloantigen-specific reaction, resulting in cytokine production and T cell activation / proliferation. Dendritic cells (DCs) were cultured with 20 ng / ml granulocyte-macrophage colony-stimulating factor (GM-CSF) and 20 ng / ml interleukin-4 (IL-4) for 6 days to obtain CD14 + CD14 cells were differentiated from monocytes and isolated from peripheral blood mononuclear cells (PBMCs) of healthy human donor material. + T cells were mixed with the antibody in a 1:10 ratio. The antibody or antibody mixture was added at a final concentration of 10 μg / mL. The antibody mixture contained anti-PD-1 and anti-TIM-3 antibodies in a 1:1 ratio. After 5 days of culture, the supernatant was collected and IFN-γ levels were determined using a Meso Scale electron chemiluminescence cytokine assay. Student's unpaired t-test was used for statistical analysis, and Bonferroni correction was used to adjust for multiple comparisons. A corrected p-value < 0.05 is considered statistically significant.

[0333] Figure 1 shows the effects of anti-PD-1 and anti-TIM-3 antibodies on IFN-γ secretion in a unidirectional MLR assay of three independent donor pairs. Both anti-PD-1 (12819) and anti-TIM-3 (15086) antibodies were functional in the unidirectional MLR assay and increased IFN-γ secretion in all three donor pairs. Furthermore, the combination of anti-PD-1 and anti-TIM-3 antibodies showed enhanced activity by inducing a statistically significant increase in IFN-γ compared to the individual antibodies.

[0334] Example 2 Enhancement of in vitro activity in bidirectional MLR assays by targeting PD-1 / TIM-3 in combination. This example demonstrates that a combination of PD-1 and TIM-3 targeting using anti-PD-1 antibody 12819 and anti-TIM-3 antibody 15086.17145 enhances IFN-γ secretion in a bidirectional MLR assay.

[0335] In a bidirectional MLR assay, PBMCs from two different healthy human donors were co-cultured to induce an alloantigen-specific response, resulting in cytokine production and T cell activation / proliferation. PBMCs from the two different donors were mixed in a 1:1 ratio. Antibodies were tested at a final total antibody concentration of 10 μg / mL. After 5 days of culture, the supernatant was collected, and IFN-γ levels were determined using a Meso-Scale electron chemiluminescence cytokine assay. Student's unpaired t-test was used for statistical analysis, and Bonferroni correction was used to adjust for multiple comparisons. A corrected p-value < 0.05 is considered statistically significant.

[0336] Figure 2 shows the effects of anti-PD-1 and anti-TIM-3 antibodies on IFN-γ secretion in a bidirectional MLR assay of three independent human donor pairs. Both anti-PD-1 and anti-TIM-3 antibodies increased IFN-γ secretion in the bidirectional MLR assay. In addition, the combination of anti-PD-1 and anti-TIM-3 antibodies showed enhanced activity by inducing increased IFN-γ secretion compared to the individual antibodies.

[0337] Example 3 Anti-TIM-3 antibodies induce T cell proliferation in vitro in a unidirectional MLR assay. The ability of the anti-TIM-3 antibody 15086.17145 to induce T cell proliferation was investigated using a unidirectional MLR assay. Anti-TIM-3 antibody, a positive control antibody against PD-1 (12819), or a negative control antibody against IgG2 was added at a final concentration of 25 μg / mL and incubated for 5 days. Following incubation, 1 μCi / well of 3H-thymidine was added for a further 18 hours. Cells were harvested, and 3H-thymidine uptake was determined by liquid scintillation counting (MicroBeta2).

[0338] As shown in Figure 3, the anti-TIM-3 antibody 15086.17145 induced proliferation of both CD4+ and CD8+ T cells to a similar extent as the positive control antibody against PD-1 (Panel A). The addition of soluble anti-CD3 enhanced the proliferation induced by the anti-TIM-3 antibody (Panel B).

[0339] Example 4 Anti-TIM-3 antibodies induce IL-12p40 secretion from dendritic cells. Monocyte-derived dendritic cells were generated from healthy donor material as already described in Example 1. The dendritic cells were incubated with 10 μg / mL anti-TIM-3 antibody 15086.17145 or a negative control IgG2 antibody for 5 days, or left untreated, and the IL-12p40 level in the supernatant was determined using a standard ELISA cytokine assay.

[0340] As shown in Figure 4, ligation of TIM-3 with the anti-TIM-3 antibody 15086.17145 resulted in increased IL-12p40 secretion from dendritic cells.

[0341] Example 5 Anti-TIM-3 antibodies induce the expression of activation markers in dendritic cells. To further investigate the functional role of TIM-3 targeting, the expression levels of selected activation markers in monocyte-derived dendritic cells were determined after treatment with the anti-TIM-3 antibody 15086.17145.

[0342] Dendritic cells were treated with 25 μg / mL anti-TIM-3 antibody for 24 hours, and the gene expression of costimulatory molecules was determined using NanoString Technologies. Gene expression levels were standardized against 30 housekeeping genes with uniform expression. Data for selected related genes are expressed as a change ratio compared to untreated control cells.

[0343] Gene expression analysis showed upregulation of several activation markers and costimulatory molecules, including MHC-II (HLA-DQB1 and HLA-DQA1), CD80, and CD86 (Figure 5, Panel A). Increased cell surface expression of CD80 and CD86 following treatment with anti-TIM-3 antibody 15086.17145 was validated using FACS analysis (Figure 5, Panel B). The histogram overlay shown represents CD11c+ dendritic cells, and the numbers next to the histogram indicate MFI values.

[0344] Example 6 In vivo efficacy of anti-TIM-3 antibody 15086 on human lung PDX tumor growth. This embodiment is CD34 + This study demonstrates the in vivo efficacy of the anti-TIM-3 antibody 15086.17145 in a humanized NSG-SGM3 mouse xenograft (PDX) tumor model derived from human lung patients.

[0345] NSG-SGM3 mice were subjected to umbilical cord blood-derived CD34 + Humanized cells were used, and a patient-derived lung tumor fragment (LG1306) was transplanted into the right flank. The tumor size was 50-150 mm. 3Once the mice reached [the specified condition], they were randomized and the treatment was initiated. After intraperitoneal injection of the vehicle or the first dose of 10 mg / kg of anti-TIM-3 antibody 15086 to the mice, 5 mg / kg was administered 5 times every 5 days. Tumors were measured two-dimensionally 3 times a week with calipers, and the tumor volume in mm 3 was calculated according to the formula: (width) 2 × length × 0.5. The gray area indicates the treatment period. Two-way ANOVA with Bonferroni's multiple comparison was applied to compare the tumor volumes between the treatment groups at each time point. The data are presented as mean ± SEM, ** and p < 0.01.

[0346] Treatment with anti-TIM-3 antibody 15086 resulted in significant tumor growth inhibition in the CD34 + humanized NSG-SGM3 mouse human lung PDX model (Figure 6).

[0347] Example 7 Enhancement of the effects of combinations of PD-1 / LAG-3, PD-1 / TIM-3, or PD-1 / TIM-3 / LAG-3 targeting in the PBMC+SEB assay This example describes the enhancing effect of the combination of anti-PD-1 antibody 12819 and anti-LAG-3 antibody 15532 or anti-TIM-3 antibody 15086.17145 in the PBMC+SEB (Staphylococcal enterotoxin B) assay.

[0348] SEB is a superantigen that binds to MHC class II molecules and the specific Vβ region of the T cell receptor (TCR), inducing nonspecific stimulation of T cells. This leads to polyclonal T cell activation / proliferation and the release of cytokines, including IL-2. Human PBMCs isolated from the buffy coat of healthy donors were seeded in 384-well plates and either left untreated or treated with 10 ng / mL of SEB and 10 μg / mL of the indicated single antibody or antibody mixture. The antibody combinations were 1:1 or 1:1:1 mixtures of the indicated antibodies. After 48 hours in a humid incubator at 37°C, the supernatant was collected and analyzed for IL-2 levels using the IL-2 ELISA kit (Life Technologies). Data are expressed as mean ± SEM. Significance was examined using Student's t-test with Bonferroni correction.

[0349] Figure 7 shows the increase in IL-2 secretion after treatment with a single anti-PD-1, anti-LAG-3, or anti-TIM-3 antibody, a mixture of two antibodies, or a mixture of all three antibodies. The data demonstrate that IL-2 levels increased with single treatment with anti-PD-1, anti-LAG-3, or anti-TIM-3 antibodies, but IL-2 secretion increased further with treatment with anti-PD-1 and anti-LAG-3 antibodies, or a combination of anti-PD-1 and anti-TIM-3 antibodies. The combination of all three antibodies—anti-PD-1, anti-LAG-3, and anti-TIM-3—increased IL-2 secretion more than any of the two antibody combinations.

[0350] Example 8 Enhanced in vivo efficacy of anti-PD-1 and anti-LAG-3 antibody combination in two syngeneic mouse tumor models. This example demonstrates the in vivo efficacy of a combination of anti-PD-1 antibody 12819 and either anti-LAG-3 antibody C9B7W (reactive with mouse LAG-3; BioXcell) or anti-LAG-3 antibody 15011 in two syngeneic mouse tumor models.

[0351] 0.5x10 6Individual MC38 (colon cancer) or 5x10 6 Each ASB-XIV (lung cancer) cell was inoculated subcutaneously into the flank of 6-8 week old female BALB / cAnNRj (ASB-XIV) or C57BL / 6 (MC38) mice. The tumors were measured in two dimensions three times a week using calipers, and the measurements were taken in mm. 3 The tumor volume is given by the formula: (width) 2 The calculation was performed according to the formula: × length × 0.5. After vaccination, the average tumor size was 30-50 mm. 3 On day 5 (ASB-XIV) or day 13 (MC38), mice were randomized into four groups of 10 animals each, and treatment was initiated. Mice were treated three times a week for a total of six times by intraperitoneal injection of medium buffer, anti-PD-1 antibody 12819, anti-LAG-3 antibody C9B7W, anti-LAG-3 antibody 15011, or a combination of anti-PD-1 and anti-LAG-3 antibodies. Antibody treatment was administered at a dose of 10 mg / kg / target. Tumor volume at each time point was compared between treatment groups using a two-way ANOVA with Bonferroni's multiple comparison test. Statistical analysis was performed using GraphPad Prism version 5.0 (GraphPad Software, Inc.).

[0352] MC38 syngeneic tumors treated with monoclonal anti-PD-1 antibody 12819 showed sustained tumor growth, although their growth kinetics were slower than those treated with the media (Figure 8). No effect was observed with treatment using anti-LAG-3 antibody C9B7W alone. The combination of anti-PD-1 and anti-LAG-3 antibodies showed enhanced inhibition of tumor growth compared to anti-PD-1 treatment and significantly inhibited tumor growth compared to those treated with the media (p<0.001) (Figure 8).

[0353] ASB-XIV syngeneic tumors treated with anti-PD-1 antibody 12819 showed slower tumor growth compared to vehicle-treated tumors (Figures 8 and 9). The effect of treatment with anti-LAG-3 antibody C9B7W alone was not observed (Figure 8), while anti-LAG-3 antibody 15011 significantly inhibited tumor growth compared to vehicle treatment (p<0.05) (Figure 9). Combining anti-PD-1 antibody and anti-LAG-3 antibody C9B7W significantly enhanced antitumor efficacy compared to vehicle treatment (Figure 8). A significant enhancement of the tumor inhibitory effect was also observed with the combination of anti-PD-1 antibody and anti-LAG-3 antibody 15011 compared to single treatment with anti-PD-1 antibody (p<0.0001) or anti-LAG-3 antibody (p<0.05) (Figure 9).

[0354] Example 9 Enhanced in vivo efficacy of combinations of anti-PD-1 and anti-TIM-3 antibodies in two syngeneic mouse tumor models This example demonstrates the in vivo efficacy of the combination of anti-PD-1 antibody 12819 and anti-TIM-3 antibody 5D12 (reactive with mouse TIM-3; Anderson et al., Science 318:1141-43 (2007)) in two syngeneic mouse tumor models.

[0355] 0.2×10 6 individual Sa1N (fibrosarcoma) or 5×10 6 individual ASB-XIV (lung cancer) cells were each subcutaneously inoculated into the flanks of 6- to 8-week-old female A / J (Sa1N) and BALB / cAnNRj (ASB-XIV) mice. Tumors were measured in two dimensions three times a week with calipers, and the tumor volume in mm 3 was calculated according to the formula: (width) 2 × length × 0.5. The mean tumor size was 60-110 mm 3Upon reaching a certain threshold, mice were randomized and treatment was initiated. Mice were treated either with a single dose (Sa1N) or three times a week for a total of six doses (ASB-XIV) by intraperitoneal injection of media buffer, anti-PD-1 antibody 12819, and / or anti-TIM-3 antibody 5D12. Antibody treatment was administered at a dose of 10 mg / kg / target in mice with ASB-XIV tumors. Mice with Sa1N tumors were administered anti-PD-1 and anti-TIM-3 antibodies at doses of 1 mg / kg and 10 mg / kg, respectively. Two-way ANOVA with Bonferroni's multiple comparison test was applied to compare tumor volume at each time point between treatment groups. Statistical analysis was performed using GraphPad Prism version 5.0 (GraphPad Software, Inc.).

[0356] Six days after inoculation with ASB-XIV tumor cells, the average tumor size was 56 mm. 3 Upon reaching a certain stage, mice were randomized into four groups of 10 animals each, and treatment was initiated. Treatment with anti-PD-1 antibody 12819 slowed tumor growth, but treatment with anti-TIM-3 antibody 5D12 had no effect on tumor growth compared to the medium treatment. Combining anti-PD-1 antibody with anti-TIM-3 antibody showed a significant tumor inhibitory effect compared to treatment with either antibody alone (p<0.001) (Figure 10).

[0357] 13 days after inoculation with Sa1N tumor cells, the average tumor size was 110 mm. 3 Mice were randomized into four groups of 10 animals each and treated with a single antibody. The results showed that single antibody treatment with either anti-PD-1 antibody 12819 or anti-TIM-3 antibody 5D12 delayed initial tumor growth. Enhanced antitumor effects were observed by combining the anti-PD-1 antibody with the anti-TIM-3 antibody compared to single antibody treatment. The combination treatment also significantly inhibited tumor growth compared to the single antibody treatment (p<0.0001) (Figure 10).

[0358] Example 10 In vivo efficacy of a combination of anti-PD-1 and anti-TIM-3 antibodies in a human xenograft tumor model. This example demonstrates the enhanced in vivo efficacy of the combination of anti-PD-1 antibody 12819 and anti-TIM-3 antibodies 15086.17145 in a human xenograft tumor model in which A375 cells (human melanoma) were transplanted into mice reconstituted with human PBMCs.

[0359] Human PBMCs were intraperitoneally injected into NOG (Donor 1 and Donor 2) or NOG-EXL (hGM-CSF / hIL-3-NOG) (Donor 3) mice, and human A375 melanoma cells were subcutaneously transplanted one day later. Treatment was initiated on the day of PBMC injection, and mice were treated three times a week for a total of six times by intraperitoneal injection of medium buffer, anti-PD-1 antibody 12819, anti-TIM-3 antibody 15086.17145, or a combination of anti-PD-1 and anti-TIM-3 antibodies. Antibody treatment was administered at a dose of 10 mg / kg / target. Tumors were measured two-dimensionally with calipers three times a week, and mm 3 The tumor volume is given by the formula: (width) 2 The calculation was performed according to the formula: ×length ×0.5. Two-way ANOVA with Bonferroni multiple comparisons was applied to compare tumor volume among treatment groups at each time point. Statistical analysis was performed using GraphPad Prism version 5.0 (GraphPad Software, Inc.).

[0360] As shown in Figure 11, treatment with a combination of anti-PD-1 and anti-TIM-3 antibodies resulted in a significant (and at least synergistic in donor 3) delay in tumor growth (p<0.05 and p<0.01), while single antibody treatment (anti-PD-1 or anti-TIM-3) showed a limited effect on tumor growth compared to the media treatment group.

[0361] Example 11 The combination of PD-1 and TIM-3 targeting results in increased survival in mice co-transplanted with human immune cells and tumor cells. This example demonstrates the in vivo efficacy of the anti-PD-1 antibody 12819 in combination with the anti-TIM-3 antibody 15086.17145 in a mouse-human tumor model.

[0362] 2 x 10 6 2 × 10⁶ A375 (melanoma) cells 6 Human PBMCs were mixed and subcutaneously inoculated into the flanks of 6-8 week old female NOD-scid mice. Treatment was initiated at the time of cell inoculation. Mice were treated three times a week for a total of six times by intraperitoneal injection of medium buffer, anti-PD-1 antibody 12819, anti-TIM-3 antibody 15086.17145, or a combination of anti-PD-1 and anti-TIM-3. Antibody treatment was administered at a dose of 10 mg / kg for each antibody. Tumors were measured two-dimensionally with calipers three times a week, and mm 3 The tumor volume is given by the formula: (width) 2 The calculation was performed according to the formula: × length × 0.5.

[0363] Survival is associated with tumor size <400mm. 3 This was defined as having [specific characteristic]. The results showed increased survival in mice treated with a combination of anti-PD-1 and anti-TIM-3 antibodies compared to treatment with either single antibody (Figure 12, panels A-E).

[0364] Example 12 The combination of PD-1, LAG-3, and TIM-3 targeting results in increased survival in syngeneic mouse tumor models. This example demonstrates the in vivo efficacy of a combination of anti-PD-1 antibody 12819, anti-LAG-3 antibody C9B7W, and anti-TIM-3 antibody 5D12 in a syngeneic mouse tumor model.

[0365] 5 x 10 6 ASB-XIV (lung cancer) cells were subcutaneously inoculated into the flanks of 6-8 week old female BALB / cAnNRj mice. Tumors were measured two-dimensionally three times a week using calipers, and mm 3 The tumor volume is given by the formula: (width) 2 The calculation was performed according to the formula: × length × 0.5. Five days after vaccination, the average tumor size was 50 mm. 3When the target was reached, the mice were randomized into seven groups of 10 animals each, and treatment was initiated. The mice were treated three times a week for a total of six times by intraperitoneal injection of a medium buffer, anti-PD-1 antibody 12819, anti-LAG-3 antibody C9B7W, anti-TIM-3 antibody 5D12, or a combination of anti-PD-1 and anti-LAG-3, a combination of anti-PD-1 and anti-TIM-3, or a combination of anti-PD-1, anti-LAG-3, and anti-TIM-3 antibodies. Antibody treatment was administered at a dose of 10 mg / kg for each antibody.

[0366] Survival is associated with tumor size <400mm. 3 This was defined as having [specific characteristic]. The results showed increased survival in mice treated with a combination of anti-PD-1, anti-TIM-3, and anti-TIM-3 antibodies compared to treatment with any one or two antibodies (Figure 13, panels A-H).

[0367] Table 1: Identifiers for antibody sequences [Table 1]

[0368] Table 2: Steady Region Array [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0369] Table 3: Target protein sequences [Table 3]

[0370] Table 4: Heavy and light chain variable domain nucleotide sequences of anti-TIM-3 antibodies [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6]

[0371] Table 5: Amino acid sequences of the heavy and light chain variable domains of anti-TIM-3 antibodies (CDRs are shown in bold and italics) [Table 5-1] [Table 5-2] [Table 5-3]

[0372] Table 6: Nucleotide sequences of the heavy and light chain variable domains of anti-PD-1 antibodies [Table 6-1] [Table 6-2] [Table 6-3]

[0373] Table 7: Amino acid sequences of the heavy and light chain variable domains of anti-PD-1 antibodies (CDRs are shown in bold and italics) [Table 7]

[0374] Table 8: Nucleotide sequences of the heavy and light chain variable domains of anti-LAG-3 antibodies [Table 8-1] [Table 8-2]

[0375] Table 9: Amino acid sequences of the heavy and light chain variable domains of anti-LAG-3 antibodies (CDRs are shown in bold and italics) [Table 9]

Claims

1. A pharmaceutical composition comprising an anti-human TIM-3 antibody or its antigen-binding moiety and an anti-human PD-1 antibody or its antigen-binding moiety, for enhancing immunity in a human patient in need, or for treating cancer, Here, the anti-TIM-3 antibody, Heavy chain complementarity determination region 1 (H-CDR1) including SEQ ID NO: 8, H-CDR2 containing Sequence ID 9, H-CDR3 containing sequence number 10, Light chain complementarity determination region 1 (L-CDR1) including sequence number 11, L-CDR2 including sequence number 12, and Includes L-CDR3 containing Sequence ID 13, Here, the anti-PD-1 antibody, a) H-CDR1 containing sequence number 228 H-CDR2 containing Sequence ID No. 229, H-CDR3 containing sequence number 230, L-CDR1 containing sequence number 231, L-CDR2 including sequence number 232, and Includes L-CDR3 containing sequence number 233; b) Pembrolizumab; or c) Nivolumab, Pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein the anti-TIM-3 antibody comprises a heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 4, and the anti-PD-1 antibody comprises a VH containing the amino acid sequence of SEQ ID NO: 226 and a VL containing the amino acid sequence of SEQ ID NO:

227.

3. The pharmaceutical composition according to claim 1, wherein the anti-TIM-3 antibody comprises VH containing the amino acid sequence of SEQ ID NO: 7 and VL containing the amino acid sequence of SEQ ID NO: 4, and the anti-PD-1 antibody is pembrolizumab or nivolumab.

4. The pharmaceutical composition according to claim 1, wherein the anti-TIM-3 antibody comprises a heavy chain (HC) containing the amino acid sequences of SEQ ID NOs. 7 and 377, and a light chain (LC) containing the amino acid sequences of SEQ ID NOs. 4 and 378; and the anti-PD-1 antibody comprises an HC containing the amino acid sequences of SEQ ID NOs. 226 and 375, and an LC containing the amino acid sequences of SEQ ID NOs. 227 and 379.

5. A pharmaceutical composition for enhancing immunity in a human patient in need thereof, or for treating cancer, comprising an anti-human TIM-3 antibody comprising a heavy chain (HC) comprising the amino acid sequences of SEQ ID NOs. 7 and 377, and a light chain (LC) comprising the amino acid sequences of SEQ ID NOs. 4 and 378, and pembrolizumab.

6. A pharmaceutical composition for enhancing immunity in a human patient in need thereof, or for treating cancer, comprising an anti-human TIM-3 antibody comprising a heavy chain (HC) comprising the amino acid sequences of SEQ ID NOs. 7 and 377, and a light chain (LC) comprising the amino acid sequences of SEQ ID NOs. 4 and 378, and nivolumab.

7. The anti-TIM-3 antibody has the following characteristics: a) K23 nM or less when measured by surface plasmon resonance Ｄ To bind to human TIM-3; b) K22 nM or less when measured by surface plasmon resonance Ｄ To bind to cynomolgus monkey TIM-3; c) Binding to human TIM-3 at an EC50 of 1.2 nM or less as measured by ELISA; d) Binding to cynomolgus monkey TIM-3 at an EC50 of 46 nM or less when measured by ELISA; e) Increasing IFN-γ secretion in a unidirectional mixed lymphocyte reaction assay; f) Increasing IFN-γ secretion in a bidirectional mixed lymphocyte reaction assay; g) Increasing TNF-α secretion in a unidirectional mixed lymphocyte reaction assay; h) Increasing TNF-α secretion from dendritic cells; and i) Inhibiting the interaction between TIM-3 and phosphatidylserine, Having at least one of the following, A pharmaceutical composition according to any one of claims 1 to 6.

8. Anti-PD-1 antibodies, H-CDR1 containing sequence number 228 H-CDR2 containing Sequence ID No. 229, H-CDR3 containing sequence number 230, L-CDR1 containing sequence number 231, L-CDR2 including sequence number 232, and Includes L-CDR3 containing Sequence ID No. 233, and has the following characteristics: a) K₀ 750 pM or less Ｄ Binding to human PD-1; b) K 7 nM or less Ｄ It binds to cynomolgus monkey PD-1; c) K of 1 nM or less Ｄ To bind to mouse PD-1; d) Does not bind to rat PD-1; e) Increasing IL-2 secretion in a Staphylococcus enterotoxin B (SEB) whole blood assay; f) Increasing IFN-γ secretion in a unidirectional mixed lymphocyte reaction assay; g) Inhibit the interaction between PD-1 and PD-L1 by at least 60% at a concentration of 10 μg / mL in a flow cytometry competitive assay; h) Blocking at least 90% of the binding of PD-L1 and PD-L2 to PD-1 at a concentration of 10 μg / mL as determined by biolayer interferometry analysis; and i) Inhibiting tumor growth in vivo A pharmaceutical composition according to any one of claims 1 to 7, having at least one of the following.

9. A pharmaceutical composition for treating cancer in a human patient requiring the same, a) Anti-human TIM-3 antibodies comprising HC containing the amino acid sequences of SEQ ID NOs. 7 and 377, and LC containing the amino acid sequences of SEQ ID NOs. 4 and 378; and b) Pembrolizumab or nivolumab, A pharmaceutical composition containing the following:

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the cancer is selected from the group consisting of hematological malignancies, solid tumors, melanoma, non-small cell lung cancer, bladder cancer, gallbladder cancer, head and neck squamous cell carcinoma, esophageal cancer, gastroesophageal cancer, stomach cancer, ovarian cancer, colorectal cancer, renal cell carcinoma, pancreatic cancer, Merkel cell carcinoma, fibrosarcoma, gliosarcoma, mesothelioma, cholangiocarcinoma, and glioblastoma.

11. A pharmaceutical composition according to any one of claims 1 to 10, for use in combination with at least one of radiotherapy, chemotherapeutic agents, antineoplastic agents, and anti-angiogenic agents.

12. The pharmaceutical composition according to claim 11, wherein the chemotherapeutic agent is irinotecan.

13. A pharmaceutical composition according to any one of claims 1 to 12, wherein an equal amount of the antibody or its antigen-binding portion is present in the composition.

14. A pharmaceutical composition according to any one of claims 1 to 13, wherein an antibody or its antigen-binding portion is administered simultaneously.

15. A pharmaceutical composition according to any one of claims 1 to 13, wherein an antibody or its antigen-binding portion is administered continuously.

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