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

Abstract

The present disclosure relates to combination therapies targeting two or all of PD-1, TIM-3, and LAG-3 using antibodies specific for these targets in patients in need of improved immunity. The present disclosure also includes compositions that can be used in the therapy. The therapy can be used to treat diseases such as cancer.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application date of April 5, 2018, application number: 201880032633.7, and invention name: “Combination therapy targeting PD-1, TIM-3 and LAG-3”.

[0002] Cross-reference to related applications

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 481,973, filed April 5, 2017, the disclosure of which is incorporated herein by reference in its entirety.

[0004] Sequence Listing

[0005] This application contains a sequence listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. An electronic copy of this sequence listing (created on March 23, 2018) is named 022675_WO058_SL.txt and is 232,360 bytes in size. Background Art

[0006] PD-1 (also known as programmed cell death protein 1 and CD279) is a cell surface receptor of 268 amino acids that belongs to the immunoglobulin superfamily. PD-1 is a member of the CD28 family of T cell regulators and is expressed on T cells, B cells, and macrophages. It binds to its 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 plays the role of an inducible immunomodulatory receptor, playing a role in, for example, the negative regulation of T cell responses to antigen stimulation.

[0007] PD-L1 is the dominant ligand for PD-1. The binding of PD-L1 to PD-1 inhibits T cell activity, reduces cytokine production, and suppresses T cell proliferation. Cancer cells expressing PD-L1 are able to exploit this mechanism to inactivate the anti-tumor activity of T cells through the binding of PD-L1 to the PD-1 receptor. Given its immune response-regulating properties, PD-1 has been investigated as a potential target for immunotherapy, including the treatment of cancer and autoimmune diseases. Two anti-PD-1 antibodies, pembrolizumab and nivolumab, have been approved in the United States and Europe for the treatment of certain cancers.

[0008] Other immune checkpoint proteins include TIM-3 (T cell immunoglobulin and mucin domain 3) and LAG-3 (lymphocyte activation gene 3). TIM-3 (also known as HAVCR2 (hepatitis A virus cellular receptor 2) or CD366) is a member of the T cell immunoglobulin and mucin domain protein family. TIM-3 is encoded by the Havcr2 gene in humans and is a 33kDa type I glycoprotein with a membrane distal IgV domain and a membrane proximal mucin domain. It contains a conserved region of five Tyr residues in the intracellular domain, which is phosphorylated immediately after ligand binding. TIM-3 is expressed by many different cells (including T cells, dendritic cells, macrophages, and natural killer (NK) cells) derived from both the adaptive and innate parts of the immune system. TIM-3 expression is low on naive T cells, but becomes highly upregulated immediately after T cell activation. In contrast to T cells, innate cells (such as dendritic cells, NK cells, and monocytes) have high basal TIM-3 expression. TIM-3 has been associated with several (mostly promiscuous) ligands, including galectin-9, phosphatidylserine, CEACAM-1, and HMGB-1, but the precise roles of these ligands are currently unknown.

[0009] Although TIM-3 has been implicated as a checkpoint inhibitor, there is relatively little evidence to support the concept that TIM-3 directly mediates inhibition of T cell activation or cytokine secretion in a manner similar to, for example, PD-1. In addition, and in contrast to PD-1, TIM-3 appears to play a role in the regulation of cells of the innate system (and particularly dendritic cells). Most functional data related to TIM-3 and its role in tumor immunology come from in vivo studies using various antibodies. In most of these studies, due to poor antibody validation, it is unclear whether the effect of TIM-3 antibodies is mediated by inhibition of ligand binding or by an agonistic effect on the target. Given its immune response-regulating properties, TIM-3 has been investigated as a potential target for immunotherapy, including the treatment of cancer and autoimmune diseases. Single anti-TIM-3 antibodies are currently in clinical development, but there are currently no approved anti-TIM-3 antibodies.

[0010] 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 on various types of cells, including activated T cells, T regulatory (Treg) cells, natural killer cells, B cells, and plasmacytoid dendritic cells. Sequence data, exon / intron organization, and chromosomal location information for LAG-3 indicate that it is closely related to CD4. Similar to CD4, LAG-3 binds to class II MHC molecules, although with higher affinity and at different sites than CD4.

[0011] LAG-3 is a co-inhibitory receptor believed to regulate T cell proliferation, activation, and homeostasis in a manner similar to CTLA-4 and PD-1. Following ligand binding to the extracellular domain, LAG-3 exerts its effects through subsequent signaling via the cytoplasmic domain. The most well-characterized LAG-3 ligand is MHC class II (MHCII), but other LAG-3 ligands have been described, including LSECtin. LAG-3 does not possess the typical ITIM or ITSM motifs, but instead possesses a conserved KIEELE motif (SEQ ID NO:397), which is believed to be essential for its inhibitory effects on T cell activity. The precise mechanism by which LAG-3 influences T cell activity is poorly understood. LAG-3 inhibits T cell expansion by blocking activated T cells from entering the growth phase of the cell cycle, resulting in accumulation of cells in the S phase. LAG-3 is also believed to play a role in enhancing the suppressive activity of regulatory T cells and regulating dendritic cell function. Cancer cells have the ability to upregulate the expression of MHCII, which binds LAG-3 on effector T cells, thereby inhibiting their activity and inducing tumor immune escape.

[0012] Given the critical roles of PD-1, LAG-3, and TIM-3 as immune regulators, there is a need for new and improved combination therapies targeting these receptors to treat cancer and certain immune system disorders. Summary of the Invention

[0013] The present invention is based on the discovery that when anti-PD-1 antibodies (such as those described herein) are used in combination with anti-TIM-3 antibodies and / or anti-LAG-3 antibodies, the immunity-enhancing efficacy of the antibodies is significantly increased. The inventors have found that the combination therapy of the present invention is particularly effective in treating cancer in human patients by activating their own anti-cancer immunity. Compared to currently available treatments for cancer (including antibody therapies), it is contemplated that the combination therapy of the present invention may provide superior clinical responses.

[0014] Thus, the present invention provides a method for enhancing immunity in a human patient in need thereof, such as a cancer patient, by administering to the patient: (1) an anti-PD-1 antibody, or an antigen-binding portion thereof, that competes for binding to human PD-1 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; and (2) an anti-TIM-3 antibody, or an antigen-binding portion thereof, and / or an anti-LAG-3 antibody, or an antigen-binding portion thereof. In certain embodiments, the method comprises administering the anti-PD-1 antibody, or an antigen-binding portion thereof, the anti-TIM-3 antibody, or an antigen-binding portion thereof, and the anti-LAG-3 antibody, or an antigen-binding portion thereof.

[0015] In some embodiments, the anti-PD-1 antibody binds to an epitope of human PD-1 comprising:

[0016] a) residues V64, L128, P130, K131, and A132 of SEQ ID NO: 388;

[0017] b) residues V44 and T145 of SEQ ID NO: 388;

[0018] c) residues K131 and E136 of SEQ ID NO: 388; or

[0019] d) residues V44 and T145 of SEQ ID NO: 388.

[0020] In some embodiments, the anti-PD-1 antibody binds to an epitope of human PD-1 comprising:

[0021] a) residues 56-64, 69-90, and 122-140 of SEQ ID NO:388;

[0022] b) residues 69-90 and 122-140 of SEQ ID NO:388;

[0023] c) residues 69-75 of SEQ ID NO:388;

[0024] d) residues 136-140 of SEQ ID NO: 388; or

[0025] e) residues 69-75 and 136-140 of SEQ ID NO:388.

[0026] In some embodiments, the anti-PD-1 antibody has at least one of the following properties:

[0027] a) With a K of 750 pM or lower D Binds to human PD-1;

[0028] b) with a K of 7 nM or lower D Binds to cynomolgus monkey PD-1;

[0029] c) with a K of 1 nM or lower D Binds to mouse PD-1;

[0030] d) does not bind to rat PD-1;

[0031] e) increased IL-2 secretion in a Staphylococcal enterotoxin B (SEB) whole blood assay;

[0032] f) increased IFN-γ secretion in a one-way mixed lymphocyte reaction assay;

[0033] g) inhibits the interaction of PD-1 and PD-L1 by at least 60% at a concentration of 10 μg / mL in a flow cytometry competition assay;

[0034] h) blocks the binding of PD-L1 and PD-L2 to PD-1 by at least 90% at a concentration of 10 μg / mL as determined by Bio-Layer interferometry analysis; and

[0035] i) Inhibition of tumor growth in vivo.

[0036] In certain embodiments, the anti-PD-1 antibody has all of the described properties.

[0037] In some embodiments, the heavy chain complementarity determining regions (H-CDRs) 1-3 and the light chain complementarity determining regions (L-CDRs) 1-3 of the anti-PD-1 antibody comprise the following amino acid sequences:

[0038] a) SEQ ID NOs: 228-233, respectively;

[0039] b) SEQ ID NOs: 238-243, respectively;

[0040] c) SEQ ID NOs: 248-253, respectively;

[0041] d) SEQ ID NOs: 258-263, respectively;

[0042] e) SEQ ID NOs: 268-273, respectively;

[0043] f) SEQ ID NOs: 278-283, respectively;

[0044] g) SEQ ID NO: 288-293, respectively; or

[0045] h) SEQ ID NOs: 298-303, respectively.

[0046] In some embodiments, the heavy and light chain variable domains of the anti-PD-1 antibody comprise the following amino acid sequences:

[0047] a) SEQ ID NOs: 226 and 227, respectively;

[0048] b) SEQ ID NOs: 236 and 237, respectively;

[0049] c) SEQ ID NOs: 236 and 392, respectively;

[0050] d) SEQ ID NOs: 246 and 247, respectively;

[0051] e) SEQ ID NOs: 256 and 257, respectively;

[0052] f) SEQ ID NOs: 266 and 267, respectively;

[0053] g) SEQ ID NOs: 276 and 277, respectively;

[0054] h) SEQ ID NOs: 286 and 287, respectively; or

[0055] i) SEQ ID NO: 296 and 297, respectively.

[0056] In some embodiments, the anti-PD-1 antibody comprises:

[0057] a) a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 226 and the amino acid sequence of SEQ ID NO: 375, and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 227 and the amino acid sequence of SEQ ID NO: 379;

[0058] b) an HC comprising the amino acid sequence of SEQ ID NO: 236 and the amino acid sequence of SEQ ID NO: 375, and an LC comprising the amino acid sequence of SEQ ID NO: 237 and the amino acid sequence of SEQ ID NO: 379;

[0059] c) an HC comprising the amino acid sequence of SEQ ID NO: 236 and the amino acid sequence of SEQ ID NO: 375, and an LC comprising the amino acid sequence of SEQ ID NO: 392 and the amino acid sequence of SEQ ID NO: 379;

[0060] d) an HC comprising the amino acid sequence of SEQ ID NO: 246 and the amino acid sequence of SEQ ID NO: 375, and an LC comprising the amino acid sequence of SEQ ID NO: 247 and the amino acid sequence of SEQ ID NO: 379;

[0061] e) an HC comprising the amino acid sequence of SEQ ID NO: 256 and the amino acid sequence of SEQ ID NO: 375, and an LC comprising the amino acid sequence of SEQ ID NO: 257 and the amino acid sequence of SEQ ID NO: 379;

[0062] f) an HC comprising the amino acid sequence of SEQ ID NO: 266 and the amino acid sequence of SEQ ID NO: 375, and an LC comprising the amino acid sequence of SEQ ID NO: 267 and the amino acid sequence of SEQ ID NO: 379;

[0063] g) an HC comprising the amino acid sequence of SEQ ID NO: 276 and the amino acid sequence of SEQ ID NO: 375, and an LC comprising the amino acid sequence of SEQ ID NO: 277 and the amino acid sequence of SEQ ID NO: 379;

[0064] h) a HC comprising the amino acid sequence of SEQ ID NO: 286 and the amino acid sequence of SEQ ID NO: 375, and a LC comprising the amino acid sequence of SEQ ID NO: 287 and the amino acid sequence of SEQ ID NO: 379; or

[0065] i) HC comprising the amino acid sequence of SEQ ID NO: 296 and the amino acid sequence of SEQ ID NO: 375, and LC comprising the amino acid sequence of SEQ ID NO: 297 and the amino acid sequence of SEQ ID NO: 379.

[0066] In some embodiments, the anti-TIM-3 antibody competes for binding to human TIM-3 with, or binds to the same epitope of human TIM-3 as, 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.

[0067] In some embodiments, the anti-TIM-3 antibody binds to an epitope of human TIM-3 comprising:

[0068] a) residues P50, V60, F61, E62, G64, R69, I117, M118, and D120 of SEQ ID NO: 389;

[0069] b) residues F61, R69, and 1117 of SEQ ID NO: 389; or

[0070] c) residues P50, F61, E62, 1117, M118, and D120 of SEQ ID NO: 389.

[0071] In some embodiments, the anti-TIM-3 antibody binds to an epitope of human TIM-3 comprising:

[0072] a) residues 62-67 of SEQ ID NO: 389; or

[0073] b) Residues 114-117 of SEQ ID NO:389.

[0074] In some embodiments, the anti-TIM-3 antibody has at least one of the following properties:

[0075] a) with a K of 23 nM or lower D Binding to human TIM-3, as measured by surface plasmon resonance;

[0076] b) with a K of 22 nM or lower D Binding to cynomolgus monkey TIM-3, as measured by surface plasmon resonance;

[0077] c) binds to human TIM-3 with an EC50 of 1.2 nM or less, as measured by ELISA;

[0078] d) binds to cynomolgus monkey TIM-3 with an EC50 of 46 nM or less, as measured by ELISA;

[0079] e) increased IFN-γ secretion in a one-way mixed lymphocyte reaction assay;

[0080] f) increased IFN-γ secretion in a two-way mixed lymphocyte reaction assay;

[0081] g) increased TNF-α secretion in a one-way mixed lymphocyte reaction assay;

[0082] h) increasing TNF-α secretion from dendritic cells; and

[0083] i) Inhibits the interaction between TIM-3 and phosphatidylserine.

[0084] In certain embodiments, the anti-TIM-3 antibody has at least properties a), c), d), e), g), h), and i).

[0085] In some embodiments, the H-CDR1-3 and L-CDR1-3 of the anti-TIM-3 antibody comprise the following amino acid sequences:

[0086] a) SEQ ID NO: 8-13, respectively;

[0087] b) SEQ ID NO: 18-23, respectively;

[0088] c) SEQ ID NOs: 28-33, respectively;

[0089] d) SEQ ID NOs: 38-43, respectively;

[0090] e) SEQ ID NOs: 48-53, respectively;

[0091] f) SEQ ID NOs: 58-63, respectively;

[0092] g) SEQ ID NOs: 68-73, respectively;

[0093] h) SEQ ID NOs: 78-83, respectively;

[0094] i) SEQ ID NOs: 88-93, respectively;

[0095] j) SEQ ID NOs: 98-103, respectively;

[0096] k) SEQ ID NOs: 108-113, respectively;

[0097] 1) SEQ ID NO: 118-123, respectively;

[0098] m) SEQ ID NOs: 128-133, respectively;

[0099] n) SEQ ID NO: 138-143, respectively;

[0100] o) SEQ ID NOs: 148-153, respectively;

[0101] p) SEQ ID NO: 158-163, respectively;

[0102] q) SEQ ID NOs: 168-173, respectively;

[0103] r) SEQ ID NO: 178-183, respectively;

[0104] s) are SEQ ID NOs: 188-193, respectively;

[0105] t) are SEQ ID NOs: 198-203, respectively;

[0106] u) SEQ ID NO: 208-213, respectively; or

[0107] v) SEQ ID NOs: 218-223, respectively.

[0108] In some embodiments, the heavy and light chain variable domains of the anti-TIM-3 antibody comprise the following amino acid sequences:

[0109] a) SEQ ID NO: 7 and 4, respectively;

[0110] b) SEQ ID NOs: 3 and 4, respectively;

[0111] c) SEQ ID NOs: 16 and 17, respectively;

[0112] d) SEQ ID NOs: 26 and 27, respectively;

[0113] e) SEQ ID NOs: 36 and 37, respectively;

[0114] f) SEQ ID NOs: 46 and 47, respectively;

[0115] g) SEQ ID NOs: 56 and 57, respectively;

[0116] h) SEQ ID NOs: 66 and 67, respectively;

[0117] i) SEQ ID NOs: 76 and 77, respectively;

[0118] j) SEQ ID NOs: 86 and 87, respectively;

[0119] k) SEQ ID NOs: 96 and 97, respectively;

[0120] 1) SEQ ID NO: 106 and 107, respectively;

[0121] m) SEQ ID NOs: 116 and 117, respectively;

[0122] n) SEQ ID NOs: 126 and 127, respectively;

[0123] o) SEQ ID NOs: 136 and 137, respectively;

[0124] p) SEQ ID NOs: 146 and 147, respectively;

[0125] q) SEQ ID NOs: 156 and 157, respectively;

[0126] r) SEQ ID NOs: 166 and 167, respectively;

[0127] s) SEQ ID NOs: 176 and 177, respectively;

[0128] t) SEQ ID NOs: 186 and 187, respectively;

[0129] u) SEQ ID NOs: 196 and 197, respectively;

[0130] v) SEQ ID NOs: 206 and 207, respectively; or

[0131] w) are SEQ ID NOs: 216 and 217, respectively.

[0132] In some embodiments, the anti-TIM-3 antibody comprises:

[0133] a) an HC comprising the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 4 and the amino acid sequence of SEQ ID NO: 378;

[0134] b) an HC comprising the amino acid sequence of SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 4 and the amino acid sequence of SEQ ID NO: 378;

[0135] c) an HC comprising the amino acid sequence of SEQ ID NO: 16 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 17 and the amino acid sequence of SEQ ID NO: 378;

[0136] d) an HC comprising the amino acid sequence of SEQ ID NO: 26 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 27 and the amino acid sequence of SEQ ID NO: 378;

[0137] e) an HC comprising the amino acid sequence of SEQ ID NO: 36 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 37 and the amino acid sequence of SEQ ID NO: 378;

[0138] f) an HC comprising the amino acid sequence of SEQ ID NO: 46 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 47 and the amino acid sequence of SEQ ID NO: 378;

[0139] g) an HC comprising the amino acid sequence of SEQ ID NO: 56 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 57 and the amino acid sequence of SEQ ID NO: 378;

[0140] h) an HC comprising the amino acid sequence of SEQ ID NO: 66 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 67 and the amino acid sequence of SEQ ID NO: 378;

[0141] i) an HC comprising the amino acid sequence of SEQ ID NO: 76 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 77 and the amino acid sequence of SEQ ID NO: 378;

[0142] j) an HC comprising the amino acid sequence of SEQ ID NO: 86 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 87 and the amino acid sequence of SEQ ID NO: 378;

[0143] k) an HC comprising the amino acid sequence of SEQ ID NO: 96 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 97 and the amino acid sequence of SEQ ID NO: 378;

[0144] 1) an HC comprising the amino acid sequence of SEQ ID NO: 106 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 107 and the amino acid sequence of SEQ ID NO: 378;

[0145] m) an HC comprising the amino acid sequence of SEQ ID NO: 116 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 117 and the amino acid sequence of SEQ ID NO: 378;

[0146] n) an HC comprising the amino acid sequence of SEQ ID NO: 126 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 127 and the amino acid sequence of SEQ ID NO: 378;

[0147] o) an HC comprising the amino acid sequence of SEQ ID NO: 136 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 137 and the amino acid sequence of SEQ ID NO: 378;

[0148] p) an HC comprising the amino acid sequence of SEQ ID NO: 146 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 147 and the amino acid sequence of SEQ ID NO: 378;

[0149] q) an HC comprising the amino acid sequence of SEQ ID NO: 156 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 157 and the amino acid sequence of SEQ ID NO: 378;

[0150] r) an HC comprising the amino acid sequence of SEQ ID NO: 166 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 167 and the amino acid sequence of SEQ ID NO: 378;

[0151] s) a HC comprising the amino acid sequence of SEQ ID NO: 176 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and a LC comprising the amino acid sequence of SEQ ID NO: 177 and the amino acid sequence of SEQ ID NO: 378;

[0152] t) an HC comprising the amino acid sequence of SEQ ID NO: 186 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 187 and the amino acid sequence of SEQ ID NO: 378;

[0153] u) a HC comprising the amino acid sequence of SEQ ID NO: 196 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and a LC comprising the amino acid sequence of SEQ ID NO: 197 and the amino acid sequence of SEQ ID NO: 378;

[0154] v) a HC comprising the amino acid sequence of SEQ ID NO: 206 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and a LC comprising the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 378; or

[0155] w) an HC comprising the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 378.

[0156] In some embodiments, the anti-LAG-3 antibody competes for binding to human LAG-3 with an antibody selected from the group consisting of 15532, 15646, 15723, 15595, 15431, 15572, and 15011.

[0157] In some embodiments, the anti-LAG-3 antibody binds to an epitope of human LAG-3 comprising:

[0158] a) residues H85, P86, A87, P89, S91, W92, and G93 of SEQ ID NO:68;

[0159] 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;

[0160] 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

[0161] d) residues G107, L109, R110, and S111 of SEQ ID NO:68.

[0162] In some embodiments, the anti-LAG-3 antibody binds to an epitope of human LAG-3 comprising:

[0163] a) residues 98-105 of SEQ ID NO:68;

[0164] b) residues 78-105 and 123-131 of SEQ ID NO:68;

[0165] c) residues 23-30, 40-66, 88-105, 123-137, and 148-152 of SEQ ID NO: 68; or

[0166] d) residues 23-30, 40-66, 98-105, 118-137, and 148-161 of SEQ ID NO: 68.

[0167] In some embodiments, the anti-LAG-3 antibody has at least one of the following properties:

[0168] a) reduces human LAG-3 binding to human MHC class II on A375 cells by greater than 85% compared to a negative control antibody at a concentration of 20 μg / mL, as determined by a flow cytometry competition assay;

[0169] b) reduces human LAG-3 binding to human MHC class II on A375 cells by between 35% and 85% compared to a negative control antibody at a concentration of 20 μg / mL, as determined by a flow cytometry competition assay;

[0170] c) blocking the binding between human LAG-3 expressed on Jurkat cells and human MHC class II expressed on Raji cells;

[0171] d) binds to human LAG-3 with an EC50 of 0.1 nM or less, as measured by flow cytometry;

[0172] e) binds to cynomolgus monkey LAG-3 with an EC50 of 0.3 nM or less, as measured by flow cytometry;

[0173] f) With a K of 3.0x 10-8 or lower D Binding to human LAG-3, as measured by surface plasmon resonance;

[0174] g) With a K of 1.5x 10-7 or lower D Binding to cynomolgus monkey LAG-3, as measured by surface plasmon resonance;

[0175] h) With K of 3.5x 10-8 or lower D Binding to mouse LAG-3, as measured by surface plasmon resonance;

[0176] i) stimulates IL-2 production in human peripheral blood mononuclear cells (PBMCs) treated with Staphylococcal enterotoxin B (SEB);

[0177] j) reducing cellular levels of LAG-3 in human T cells;

[0178] k) reducing soluble levels of LAG-3 in cultures of human T cells;

[0179] l) inducing tumor growth regression in vivo;

[0180] m) delaying tumor growth in vivo; and

[0181] n) does not bind to the same epitope of human LAG-3 as antibody 25F7-Lag3.5.

[0182] In certain embodiments, the anti-LAG-3 antibody has at least properties a), c), d), e), f), g), i), j), k), m), and n).

[0183] In some embodiments, the H-CDR1-3 and L-CDR1-3 of the anti-LAG-3 antibody comprise the following amino acid sequences:

[0184] a) SEQ ID NOs: 318-323, respectively;

[0185] b) SEQ ID NOs: 308-313, respectively;

[0186] c) SEQ ID NOs: 328-333, respectively;

[0187] d) SEQ ID NOs: 338-343, respectively;

[0188] e) SEQ ID NOs: 348-353, respectively;

[0189] f) SEQ ID NO: 358-363, respectively; or

[0190] g) SEQ ID NOs: 368-373, respectively.

[0191] In some embodiments, the heavy and light chain variable domains of the anti-LAG-3 antibody comprise the following amino acid sequences:

[0192] a) SEQ ID NOs: 316 and 317, respectively;

[0193] b) SEQ ID NOs: 306 and 307, respectively;

[0194] c) SEQ ID NOs: 326 and 327, respectively;

[0195] d) SEQ ID NOs: 336 and 337, respectively;

[0196] e) SEQ ID NOs: 346 and 347, respectively;

[0197] f) SEQ ID NOs: 356 and 357, respectively; or

[0198] g) SEQ ID NOs: 366 and 367, respectively.

[0199] In some embodiments, the anti-LAG-3 antibody comprises:

[0200] a) an HC comprising the amino acid sequence of SEQ ID NO: 316 and the amino acid sequence of SEQ ID NO: 375, and an LC comprising the amino acid sequence of SEQ ID NO: 317 and the amino acid sequence of SEQ ID NO: 378;

[0201] b) an HC comprising the amino acid sequence of SEQ ID NO: 306 and the amino acid sequence of SEQ ID NO: 375, and an LC comprising the amino acid sequence of SEQ ID NO: 307 and the amino acid sequence of SEQ ID NO: 378;

[0202] c) an HC comprising the amino acid sequence of SEQ ID NO: 326 and the amino acid sequence of SEQ ID NO: 375, and an LC comprising the amino acid sequence of SEQ ID NO: 327 and the amino acid sequence of SEQ ID NO: 378;

[0203] d) an HC comprising the amino acid sequence of SEQ ID NO: 336 and the amino acid sequence of SEQ ID NO: 375, and an LC comprising the amino acid sequence of SEQ ID NO: 337 and the amino acid sequence of SEQ ID NO: 378;

[0204] e) an HC comprising the amino acid sequence of SEQ ID NO: 346 and the amino acid sequence of SEQ ID NO: 375, and an LC comprising the amino acid sequence of SEQ ID NO: 347 and the amino acid sequence of SEQ ID NO: 378;

[0205] f) HC comprising the amino acid sequence of SEQ ID NO: 356 and the amino acid sequence of SEQ ID NO: 375, and LC comprising the amino acid sequence of SEQ ID NO: 357 and the amino acid sequence of SEQ ID NO: 378; or

[0206] g) HC comprising the amino acid sequence of SEQ ID NO: 366 and the amino acid sequence of SEQ ID NO: 375, and LC comprising the amino acid sequence of SEQ ID NO: 367 and the amino acid sequence of SEQ ID NO: 379.

[0207] In some embodiments, the method comprises administering to the patient:

[0208] a) an anti-PD-1 antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequence of SEQ ID NOs: 228-233; and an anti-TIM-3 antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequence of SEQ ID NOs: 8-13;

[0209] b) an anti-PD-1 antibody whose VH and VL comprise the amino acid sequences of SEQ ID NOs: 226 and 227, respectively; or an anti-TIM-3 antibody whose VH and VL comprise the amino acid sequences of SEQ ID NOs: 7 and 4, respectively; or

[0210] c) an anti-PD-1 antibody comprising a HC comprising the amino acid sequence of SEQ ID NOs: 226 and 375 and a LC comprising the amino acid sequence of SEQ ID NOs: 227 and 379; and an anti-TIM-3 antibody comprising a HC comprising the amino acid sequence of SEQ ID NOs: 7 and 377 and a LC comprising the amino acid sequence of SEQ ID NOs: 4 and 378.

[0211] In some embodiments, the method comprises administering to the patient:

[0212] a) an anti-PD-1 antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequence of SEQ ID NOs: 228-233; and an anti-LAG-3 antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequence of SEQ ID NOs: 318-323;

[0213] b) an anti-PD-1 antibody whose VH and VL comprise the amino acid sequences of SEQ ID NOs: 226 and 227, respectively; and an anti-LAG-3 antibody whose VH and VL comprise the amino acid sequences of SEQ ID NOs: 316 and 317, respectively; or

[0214] c) an anti-PD-1 antibody comprising a HC comprising the amino acid sequence of SEQ ID NOs: 226 and 375 and a LC comprising the amino acid sequence of SEQ ID NOs: 227 and 379; and an anti-LAG-3 antibody comprising a HC comprising the amino acid sequence of SEQ ID NOs: 316 and 375 and a LC comprising the amino acid sequence of SEQ ID NOs: 317 and 378.

[0215] In some embodiments, the method comprises administering to the patient:

[0216] a) an anti-PD-1 antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequence of SEQ ID NOs: 228-233; an anti-TIM-3 antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequence of SEQ ID NOs: 8-13; and an anti-LAG-3 antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequence of SEQ ID NOs: 318-323;

[0217] b) an anti-PD-1 antibody whose VH and VL comprise the amino acid sequences of SEQ ID NOs: 226 and 227, respectively; an anti-TIM-3 antibody whose VH and VL comprise the amino acid sequences of SEQ ID NOs: 7 and 4, respectively; and an anti-LAG-3 antibody whose VH and VL comprise the amino acid sequences of SEQ ID NOs: 316 and 317, respectively; or

[0218] c) an anti-PD-1 antibody comprising a HC comprising the amino acid sequence of SEQ ID NOs: 226 and 375 and a LC comprising the amino acid sequence of SEQ ID NOs: 227 and 379; an anti-TIM-3 antibody comprising a HC comprising the amino acid sequence of SEQ ID NOs: 7 and 377 and a LC comprising the amino acid sequence of SEQ ID NOs: 4 and 378; and an anti-LAG-3 antibody comprising a HC comprising the amino acid sequence of SEQ ID NOs: 316 and 375 and a LC comprising the amino acid sequence of SEQ ID NOs: 317 and 378.

[0219] The antibodies or antigen-binding portions can be administered to a patient simultaneously (eg, in a single pharmaceutical composition) or sequentially.

[0220] Therapies of the present invention can be used to treat patients with cancers such as hematological malignancies (e.g., leukemia, Hodgkin's lymphoma, or non-Hodgkin's lymphoma) or solid tumors. In some embodiments, the patient 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. Therapies of the present invention may additionally include radiation, or at least one of a chemotherapeutic agent, an anti-neoplastic agent, and an anti-angiogenic agent.

[0221] Also provided herein are multispecific (e.g., bispecific or trispecific) antibodies that specifically bind to: a) human PD-1 and human TIM-3; b) human PD-1 and human LAG-3; or c) human PD-1, human anti-TIM-3, and human LAG-3. In certain embodiments, the multispecific antibody comprises an antigen-binding portion of an anti-PD-1 antibody as described herein, an antigen-binding portion of an anti-TIM-3 antibody as described herein, and / or an antigen-binding portion of an anti-LAG-3 antibody as described herein.

[0222] The present invention also provides a pharmaceutical composition comprising: 1) an anti-PD-1 antibody or an antigen-binding portion thereof as described herein, (2) an anti-TIM-3 antibody or an antigen-binding portion thereof and / or an anti-LAG-3 antibody or an antigen-binding portion thereof, and (3) a pharmaceutically acceptable excipient. The anti-TIM-3 antibody and the anti-LAG-3 antibody can be selected from those described herein.

[0223] In some embodiments, the pharmaceutical composition comprises:

[0224] a) an anti-PD-1 antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequence of SEQ ID NOs: 228-233; an anti-TIM-3 antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequence of SEQ ID NOs: 8-13; and an anti-LAG-3 antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequence of SEQ ID NOs: 318-323;

[0225] b) an anti-PD-1 antibody whose VH and VL comprise the amino acid sequences of SEQ ID NOs: 226 and 227, respectively; an anti-TIM-3 antibody whose VH and VL comprise the amino acid sequences of SEQ ID NOs: 7 and 4, respectively; and an anti-LAG-3 antibody whose VH and VL comprise the amino acid sequences of SEQ ID NOs: 316 and 317, respectively; or

[0226] c) an anti-PD-1 antibody comprising a HC comprising the amino acid sequence of SEQ ID NOs: 226 and 375 and a LC comprising the amino acid sequence of SEQ ID NOs: 227 and 379; an anti-TIM-3 antibody comprising a HC comprising the amino acid sequence of SEQ ID NOs: 7 and 377 and a LC comprising the amino acid sequence of SEQ ID NOs: 4 and 378; and an anti-LAG-3 antibody comprising a HC comprising the amino acid sequence of SEQ ID NOs: 316 and 375 and a LC comprising the amino acid sequence of SEQ ID NOs: 317 and 378.

[0227] The antibodies in the composition can be present in the same amount. In some embodiments, the pharmaceutical composition is used to treat human patients in the methods described herein. In specific embodiments, the pharmaceutical composition is used to improve immunity (for example) to treat cancer in human patients in need.

[0228] The present invention also provides an anti-PD-1 antibody, or an antigen-binding portion thereof, as described herein, for use in combination with an anti-TIM-3 antibody, or an antigen-binding portion thereof, and / or an anti-LAG-3 antibody, or an antigen-binding portion thereof, in the treatment methods described herein, e.g., to enhance immunity and / or treat cancer in a human patient in need thereof. The anti-TIM-3 antibody and the anti-LAG-3 antibody may be selected from those described herein.

[0229] The present invention also provides an anti-PD-1 antibody, or antigen-binding portion thereof, as described herein for use in treating a human patient in the methods described herein.

[0230] The present invention also provides the use of an anti-PD-1 antibody or an antigen-binding portion thereof as described herein for the preparation of a medicament for use in combination with an anti-TIM-3 antibody or an antigen-binding portion thereof and / or an anti-LAG-3 antibody or an antigen-binding portion thereof in a patient in need thereof (e.g., a human patient) to enhance immunity and / or treat cancer. In some embodiments, the present invention provides the use of an anti-PD-1 antibody or an antigen-binding portion thereof, and an anti-TIM-3 antibody or an antigen-binding portion thereof and / or an anti-LAG-3 antibody or an antigen-binding portion thereof as described herein for the preparation of a medicament for enhancing immunity (e.g., to treat cancer) in a human patient in need thereof. The anti-TIM-3 antibody and the anti-LAG-3 antibody can be selected from those described herein.

[0231] The present invention also provides the use of an anti-PD-1 antibody, or an antigen-binding portion thereof, as described herein for the preparation of a medicament for treating a human patient in the methods described herein.

[0232] The present invention further provides an article of manufacture comprising an anti-PD-1 antibody, or an antigen-binding portion thereof, as described herein, in combination with an anti-TIM-3 antibody, or an antigen-binding portion thereof, and / or an anti-LAG-3 antibody, or an antigen-binding portion thereof, wherein the article of manufacture is suitable for use, for example, in the therapeutic methods described herein, in patients (such as human patients) to enhance immunity and / or treat cancer. The anti-TIM-3 antibody and the anti-LAG-3 antibody may be selected from those described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0233] Figure 1The following is a set of graphs showing the percentage increase in IFN-γ levels in three donor pairs after 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 after 5 days of culture in a one-way MLR (mixed lymphocyte reaction) assay (panels A to C). Each point in the graph represents replicates, and the mean is indicated by a horizontal bar.

[0234] Figure 2 Figure 1 is a set of graphs showing the percent increase in IFN-γ levels in three donor pairs after 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 in a two-way MLR assay after 5 days of culture (panels AC). Each point in the graph represents replicates, and the mean is indicated by a horizontal bar.

[0235] Figure 3 Figures 1 and 2 show the efficacy of anti-TIM-3 antibody 15086.17145 on T cell proliferation in a one-way MLR assay compared to the positive control anti-PD-1 antibody 12819. Antibodies were added to a final concentration of 25 μg / mL and cultured for 5 days in the absence (panel A) or presence (panel B) of soluble anti-CD3. 3H-thymidine was then added at 1 μCi / well for an additional 18 hours. 3H-thymidine incorporation in the harvested cells was measured by liquid scintillation counting.

[0236] Figure 4 This graph shows the effect of anti-TIM-3 antibody 15086.17145 on IL-12p40 secretion from monocyte-derived dendritic cells incubated with 10 μg / mL of the antibody for 5 days. IL-12p40 levels in cell supernatants were measured using ELISA.

[0237] Figure 5 Figure 1 is a pair of graphs showing the expression levels of selected activation markers on monocyte-derived dendritic cells after 24-hour treatment with 25 μg / mL of the anti-TIM-3 antibody 15086.17145. Panel A shows gene expression analysis of several activation markers and co-stimulatory molecules. Panel B shows verification of increased CD80 and CD86 levels using FACS. The displayed histogram overlay represents CD11c+ dendritic cells, and the numbers adjacent to the histograms indicate MFI (mean fluorescence intensity) values.

[0238] Figure 6is a graph showing the following: CD34 + Efficacy of TIM-3 targeting on tumor growth in the humanized NSG-SGM3 mouse model. Mice were treated with the anti-TIM-3 antibody 15086.17145 at an initial dose of 10 mg / kg followed by 5 mg / kg 5x Q5D. Gray areas indicate treatment periods. Data are presented as mean ± SEM. **p < 0.01.

[0239] Figure 7 Figure 1 is a pair of graphs showing the efficacy of combining anti-PD-1 antibody 12819 with anti-LAG-3 antibody 15532 and / or anti-TIM-3 antibody 15086.17145 in increasing IL-2 levels in the SEB (Staphylococcal Enterotoxin B) + PBMC (Peripheral Blood Mononuclear Cell) assay for two donor pairs (panels A and B). The horizontal bar indicates IL-2 secretion from PBMCs treated with 10 μg / mL of the indicated antibody or antibody mixture (and SEB) for 48 hours. Symbols above the horizontal bar indicate single treatments (*) that were significantly different from the control antibody (p < 0.05) or a mixture of two antibodies (#) that were significantly different from the constitutive single antibody treatment.

[0240] Figure 8 Figure 1 shows the efficacy of 10 mg / kg of the anti-PD-1 antibody 12819, the anti-LAG-3 antibody C9B7W (which is reactive against mouse LAG-3), or a combination of anti-PD-1 and anti-LAG-3 antibodies, or vehicle treatment on tumor growth in two syngeneic mouse tumor models: MC38 (colon cancer, panel A) and ASB-XIV (lung cancer, panel B). Gray areas indicate treatment periods. Data are presented as mean ± SEM (standard error of the mean). **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0241] Figure 9 Figure 1 shows the efficacy 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 vehicle treatment on tumor growth in the ASB-XIV syngeneic mouse tumor model. Gray areas indicate treatment periods. Data are presented as mean ± SEM. *p < 0.05, ****p < 0.0001.

[0242] Figure 10Figure 1 is a pair of graphs showing the efficacy of anti-PD-1 antibody 12819, anti-TIM-3 antibody 5D12, or a combination of anti-PD-1 and anti-TIM-3 antibodies, or vehicle treatment, on tumor growth in two syngeneic mouse tumor models: ASB-XIV (lung cancer, panel A) and SalN (fibrosarcoma, panel B). Antibody treatment was administered at a dose of 10 mg / kg / target in mice bearing ASB-XIV tumors. Mice bearing SalN tumors were dosed with anti-PD-1 and anti-TIM-3 antibodies at 1 mg / kg and 10 mg / kg, respectively. Gray areas indicate treatment periods. Data are presented as mean ± SEM. **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0243] Figure 11 : is a set of graphs showing the efficacy 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 vehicle treatment on tumor growth in a human xenograft tumor model (where the human melanoma cell line A375 is implanted into mice reconstituted with human PBMCs). One human PBMC donor was used in each experiment, and three graphs represent three different donors (subgraphs AC). The gray area represents the treatment period. Data are presented as mean ± SEM. *p < 0.05 and **p < 0.01.

[0244] Figure 12 : is a set of graphs showing the efficacy of single and dual targeting of PD-1 and TIM-3 on survival percentage (sub-graph A) and tumor growth (sub-graphs BE) in NOD-scid mice implanted 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. For each antibody, antibody treatment was administered three times a week at a dose of 10 mg / kg. The gray area represents the treatment period. The data in sub-graph A are based on the time course of mice with <400 mm Hg in each treatment group. 3 The tumor sizes are presented as percentages of mice.

[0245] Figure 13Figure 1 is a set of graphs showing the efficacy of single, dual, and triple targeting of PD-1, LAG-3, and TIM-3 on survival percentage (panel A) and tumor growth (panels BH) in the 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-PD-1 and anti-LAG3 antibodies, a combination of anti-PD-1 and anti-TIM-3 antibodies, or a "triple combination" (which refers to a combination of anti-PD-1, anti-LAG-3, and anti-TIM-3 antibodies). For each antibody, antibody treatment was administered at a dose of 10 mg / kg. The gray area represents the treatment period. DETAILED DESCRIPTION

[0246] The present invention provides novel combination therapies and compositions targeting human PD-1, human TIM-3, and / or human LAG-3 using antibodies that bind to these targets. The therapies and compositions can be used to boost the immune system in human patients, such as cancer patients. Unless otherwise stated, as used herein, "PD-1" refers to human PD-1. The human PD-1 polypeptide sequence is available as Uniprot Accession No. Q15116 and is shown herein as SEQ ID NO: 388. Unless otherwise stated, as used herein, "TIM-3" refers to human TIM-3. The human TIM-3 polypeptide sequence is available as Uniprot Accession No. Q8TDQ0 and is shown herein as SEQ ID NO: 389. Unless otherwise stated, as used herein, "LAG-3" refers to human LAG-3. The human LAG-3 polypeptide sequence is available as Uniprot Accession No. P18627 and is shown herein as SEQ ID NO: 390.

[0247] As used herein, the term "antibody" (Ab) or "immunoglobulin" (Ig) refers to a tetramer comprising two heavy (H) chains (about 50-70 kDa) and two light (L) chains (about 25 kDa) interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable domain (VH) and a heavy chain constant region (CH). Each light chain is composed of a light chain variable domain (VL) and a light chain constant region (CL). The VH and VL domains can be further subdivided into highly variable regions called "complementarity determining regions" (CDRs), interspersed with more conserved regions called "framework regions" (FRs). Each VH and VL is composed of three CDRs (H-CDR refers to CDRs from the heavy chain in this article; and L-CDR refers to CDRs from the light chain in this article) and four FRs, which are arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The assignment of amino acid numbers in the heavy chain or light chain can be based on Definitions (Lefranc et al., Dev Comp Immunol 27(1):55-77 (2003)); or 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 indicated, all antibody amino acid residue numbers referred to in the disclosure herein are according to Those with numbering schemes.

[0248] The term "recombinant antibody" refers to an antibody that is expressed from a cell or cell line that contains a nucleotide sequence encoding the antibody, where the nucleotide sequence is not naturally associated with the cell.

[0249] The terms "isolated protein," "isolated polypeptide," or "isolated antibody" refer to a protein, polypeptide, or antibody that, by reason of its origin or source of derivation, (1) is not associated with naturally associated components that accompany it in its native state, (2) is free of other proteins from the same species, (3) is expressed by cells from a different species, and / or (4) does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it is naturally derived is "isolated" from its naturally associated components. A protein may also be rendered substantially free of naturally associated components by isolation using protein purification techniques well known in the art.

[0250] The term "affinity" refers to a measure of the attractive force between an antigen and an antibody. The intrinsic attractiveness of an antibody to an antigen is typically expressed as the equilibrium constant (KD) for the binding affinity of a particular antibody-antigen interaction. D When the concentration is ≤1 mM, preferably ≤100 nM, the antibody is said to specifically bind to the antigen. D Binding affinity constants can be determined, for example, by surface plasmon resonance (SPR) (BIAcore TM ) or biofilm interferometry, for example using the IBIS MX96 SPR system from IBIS Technologies, the ProteOn TM XPR36 SPR system, or Octet from ForteBio TM System measurement.

[0251] 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 biofilm interferometry, eg, using one of the systems listed above.

[0252] The term "epitope" is used herein to refer to the portion (determinant) of an antigen that specifically binds to an antibody or related molecule (such as a bispecific binding molecule). Epitope determinants are generally composed of chemically active surface groupings of molecules such as amino acids or carbohydrates or sugar side chains and generally have special three-dimensional spatial structural characteristics, as well as special charge characteristics. Epitopes can be "linear" or "conformational". In a linear epitope, all interactions between a protein (e.g., an antigen) and an interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, the interaction point occurs across amino acid residues on the protein that are separated from each other in terms of the primary amino acid sequence. Once the desired epitope on the antigen is determined, it is possible to generate antibodies against the epitope using techniques well known in the art. For example, antibodies against linear epitopes can be generated, for example, by immunizing an animal with a peptide having the amino acid residues of a linear epitope. Antibodies against conformational epitopes can be generated, for example, by immunizing an animal with a mini-domain containing the relevant amino acid residues of the conformational epitope. Antibodies directed against a specific epitope can also be generated, for example, by immunizing an animal with the target molecule of interest or a relevant portion thereof, followed by screening for binding to the epitope.

[0253] Whether an antibody binds to the same epitope as an antibody as described herein or competes for binding with an antibody as 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 the antibody as described herein bind to at least one common residue (e.g., at least two, three, four, five, six, seven, eight, or nine residues) on the target protein (i.e., TIM-3, PD-1, or LAG-3). In another embodiment, the contact residues on the target protein are exactly the same between the test antibody and the antibody as described herein. In one embodiment, the antibody as described herein is allowed to bind to the target protein under saturating conditions and then the ability of the test antibody to bind to the target protein is measured. If the test antibody and the reference antibody are able to bind to the target protein at the same time, the test antibody and the reference antibody bind to different epitopes. However, if the test antibody cannot bind to the target protein at the same time, the test antibody binds to the same epitope, an overlapping epitope, or an epitope very close to the epitope bound by the antibody as described herein. This experiment can be performed using (for example) ELISA, RIA, BIACORE TMTo test whether an antibody cross-competes with another antibody, the competition method described above can be used in two directions, i.e., to determine whether a known antibody blocks the test antibody and vice versa. Such cross-competition experiments can be performed, for example, using an IBIS MX96 SPR instrument or an Octet TM System carried out.

[0254] The term "chimeric antibody" in its broadest sense refers to an antibody containing one or more regions from an antibody and one or more regions from one or more other antibodies, typically partly human and partly non-human, i.e., partly derived from non-human animals, such as mice, rats or other rodents, or birds, such as chicken antibodies. In order to reduce the risk of human anti-antibody responses (such as human anti-mouse antibody responses in the example of murine antibodies), chimeric antibodies are preferred over non-human antibodies. Typical examples of chimeric antibodies are antibodies in which the variable domain sequence is murine and the constant region sequence is human. In the example of chimeric antibodies, the non-human portion can undergo additional changes to humanize the antibody. The chimeric antibody described herein has a chicken variable domain sequence and a human constant region sequence.

[0255] The term "humanization" refers to the fact that when an antibody is of fully or partially non-human origin (e.g., a murine or chicken antibody obtained by immunizing a mouse or chicken, respectively, with an antigen of interest, or a chimeric antibody based on such a murine or chicken antibody), it is possible to replace certain amino acids (particularly in the framework and constant regions of the heavy and light chains) to avoid or minimize an immune response in humans. Although it is impossible to accurately predict the immunogenicity of a particular antibody and thus its human anti-antibody response, non-human antibodies tend to be more immunogenic in humans than human antibodies. Chimeric antibodies (in which a foreign (e.g., rodent or bird) constant region has been replaced with a sequence of human origin) have been shown to be generally less immunogenic than antibodies of completely foreign origin, and the trend in therapeutic antibodies is towards humanized or fully human antibodies. Therefore, chimeric antibodies or other antibodies of non-human origin can be humanized to reduce the risk of human anti-antibody response.

[0256] For chimeric antibodies, humanization typically involves modification of the framework regions of the variable domain sequence. Amino acid residues that are part of the complementarity determining regions (CDRs) are generally not altered for humanization, although in some cases it may be desirable to alter individual CDR amino acid residues, for example to remove glycosylation sites, deamidation sites, aspartate isomerization sites, or unwanted cysteine ​​or methionine residues. N-linked glycosylation occurs by the attachment of oligosaccharide chains to asparagine residues in the tripeptide sequences Asn-X-Ser or Asn-X-Thr (where X can be any amino acid except Pro). Removal of N-glycosylation positions can be achieved by mutating the Asn or Ser / Thr residues to different residues, preferably by conservative substitutions. Deamidation of asparagine and glutamine residues can occur based on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation, primarily when present in the sequence Asn-Gly, and to a lesser extent when present in other dipeptide sequences such as Asn-Ala. When such a deamidation site (particularly Asn-Gly) is present in a CDR sequence, it may therefore be desirable to remove the site (typically by conservative substitution to remove one of the residues involved).

[0257] A large number of methods for humanizing antibody sequences are known in the art; see, for example, Almagro & Fransson, Front Biosci. 13: 1619-1633 (2008) for a review. One commonly used method is CDR grafting, which, for example, involves the identification of human germline gene counterparts of murine variable domain genes and the grafting of murine CDR sequences onto this framework for murine-derived chimeric antibodies. The specificity of the interaction between an antibody and a target antigen resides primarily in the amino acid residues located in the six CDRs of the heavy and light chains. Therefore, the amino acid sequences within the CDRs are much more variable between individual antibodies than the sequences located outside the CDRs. Since the CDR sequences are responsible for most of the antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a specific naturally occurring antibody, or more generally, any specific antibody having a given amino acid sequence, for example, by constructing an expression vector that expresses the CDR sequences from that specific antibody grafted onto framework sequences from a different antibody. Thus, it is possible to "humanize" a non-human antibody and still substantially maintain the binding specificity and affinity of the source antibody. CDR grafting can be based on the Kabat CDR definition, although a recent publication (Magdelaine-Beuzelin et al., Crit Rev. Oncol Hematol. 64:210-225 (2007)) has suggested that Definition (International ImMunoGeneTics Information System) , www.imgt.org) can improve the results of humanization (see Lefranc et al., Dev. Comp Immunol. 27:55-77 (2003)).

[0258] In some cases, CDR grafting can reduce the binding specificity and affinity of the non-human antibody through the grafted CDR, and thus reduce its biological activity (compared to the parent antibody from which the CDR is obtained). Back mutations (sometimes referred to as "framework repair") can be introduced into the selected positions of the antibody through the grafted CDR (typically in the framework region) to rebuild the binding specificity and affinity of the parent antibody. The position of the possible back mutation can be identified using the information available in the literature and in the antibody database. The amino acid residues that are candidates for back mutations are typically those located on the surface of the antibody molecule, and generally do not change the embedded or low surface exposure residues.

[0259] An alternative humanization technique to CDR grafting and backmutation is resurfacing, in which non-surface exposed residues of non-human origin are retained, while surface residues are changed to human residues.

[0260] In some cases, it may be desirable to alter one or more CDR amino acid residues to improve binding affinity for the target epitope. This is known as "affinity maturation." 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 of Wu et al., Proc Natl Acad Sci USA 95:6037–6042 (1998).

[0261] The term "human antibody" refers to antibodies whose variable and constant region sequences are derived from human sequences. The term encompasses antibodies having sequences derived from human genes but that have been modified, for example, to reduce immunogenicity, increase affinity, and / or increase stability. In addition, the term encompasses antibodies recombinantly produced in non-human cells using human-derived sequences that confer glycosylation that is atypical for human cells. The term also encompasses transgenic non-human organisms (e.g., rat).

[0262] As used herein, the term "antigen-binding portion" of an antibody (or simply "antibody portion") 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). Certain fragments of full-length antibodies have been shown to perform the antigen-binding function of an antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" include (i) Fab fragments: consisting of V L 、V H 、C L and C H 1 domain; (ii) F(ab')2 fragment: a bivalent fragment consisting of two Fab fragments connected by a disulfide bond located in the hinge region; (iii) H and C H 1 domain; (iv) a V fragment consisting of a single arm of the antibody L and V H Fv fragment composed of V domain, (v)dAb fragment, which consists of V H domains; and (vi) isolated complementary determining regions (CDRs) capable of specifically binding to an antigen. L and V H ) are encoded by separate genes that can be expressed using recombinant methods by enabling them to express V L and V H The domains are paired and connected by a synthetic linker to form a single protein chain that forms a monovalent molecule known as a single-chain Fv (scFv). Also included in the present invention are proteins comprising V H and / or V L Antigen binding molecules. H In the case of , the molecule may also comprise one or more of the CH1, hinge, CH2, or CH3 regions. Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Other forms of single-chain antibodies (such as diabodies) are also encompassed. Diabodies are bivalent bispecific antibodies in which V H and V L The domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites.

[0263] Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from intact antibodies using conventional techniques, such as papain or pepsin digestion of intact antibodies. In addition, antibodies, antibody portions, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, e.g., as described herein.

[0264] The type (isotype) and subclass of the antibodies described herein can be determined by any method known in the art. In general, the type and subclass of an antibody can be determined using antibodies specific for a particular type and subclass of the antibody. Such antibodies are commercially available. The type and subclass can be determined by ELISA and Western blotting, as well as other techniques. Alternatively, the type and subclass can be determined by sequencing all or part of the heavy chain and / or light chain constant regions of the antibody, comparing their amino acid sequences with known amino acid sequences of various types and subclasses of immunoglobulins, and determining the type and subclass of the antibody. The preferred isotype of the present invention is the IgG isotype.

[0265] When referring to a specific amino acid residue at a given position of an antibody sequence, the designation "35S" for example refers to both the position and the residue, i.e., in this case, the serine residue (S) is present at position 35 of the sequence. Similarly, the designation "13Q+35S" for example refers to two residues at the respective position. Unless otherwise indicated, all references to antibody amino acid residue numbering in this disclosure are based on Those with numbering schemes.

[0266] Anti-PD-1 antibodies

[0267] In some embodiments, the anti-PD-1 antibodies disclosed herein may be chimeric (with variable domains derived from chicken and human constant regions) or may be humanized.

[0268] The anti-PD-1 antibodies disclosed herein may be referred to by a 5-digit number (e.g., "12819") or a 10-digit number (e.g., "12819.15384"). As used herein, the 5-digit number refers to all antibodies having the heavy and light chain CDR1-3 sequences shown for that number, while the use of the 10-digit number refers to a particular humanized variant. For example, 12819.15384 is a particular humanized variant having the CDR sequences of the 12819 antibody. This 5-digit number encompasses 10-digit variants that are identical except for some changes in the FRs (e.g., lack of residue SY at the N-terminus of the mature light chain, or having residue SS in place of SY). These modifications do not alter the functional (e.g., antigen binding) properties of the antibody.

[0269] In some embodiments, the combination therapy or composition comprises an anti-PD-1 antibody, or an antigen-binding portion thereof, wherein the anti-PD-1 antibody is 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 a variant of any of these, wherein the variant may, for example, contain certain minimal amino acid changes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes, which may, for example, be located in the framework regions) relative to the antibody without losing the antigen-binding specificity of the antibody.

[0270] In some embodiments, the anti-PD-1 antibody competes for binding to human PD-1 with, or binds to the same epitope of human PD-1 as, any of antibodies 12819.15384, 12748.15381, 12748.16124, 12865.15377, 12892.15378, 12796.15376, 12777.15382, 12760.15375, and 13112.15380.

[0271] In some embodiments, any anti-PD-1 antibody or antigen-binding portion described herein can compete or cross-compete for binding to PD-1 with the following: 12865, 12892, and 12777 antibodies (e.g., antibodies 12865.15377, 12892.15378, and 12777.15382). In some embodiments, any anti-PD-1 antibody or antigen-binding portion described herein can compete or cross-compete for binding to PD-1 with the following: 12819 antibody (e.g., antibody 12819.15384). In some embodiments, any anti-PD-1 antibody or antigen-binding portion described herein can compete or cross-compete for binding to PD-1 with the following: 12760 and 13112 antibodies (e.g., antibodies 12760.15375 and 13112.15380). In some embodiments, the antibody has an IgG1 or IgG2 format. In certain embodiments, the antibody has an IgG1 format.

[0272] In some embodiments, the anti-PD-1 antibody competes or cross-competes for binding to human PD-1 or binds to the same epitope of human PD-1 as an antibody whose heavy chain (H) CDR1-3 and light chain (L) CDR1-3 comprise SEQ ID NOs: 228-233, 238-243, 248-253, 258-263, 268-273, 278-283, 288-293, or 298-303, respectively.

[0273] In some embodiments, the anti-PD-1 antibody comprises an H-CDR3 comprising the following H-CDR3 amino acid sequence: SEQ ID NO: 230, 240, 250, 260, 270, 280, 290, or 300.

[0274] In some embodiments, the anti-PD-1 antibody contains H-CDR1-3 comprising the following H-CDR1-3 amino acid sequences, respectively: SEQ ID NO: 228-230, 238-240, 248-250, 258-260, 268-270, 278-280, 288-290, or 298-300.

[0275] In some embodiments, the anti-PD-1 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 NO: 226, 236, 246, 256, 266, 276, 286, or 296.

[0276] In some embodiments, the anti-PD-1 antibody has a VH comprising SEQ ID NO: 226, 236, 246, 256, 266, 276, 286, or 296.

[0277] In some embodiments, the anti-PD-1 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 sequence to SEQ ID NO: 226, 236, 246, 256, 266, 276, 286, or 296; and a CH 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 sequence to SEQ ID NO: 375.

[0278] In some embodiments, the anti-PD-1 antibody has a 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.

[0279] In some embodiments, the anti-PD-1 antibody has an L-CDR3 comprising the following L-CDR3 amino acid sequence: SEQ ID NO: 233, 243, 253, 263, 273, 283, 293, or 303.

[0280] In some embodiments, the anti-PD-1 antibody comprises L-CDR1-3 comprising the following L-CDR1-3 amino acid sequences, respectively: SEQ ID NO: 231-233, 241-243, 251-253, 261-263, 271-273, 281-283, 291-293, or 301-303.

[0281] In some embodiments, the anti-PD-1 antibody has a VL that is at least 90% identical in sequence (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to the VL amino acid sequence of SEQ ID NO: 227, 237, 247, 257, 267, 277, 287, 297, or 392.

[0282] In some embodiments, the anti-PD-1 antibody has a VL comprising the VL amino acid sequence of SEQ ID NO: 227, 237, 247, 257, 267, 277, 287, 297, or 392.

[0283] In some embodiments, the anti-PD-1 antibody has a VL 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 to the VL amino acid sequence of SEQ ID NO: 227, 237, 247, 257, 267, 277, 287, 297, or 392; and a CL 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 to SEQ ID NO: 379.

[0284] In some embodiments, the anti-PD-1 antibody has a 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.

[0285] In some embodiments, the anti-PD-1 antibody comprises any of the above heavy chain sequences and any of the above light chain sequences.

[0286] In some embodiments, the anti-PD-1 antibody comprises H-CDR3 and L-CDR3 comprising the following H-CDR3 and L-CDR3 amino acid sequences, respectively: SEQ ID NO: 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.

[0287] In some embodiments, the anti-PD-1 antibody comprises H-CDR1-3 and L-CDR1-3 comprising the following H-CDR1-3 and L-CDR1-3 sequences, respectively: SEQ ID NO: 228-233, 238-243, 248-253, 258-263, 268-273, 278-283, 288-293, or 298-303.

[0288] In some embodiments, the anti-PD-1 antibody comprises a VH 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 to the amino acid sequence of SEQ ID NO: 226, 236, 246, 256, 266, 276, 286, or 296, and a VL 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 to the amino acid sequence of SEQ ID NO: 227, 237, 247, 257, 267, 277, 287, 297, or 392.

[0289] In some embodiments, the anti-PD-1 antibody has a VH comprising the amino acid sequence of SEQ ID NO: 226, 236, 246, 256, 266, 276, 286, or 296, and a VL comprising the amino acid sequence of SEQ ID NO: 227, 237, 247, 257, 267, 277, 287, 297, or 392.

[0290] In some embodiments, the anti-PD-1 antibody has a HC comprising the amino acid sequence of SEQ ID NO: 226, 236, 246, 256, 266, 276, 286, or 296 and SEQ ID NO: 375; and a LC comprising the amino acid sequence of SEQ ID NO: 227, 237, 247, 257, 267, 277, 287, 297, or 392 and SEQ ID NO: 379.

[0291] In some embodiments, the anti-PD-1 antibody comprises the following H-CDR1-3 and L-CDR1-3 amino acid sequences:

[0292] a) SEQ ID NOs: 228-233, respectively;

[0293] b) SEQ ID NOs: 238-243, respectively;

[0294] c) SEQ ID NOs: 248-253, respectively;

[0295] d) SEQ ID NOs: 258-263, respectively;

[0296] e) SEQ ID NOs: 268-273, respectively;

[0297] f) SEQ ID NOs: 278-283, respectively;

[0298] g) SEQ ID NO: 288-293, respectively; or

[0299] h) SEQ ID NOs: 298-303, respectively.

[0300] In some embodiments, the anti-PD-1 antibody comprises VH and VL having the following amino acid sequences:

[0301] a) SEQ ID NOs: 226 and 227, respectively;

[0302] b) SEQ ID NOs: 236 and 237, respectively;

[0303] c) SEQ ID NOs: 236 and 392, respectively;

[0304] d) SEQ ID NOs: 246 and 247, respectively;

[0305] e) SEQ ID NOs: 256 and 257, respectively;

[0306] f) SEQ ID NOs: 266 and 267, respectively;

[0307] g) SEQ ID NOs: 276 and 277, respectively;

[0308] h) SEQ ID NOs: 286 and 287, respectively; or

[0309] i) SEQ ID NO: 296 and 297, respectively.

[0310] In some embodiments, the anti-PD-1 antibody comprises:

[0311] a) an HC comprising the amino acid sequence of SEQ ID NO: 226 and the amino acid sequence of SEQ ID NO: 375, and an LC comprising the amino acid sequence of SEQ ID NO: 227 and the amino acid sequence of SEQ ID NO: 379;

[0312] b) an HC comprising the amino acid sequence of SEQ ID NO: 236 and the amino acid sequence of SEQ ID NO: 375, and an LC comprising the amino acid sequence of SEQ ID NO: 237 and the amino acid sequence of SEQ ID NO: 379;

[0313] c) an HC comprising the amino acid sequence of SEQ ID NO: 236 and the amino acid sequence of SEQ ID NO: 375, and an LC comprising the amino acid sequence of SEQ ID NO: 392 and the amino acid sequence of SEQ ID NO: 379;

[0314] d) an HC comprising the amino acid sequence of SEQ ID NO: 246 and the amino acid sequence of SEQ ID NO: 375, and an LC comprising the amino acid sequence of SEQ ID NO: 247 and the amino acid sequence of SEQ ID NO: 379;

[0315] e) an HC comprising the amino acid sequence of SEQ ID NO: 256 and the amino acid sequence of SEQ ID NO: 375, and an LC comprising the amino acid sequence of SEQ ID NO: 257 and the amino acid sequence of SEQ ID NO: 379;

[0316] f) an HC comprising the amino acid sequence of SEQ ID NO: 266 and the amino acid sequence of SEQ ID NO: 375, and an LC comprising the amino acid sequence of SEQ ID NO: 267 and the amino acid sequence of SEQ ID NO: 379;

[0317] g) an HC comprising the amino acid sequence of SEQ ID NO: 276 and the amino acid sequence of SEQ ID NO: 375, and an LC comprising the amino acid sequence of SEQ ID NO: 277 and the amino acid sequence of SEQ ID NO: 379;

[0318] h) a HC comprising the amino acid sequence of SEQ ID NO: 286 and the amino acid sequence of SEQ ID NO: 375, and a LC comprising the amino acid sequence of SEQ ID NO: 287 and the amino acid sequence of SEQ ID NO: 379; or

[0319] i) HC comprising the amino acid sequence of SEQ ID NO: 296 and the amino acid sequence of SEQ ID NO: 375, and LC comprising the amino acid sequence of SEQ ID NO: 297 and the amino acid sequence of SEQ ID NO: 379.

[0320] In some embodiments, the anti-PD-1 antibody is selected from the group consisting of:

[0321] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 228-230, respectively;

[0322] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 226;

[0323] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 226;

[0324] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NOs: 226 and 375;

[0325] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 231-233, respectively;

[0326] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 227;

[0327] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 227;

[0328] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 227 and 379;

[0329] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequence of SEQ ID NOs: 228-233;

[0330] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 226 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 227;

[0331] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 226 and whose VL comprises the amino acid sequence of SEQ ID NO: 227; and

[0332] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 226 and the amino acid sequence of SEQ ID NO: 375; and whose LC comprises the amino acid sequences of SEQ ID NOs: 227 and 379.

[0333] In some embodiments, the anti-PD-1 antibody is selected from the group consisting of:

[0334] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 238-240, respectively;

[0335] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 236;

[0336] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 236;

[0337] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NOs: 236 and 375;

[0338] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 241-243, respectively;

[0339] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 237;

[0340] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 237;

[0341] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 237 and 379;

[0342] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequence of SEQ ID NOs: 238-243;

[0343] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 236 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 237;

[0344] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 236 and whose VL comprises the amino acid sequence of SEQ ID NO: 237; and

[0345] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 236 and the amino acid sequence of SEQ ID NO: 375; and whose LC comprises the amino acid sequences of SEQ ID NOs: 237 and 379.

[0346] In some embodiments, the anti-PD-1 antibody is selected from the group consisting of:

[0347] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 238-240, respectively;

[0348] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 236;

[0349] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 236;

[0350] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NOs: 236 and 375;

[0351] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 241-243, respectively;

[0352] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 392;

[0353] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 392;

[0354] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 392 and 379;

[0355] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequence of SEQ ID NOs: 238-243;

[0356] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 236 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 392;

[0357] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 236 and whose VL comprises the amino acid sequence of SEQ ID NO: 392; and

[0358] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 236 and the amino acid sequence of SEQ ID NO: 375; and whose LC comprises the amino acid sequences of SEQ ID NOs: 392 and 379.

[0359] In some embodiments, the anti-PD-1 antibody is selected from the group consisting of:

[0360] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 248-250, respectively;

[0361] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 246;

[0362] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 246;

[0363] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NOs: 246 and 375;

[0364] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 251-253, respectively;

[0365] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 247;

[0366] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 247;

[0367] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 247 and 379;

[0368] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequence of SEQ ID NOs: 248-253;

[0369] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 246 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 247;

[0370] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 246 and whose VL comprises the amino acid sequence of SEQ ID NO: 247; and

[0371] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 246 and the amino acid sequence of SEQ ID NO: 375; and whose LC comprises the amino acid sequences of SEQ ID NOs: 247 and 379.

[0372] In some embodiments, the anti-PD-1 antibody is selected from the group consisting of:

[0373] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 258-260, respectively;

[0374] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 256;

[0375] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 256;

[0376] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NOs: 256 and 375;

[0377] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 261-263, respectively;

[0378] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 257;

[0379] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 257;

[0380] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 257 and 379;

[0381] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequence of SEQ ID NOs: 258-263;

[0382] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 256 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 257;

[0383] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 256 and whose VL comprises the amino acid sequence of SEQ ID NO: 257; and

[0384] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 256 and the amino acid sequence of SEQ ID NO: 375; and whose LC comprises the amino acid sequences of SEQ ID NOs: 257 and 379.

[0385] In some embodiments, the anti-PD-1 antibody is selected from the group consisting of:

[0386] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 268-270, respectively;

[0387] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 266;

[0388] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 266;

[0389] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NOs: 266 and 375;

[0390] e) an antibody whose L-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 271-273, respectively;

[0391] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 267;

[0392] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 267;

[0393] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 267 and 379;

[0394] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequence of SEQ ID NOs: 268-273;

[0395] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 266 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 267;

[0396] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 266 and whose VL comprises the amino acid sequence of SEQ ID NO: 267; and

[0397] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 266 and the amino acid sequence of SEQ ID NO: 375; and whose LC comprises the amino acid sequences of SEQ ID NOs: 267 and 379.

[0398] In some embodiments, the anti-PD-1 antibody is selected from the group consisting of:

[0399] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 278-280, respectively;

[0400] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 276;

[0401] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 276;

[0402] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NOs: 276 and 375;

[0403] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 281-283, respectively;

[0404] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 277;

[0405] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 277;

[0406] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 277 and 379;

[0407] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequence of SEQ ID NOs: 278-283;

[0408] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 276 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 277;

[0409] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 276 and whose VL comprises the amino acid sequence of SEQ ID NO: 277; and

[0410] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 276 and the amino acid sequence of SEQ ID NO: 375; and whose LC comprises the amino acid sequences of SEQ ID NOs: 277 and 379.

[0411] In some embodiments, the anti-PD-1 antibody is selected from the group consisting of:

[0412] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 288-290, respectively;

[0413] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 286;

[0414] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 286;

[0415] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NOs: 286 and 375;

[0416] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 291-293, respectively;

[0417] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 287;

[0418] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 287;

[0419] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 287 and 379;

[0420] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequence of SEQ ID NOs: 288-293;

[0421] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 286 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 287;

[0422] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 286 and whose VL comprises the amino acid sequence of SEQ ID NO: 287; and

[0423] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 286 and the amino acid sequence of SEQ ID NO: 375; and whose LC comprises the amino acid sequences of SEQ ID NOs: 287 and 379.

[0424] In some embodiments, the anti-PD-1 antibody is selected from the group consisting of:

[0425] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 298-300, respectively;

[0426] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 296;

[0427] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 296;

[0428] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NOs: 296 and 375;

[0429] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 301-303, respectively;

[0430] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 297;

[0431] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 297;

[0432] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 297 and 379;

[0433] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequence of SEQ ID NOs: 298-303;

[0434] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 296 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 297;

[0435] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 296 and whose VL comprises the amino acid sequence of SEQ ID NO: 297; and

[0436] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 296 and the amino acid sequence of SEQ ID NO: 375; and whose LC comprises the amino acid sequences of SEQ ID NOs: 297 and 379.

[0437] In some embodiments, any anti-PD-1 antibody or antigen-binding portion described herein may have a K of D Binding to human PD-1: 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. In certain embodiments, the KD is determined using surface plasmon resonance. In specific embodiments, the anti-PD-1 antibody, or antigen-binding portion, binds to human PD-1 with a greater affinity than nivolumab, pembrolizumab, or both.

[0438] In some embodiments, any anti-PD-1 antibody or antigen-binding portion described herein may have a K of DBinding to cynomolgus monkey PD-1: 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. In certain embodiments, the K D Surface plasmon resonance was used for determination.

[0439] In some embodiments, any anti-PD-1 antibody or antigen-binding portion described herein may have a K of D Binding to mouse PD-1: at least 1000, at least 950, at least 900, or at least 850 pM. In certain embodiments, the K D Surface plasmon resonance was used for determination.

[0440] In some embodiments, any of the anti-PD-1 antibodies or antigen-binding portions described herein can inhibit the interaction of 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% at a concentration of 10 μg / mL in a flow cytometry competition assay. In certain embodiments, the anti-PD-1 antibody or antigen-binding portion can inhibit the interaction of PD-1 and PD-L1 by at least 83%.

[0441] In some embodiments, any of the anti-PD-1 antibodies or antigen-binding portions described herein can 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 biofilm interferometry analysis. In certain embodiments, the anti-PD-1 antibody or antigen-binding portion blocks the binding of PD-L1 and PD-L2 to PD-1 by at least 90%.

[0442] In some embodiments, the anti-PD-1 antibodies or antigen-binding portions described herein have at least one of the following properties:

[0443] a) With a K of 750 pM or lower D Binds to human PD-1;

[0444] b) with a K of 7 nM or lower D Binds to cynomolgus monkey PD-1;

[0445] c) with a K of 1 nM or lower D Binds to mouse PD-1;

[0446] d) does not bind to rat PD-1;

[0447] e) increased IL-2 secretion in the SEB whole blood assay;

[0448] f) increased IFN-γ secretion in a one-way mixed lymphocyte reaction assay;

[0449] g) inhibits the interaction of PD-1 and PD-L1 by at least 60% at a concentration of 10 μg / mL in a flow cytometry competition assay;

[0450] h) blocks the binding of PD-L1 and PD-L2 to PD-1 by at least 90% at a concentration of 10 μg / mL as determined by biofilm interferometry analysis; and

[0451] i) Inhibition of tumor growth in vivo.

[0452] Examples of such antibodies include, but are not limited to, antibody 12819 (having properties ai); antibodies 12748, 12892, and 12777 (having at least properties a, b, and eh); antibodies 12865 and 12796 (having at least properties a, b, e, f, and h), and antibodies 12760 and 13112 (having at least properties a, b, e, and f). In some embodiments, the anti-PD-1 antibody or antigen-binding portion has all of the aforementioned properties. In some embodiments, the anti-PD-1 antibody or antigen-binding portion has at least properties a, b, and eh. In some embodiments, the anti-PD-1 antibody or antigen-binding portion has at least properties a, b, e, f, and h. In some embodiments, the anti-PD-1 antibody or antigen-binding portion has at least properties a, b, e, and f.

[0453] In some embodiments, an anti-PD-1 antibody or antigen-binding portion thereof as described herein binds to an epitope of PD-1 comprising at least one (e.g., at least one, at least two, at least three, at least four, or at least five) of the following residues of SEQ ID NO: 388: V44, V64, L128, P130, K131, A132, E136, and T145. In certain embodiments, the antibody or antigen-binding portion binds to an epitope of PD-1 comprising residues V64, L128, P130, K131, and A132 of SEQ ID NO: 388 (such as the 12819 antibody, e.g., antibody 12819.15384). In certain embodiments, the antibody or antigen-binding portion binds to an epitope of PD-1 comprising residues K131 and E136 of SEQ ID NO: 388 (such as the 12865 antibody, e.g., antibody 12865.15377). In certain embodiments, the antibody or antigen binding portion binds to an epitope of PD-1 that includes residues V44 and T145 of SEQ ID NO: 388 (such as the 13112 antibody, e.g., antibody 13112.15380).

[0454] In some embodiments, the combination therapy or composition comprises an anti-PD-1 antibody, or antigen-binding portion thereof, that binds to an epitope of PD-1 comprising amino acid residue K131 of SEQ ID NO: 388 (e.g., the 12819 or 12865 antibodies). In some embodiments, the epitope further comprises amino acid residues P130 and A132, and may additionally comprise amino acid residues V64 and L128 (e.g., the 12819 antibody). In some embodiments, the epitope further comprises amino acid residue E136 (e.g., the 12865 antibody).

[0455] In some embodiments, an anti-PD-1 antibody or antigen-binding portion thereof as 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 portion binds to an epitope of PD-1 comprising residues 69-90 and 122-140 of SEQ ID NO: 388 (such as the 12819 and 12865 antibodies, e.g., antibodies 12819.15384 and 12865.15377). In certain embodiments, the antibody or antigen-binding portion binds to an epitope of PD-1 comprising residues 56-64, 69-90, and 122-140 of SEQ ID NO: 388 (e.g., the 12819 antibody). In certain embodiments, the antibody or antigen binding portion binds to an epitope of PD-1 comprising residues 69-90 and 122-140 of SEQ ID NO: 388 (e.g., the 12865 antibody). In some embodiments, the antibody or portion binds to residues 69-75 of SEQ ID NO: 388 (or a fragment thereof, such as a fragment of one, two, three, four, five, or six residues) (such as the 12819 and 12865 antibodies, e.g., antibodies 12819.15384 and 12865.15377). In some embodiments, the antibody or portion binds to residues 136-140 of SEQ ID NO: 388 (or a fragment thereof, such as a fragment of one, two, three, or four residues) (such as the 12819 and 12865 antibodies, e.g., antibodies 12819.15384 and 12865.15377). In some embodiments, the antibody or portion binds to residues 69-75 (or a fragment thereof) and residues 136-140 (or a fragment thereof) of SEQ ID NO: 388 (such as the 12819 and 12865 antibodies, e.g., antibodies 12819.15384 and 12865.15377). Epitopes having any combination of the above residues are also contemplated.

[0456] In some embodiments, an anti-PD-1 antibody, or antigen-binding portion thereof, as described herein is an anti-PD-1 antibody, or antigen-binding portion thereof, described in PCT Patent Publication WO 2017 / 055547 or PCT Patent Application PCT / EP2017 / 079615, which are herein incorporated by reference in their entireties.

[0457] Anti-TIM-3 antibody

[0458] In specific embodiments, the anti-TIM-3 antibodies disclosed herein are human antibodies produced from transgenic rats capable of producing antibodies with a human idiotype.

[0459] The anti-TIM-3 antibodies disclosed herein may be referred to by a 5-digit number (e.g., "20131") or a 10-digit number (e.g., "15086.16837"). Ten-digit numbers with the same first five digits are derived from the same parent antibody, as in the case of antibodies 15086.15086, 15086.16837, 15086.17145, and 15086.17144. It is expected that such antibodies (which share the same six CDRs) will have the same or substantially the same target binding properties. As will be apparent based on the protein and DNA sequences provided herein, the 15086.16837, 15086.17145, and 15086.17144 variants have only a single amino acid difference at position 6 in the VH sequence compared to the parent 15086 antibody ("15086.15086"), namely, E instead of Q, while the VL amino acid sequence is identical. It will also be apparent that these variants differ primarily in their antibody form / subclass, namely:

[0460] 15086.15086:IgG1

[0461] 15086.16837:IgG1 LALA

[0462] 15086.17145: IgG2

[0463] 15086.17144: IgG4

[0464] The VH sequences of IgG2 and IgG4 subclass antibodies are identical to those of the IgG1 LALA variant. The VL sequences are identical for all four antibody subclasses.

[0465] In some embodiments, the combination therapy or composition comprises an anti-TIM-3 antibody, or an antigen-binding portion thereof, wherein the anti-TIM-3 antibody is represented herein by antibodies 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, or 20621, or a variant of any of these, wherein the variant may, for example, contain certain minimal amino acid changes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes) relative to the antibody, which may, for example, be located in the framework regions without losing the antigen-binding specificity of the antibody.

[0466] In some embodiments, the anti-TIM-3 antibody competes or cross-competes for binding to human TIM-3 with, or binds to the same epitope of human TIM-3 as, antibody 15086.15086 having an IgG1 format, antibody 15086.16837 having an IgG1LALA format, antibody 15086.17145 having an IgG2 format, or antibody 15086.17144 having an IgG4 format. In some embodiments, the antibody has an IgG1 or IgG2 format. In certain embodiments, the antibody has an IgG2 format.

[0467] In some embodiments, the anti-TIM-3 antibody competes or cross-competes for binding to human TIM-3 with, or binds to the same epitope of human TIM-3 as, antibodies 20131, 20293, 15105, 15107, 15109, 15174, 15175, 15260, 15284, 15299, 15353, 15354, 17244, 17245, 19324, 19416, 19568, 20185, 20300, 20362, or 20621. In some embodiments, the antibody has an IgG1 or IgG2 format. In certain embodiments, the antibody has an IgG2 format.

[0468] In some embodiments, the anti-TIM-3 antibody competes or cross-competes for binding to human TIM-3 with an antibody whose heavy chain (H) CDR1-3 and light chain (L) CDR1-3 each comprise the following, or binds to the same epitope of human TIM-3 with an antibody whose heavy chain (H) CDR1-3 and light chain (L) CDR1-3 each comprise: SEQ ID NO: NO: 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.

[0469] In some embodiments, the anti-TIM-3 antibody comprises an H-CDR3 comprising the following H-CDR3 amino acid sequence: SEQ ID NO: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, or 220.

[0470] In some embodiments, the anti-TIM-3 antibody contains H-CDR1-3 comprising the following H-CDR1-3 amino acid sequences, respectively: SEQ ID NO: 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.

[0471] 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 NO: 3, 7, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, or 216.

[0472] In some embodiments, the anti-TIM-3 antibody has a VH comprising SEQ ID NO: 3, 7, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, or 216.

[0473] 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 sequence to 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 a CH 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 sequence to SEQ ID NO: 374.

[0474] 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.

[0475] 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 sequence 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 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 sequence to SEQ ID NO: 375, 376, or 377.

[0476] In some embodiments, the anti-TIM-3 antibody has a HC comprising a 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 a 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.

[0477] In some embodiments, the anti-TIM-3 antibody contains an L-CDR3 comprising the following L-CDR3 amino acid sequence: 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.

[0478] In some embodiments, the anti-TIM-3 antibody comprises L-CDR1-3 comprising the following L-CDR1-3 amino acid sequences, respectively: SEQ ID NO: 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.

[0479] In some embodiments, the anti-TIM-3 antibody has a VL 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 sequence to the VL amino acid sequence of SEQ ID NO: 4, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, 187, 197, 207, or 217.

[0480] In some embodiments, the anti-TIM-3 antibody has a VL comprising the VL amino acid sequence of SEQ ID NO: 4, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, 187, 197, 207, or 217.

[0481] In some embodiments, the anti-TIM-3 antibody has a VL 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 sequence to the VL amino acid sequence of SEQ ID NO: 4, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, 187, 197, 207, or 217; and a CL 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 sequence to SEQ ID NO: 378.

[0482] In some embodiments, the anti-TIM-3 antibody has a LC comprising the VL amino acid sequence of SEQ ID NO: 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 NO: 378.

[0483] In some embodiments, the anti-TIM-3 antibody comprises any of the above heavy chain sequences and any of the above light chain sequences.

[0484] In some embodiments, the anti-TIM-3 antibody comprises H-CDR3 and L-CDR3 comprising the following H-CDR3 and L-CDR3 amino acid sequences, respectively: SEQ ID NO: 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.

[0485] In some embodiments, the anti-TIM-3 antibody comprises H-CDR1-3 and L-CDR1-3 comprising the following H-CDR1-3 and L-CDR1-3 sequences, respectively: SEQ ID NO: 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.

[0486] 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 sequence to the amino acid sequence of SEQ ID NO: 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 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 sequence to the amino acid sequence of SEQ ID NO: NO:4, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, 187, 197, 207, or 217.

[0487] In some embodiments, the anti-TIM-3 antibody has a VH comprising the amino acid sequence of SEQ ID NO: 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 comprising the amino acid sequence of SEQ ID NO: 4, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, 187, 197, 207, or 217.

[0488] In some embodiments, the anti-TIM-3 antibody has a HC comprising the amino acid sequence of SEQ ID NO: 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 NO: 378; and a LC comprising the amino acid sequence of SEQ ID NO: 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 NO: 374, 375, 376, or 377.

[0489] In some embodiments, the anti-TIM-3 antibody comprises the following H-CDR1-3 and L-CDR1-3 amino acid sequences:

[0490] a) SEQ ID NO: 8-13, respectively;

[0491] b) SEQ ID NO: 18-23, respectively;

[0492] c) SEQ ID NOs: 28-33, respectively;

[0493] d) SEQ ID NOs: 38-43, respectively;

[0494] e) SEQ ID NOs: 48-53, respectively;

[0495] f) SEQ ID NOs: 58-63, respectively;

[0496] g) SEQ ID NOs: 68-73, respectively;

[0497] h) SEQ ID NOs: 78-83, respectively;

[0498] i) SEQ ID NOs: 88-93, respectively;

[0499] j) SEQ ID NOs: 98-103, respectively;

[0500] k) SEQ ID NOs: 108-113, respectively;

[0501] 1) SEQ ID NO: 118-123, respectively;

[0502] m) SEQ ID NOs: 128-133, respectively;

[0503] n) SEQ ID NO: 138-143, respectively;

[0504] o) SEQ ID NOs: 148-153, respectively;

[0505] p) SEQ ID NO: 158-163, respectively;

[0506] q) SEQ ID NOs: 168-173, respectively;

[0507] r) SEQ ID NO: 178-183, respectively;

[0508] s) are SEQ ID NOs: 188-193, respectively;

[0509] t) are SEQ ID NOs: 198-203, respectively;

[0510] u) SEQ ID NO: 208-213, respectively; or

[0511] v) SEQ ID NOs: 218-223, respectively.

[0512] In some embodiments, the anti-TIM-3 antibody comprises a heavy chain variable domain and a light chain variable domain having the following amino acid sequence:

[0513] a) SEQ ID NO: 7 and 4, respectively;

[0514] b) SEQ ID NOs: 3 and 4, respectively;

[0515] c) SEQ ID NOs: 16 and 17, respectively;

[0516] d) SEQ ID NOs: 26 and 27, respectively;

[0517] e) SEQ ID NOs: 36 and 37, respectively;

[0518] f) SEQ ID NOs: 46 and 47, respectively;

[0519] g) SEQ ID NOs: 56 and 57, respectively;

[0520] h) SEQ ID NOs: 66 and 67, respectively;

[0521] i) SEQ ID NOs: 76 and 77, respectively;

[0522] j) SEQ ID NOs: 86 and 87, respectively;

[0523] k) SEQ ID NOs: 96 and 97, respectively;

[0524] 1) SEQ ID NO: 106 and 107, respectively;

[0525] m) SEQ ID NOs: 116 and 117, respectively;

[0526] n) SEQ ID NOs: 126 and 127, respectively;

[0527] o) SEQ ID NOs: 136 and 137, respectively;

[0528] p) SEQ ID NOs: 146 and 147, respectively;

[0529] q) SEQ ID NOs: 156 and 157, respectively;

[0530] r) SEQ ID NOs: 166 and 167, respectively;

[0531] s) SEQ ID NOs: 176 and 177, respectively;

[0532] t) SEQ ID NOs: 186 and 187, respectively;

[0533] u) SEQ ID NOs: 196 and 197, respectively;

[0534] v) SEQ ID NOs: 206 and 207, respectively; or

[0535] w) are SEQ ID NOs: 216 and 217, respectively.

[0536] In some embodiments, the anti-TIM-3 antibody comprises:

[0537] a) an HC comprising the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 4 and the amino acid sequence of SEQ ID NO: 378;

[0538] b) an HC comprising the amino acid sequence of SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 4 and the amino acid sequence of SEQ ID NO: 378;

[0539] c) an HC comprising the amino acid sequence of SEQ ID NO: 16 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 17 and the amino acid sequence of SEQ ID NO: 378;

[0540] d) an HC comprising the amino acid sequence of SEQ ID NO: 26 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 27 and the amino acid sequence of SEQ ID NO: 378;

[0541] e) an HC comprising the amino acid sequence of SEQ ID NO: 36 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 37 and the amino acid sequence of SEQ ID NO: 378;

[0542] f) an HC comprising the amino acid sequence of SEQ ID NO: 46 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 47 and the amino acid sequence of SEQ ID NO: 378;

[0543] g) an HC comprising the amino acid sequence of SEQ ID NO: 56 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 57 and the amino acid sequence of SEQ ID NO: 378;

[0544] h) an HC comprising the amino acid sequence of SEQ ID NO: 66 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 67 and the amino acid sequence of SEQ ID NO: 378;

[0545] i) an HC comprising the amino acid sequence of SEQ ID NO: 76 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 77 and the amino acid sequence of SEQ ID NO: 378;

[0546] j) an HC comprising the amino acid sequence of SEQ ID NO: 86 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 87 and the amino acid sequence of SEQ ID NO: 378;

[0547] k) an HC comprising the amino acid sequence of SEQ ID NO: 96 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 97 and the amino acid sequence of SEQ ID NO: 378;

[0548] 1) an HC comprising the amino acid sequence of SEQ ID NO: 106 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 107 and the amino acid sequence of SEQ ID NO: 378;

[0549] m) an HC comprising the amino acid sequence of SEQ ID NO: 116 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 117 and the amino acid sequence of SEQ ID NO: 378;

[0550] n) an HC comprising the amino acid sequence of SEQ ID NO: 126 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 127 and the amino acid sequence of SEQ ID NO: 378;

[0551] o) an HC comprising the amino acid sequence of SEQ ID NO: 136 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 137 and the amino acid sequence of SEQ ID NO: 378;

[0552] p) an HC comprising the amino acid sequence of SEQ ID NO: 146 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 147 and the amino acid sequence of SEQ ID NO: 378;

[0553] q) an HC comprising the amino acid sequence of SEQ ID NO: 156 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 157 and the amino acid sequence of SEQ ID NO: 378;

[0554] r) an HC comprising the amino acid sequence of SEQ ID NO: 166 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 167 and the amino acid sequence of SEQ ID NO: 378;

[0555] s) a HC comprising the amino acid sequence of SEQ ID NO: 176 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and a LC comprising the amino acid sequence of SEQ ID NO: 177 and the amino acid sequence of SEQ ID NO: 378;

[0556] t) an HC comprising the amino acid sequence of SEQ ID NO: 186 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 187 and the amino acid sequence of SEQ ID NO: 378;

[0557] u) a HC comprising the amino acid sequence of SEQ ID NO: 196 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and a LC comprising the amino acid sequence of SEQ ID NO: 197 and the amino acid sequence of SEQ ID NO: 378;

[0558] v) a HC comprising the amino acid sequence of SEQ ID NO: 206 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and a LC comprising the amino acid sequence of SEQ ID NO: 207 and the amino acid sequence of SEQ ID NO: 378; or

[0559] w) an HC comprising the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377, and an LC comprising the amino acid sequence of SEQ ID NO: 217 and the amino acid sequence of SEQ ID NO: 378.

[0560] In some embodiments, the anti-TIM-3 antibody is selected from the group consisting of:

[0561] a) an antibody whose H-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 8-10, respectively;

[0562] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 3 or 7;

[0563] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 3 or 7;

[0564] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NOs: 3 and 374, or the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NOs: 375, 376, or 377;

[0565] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 11-13, respectively;

[0566] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 4;

[0567] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 4;

[0568] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 4 and 378;

[0569] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequences of SEQ ID NOs: 8-13;

[0570] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 3 or 7 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 4;

[0571] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 3 or 15 and whose VL comprises the amino acid sequence of SEQ ID NO: 4; and

[0572] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NOs: 3 and 374, or the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NOs: 375, 376, or 377; and whose LC comprises the amino acid sequence of SEQ ID NOs: 4 and 378.

[0573] In some embodiments, the anti-TIM-3 antibody is selected from the group consisting of:

[0574] a) an antibody whose H-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 18-20, respectively;

[0575] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 16;

[0576] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 16;

[0577] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 16 and SEQ ID NO: 374, 375, 376, or 377;

[0578] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 21-23, respectively;

[0579] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 17;

[0580] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 17;

[0581] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 17 and 378;

[0582] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequences of SEQ ID NOs: 18-23;

[0583] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 16 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 17;

[0584] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 16 and whose VL comprises the amino acid sequence of SEQ ID NO: 17; and

[0585] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 16 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377; and whose LC comprises the amino acid sequences of SEQ ID NO: 17 and 378.

[0586] In some embodiments, the anti-TIM-3 antibody is selected from the group consisting of:

[0587] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 28-30, respectively;

[0588] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 26;

[0589] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 26;

[0590] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 26 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377;

[0591] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 31-33, respectively;

[0592] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 27;

[0593] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 27;

[0594] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 27 and 378;

[0595] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequences of SEQ ID NOs: 28-33;

[0596] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 26 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 27;

[0597] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 26 and whose VL comprises the amino acid sequence of SEQ ID NO: 27; and

[0598] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 26 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377; and whose LC comprises the amino acid sequences of SEQ ID NO: 27 and 378.

[0599] In some embodiments, the anti-TIM-3 antibody is selected from the group consisting of:

[0600] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 38-40, respectively;

[0601] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 36;

[0602] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 36;

[0603] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 36 and SEQ ID NO: 374, 375, 376, or 377;

[0604] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 41-43, respectively;

[0605] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 37;

[0606] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 37;

[0607] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 37 and 378;

[0608] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequences of SEQ ID NOs: 38-43;

[0609] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 36 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 37;

[0610] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 36 and whose VL comprises the amino acid sequence of SEQ ID NO: 37; and

[0611] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 36 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377; and whose LC comprises the amino acid sequences of SEQ ID NO: 37 and 378.

[0612] In some embodiments, the anti-TIM-3 antibody is selected from the group consisting of:

[0613] a) an antibody whose H-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 48-50, respectively;

[0614] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 46;

[0615] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 46;

[0616] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 46 and SEQ ID NO: 374, 375, 376, or 377;

[0617] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 51-53, respectively;

[0618] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 47;

[0619] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 47;

[0620] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 47 and 378;

[0621] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequences of SEQ ID NOs: 48-53;

[0622] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 46 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 47;

[0623] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 46 and whose VL comprises the amino acid sequence of SEQ ID NO: 47; and

[0624] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 46 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377; and whose LC comprises the amino acid sequences of SEQ ID NO: 47 and 378.

[0625] In some embodiments, the anti-TIM-3 antibody is selected from the group consisting of:

[0626] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 58-60, respectively;

[0627] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 56;

[0628] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 56;

[0629] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 56 and SEQ ID NO: 374, 375, 376, or 377;

[0630] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 61-63, respectively;

[0631] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 57;

[0632] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 57;

[0633] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 57 and 378;

[0634] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequences of SEQ ID NOs: 58-63;

[0635] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 56 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 57;

[0636] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 56 and whose VL comprises the amino acid sequence of SEQ ID NO: 57; and

[0637] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 56 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377; and whose LC comprises the amino acid sequences of SEQ ID NO: 57 and 378.

[0638] In some embodiments, the anti-TIM-3 antibody is selected from the group consisting of:

[0639] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 68-70, respectively;

[0640] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 66;

[0641] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 66;

[0642] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 66 and SEQ ID NO: 374, 375, 376, or 377;

[0643] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 71-73, respectively;

[0644] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 67;

[0645] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 67;

[0646] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 67 and 378;

[0647] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequences of SEQ ID NOs: 68-73;

[0648] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 66 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 67;

[0649] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 66 and whose VL comprises the amino acid sequence of SEQ ID NO: 67; and

[0650] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 66 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377; and whose LC comprises the amino acid sequences of SEQ ID NO: 67 and 378.

[0651] In some embodiments, the anti-TIM-3 antibody is selected from the group consisting of:

[0652] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 78-80, respectively;

[0653] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 76;

[0654] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 76;

[0655] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 76 and SEQ ID NO: 374, 375, 376, or 377;

[0656] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 81-83, respectively;

[0657] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 77;

[0658] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 77;

[0659] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 77 and 378;

[0660] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequences of SEQ ID NOs: 78-83;

[0661] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 76 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 77;

[0662] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 76 and whose VL comprises the amino acid sequence of SEQ ID NO: 77; and

[0663] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 76 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377; and whose LC comprises the amino acid sequences of SEQ ID NO: 77 and 378.

[0664] In some embodiments, the anti-TIM-3 antibody is selected from the group consisting of:

[0665] a) an antibody whose H-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 88-90, respectively;

[0666] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 86;

[0667] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 86;

[0668] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 86 and SEQ ID NO: 374, 375, 376, or 377;

[0669] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 91-93, respectively;

[0670] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 87;

[0671] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 87;

[0672] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 87 and 378;

[0673] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequences of SEQ ID NOs: 88-93;

[0674] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 86 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 87;

[0675] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 86 and whose VL comprises the amino acid sequence of SEQ ID NO: 87; and

[0676] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 86 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377; and whose LC comprises the amino acid sequences of SEQ ID NO: 87 and 378.

[0677] In some embodiments, the anti-TIM-3 antibody is selected from the group consisting of:

[0678] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 98-100, respectively;

[0679] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 96;

[0680] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 96;

[0681] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 96 and SEQ ID NO: 374, 375, 376, or 377;

[0682] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 101-103, respectively;

[0683] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 97;

[0684] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 97;

[0685] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 97 and 378;

[0686] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequence of SEQ ID NOs: 98-103;

[0687] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 96 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 97;

[0688] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 96 and whose VL comprises the amino acid sequence of SEQ ID NO: 97; and

[0689] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 96 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377; and whose LC comprises the amino acid sequences of SEQ ID NO: 97 and 378.

[0690] In some embodiments, the anti-TIM-3 antibody is selected from the group consisting of:

[0691] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 108-110, respectively;

[0692] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 106;

[0693] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 106;

[0694] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 106 and SEQ ID NO: 374, 375, 376, or 377;

[0695] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 111-113, respectively;

[0696] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 107;

[0697] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 107;

[0698] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 107 and 378;

[0699] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequences of SEQ ID NOs: 108-113;

[0700] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 106 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 107;

[0701] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 106 and whose VL comprises the amino acid sequence of SEQ ID NO: 107; and

[0702] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 106 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377; and whose LC comprises the amino acid sequences of SEQ ID NO: 107 and 378.

[0703] In some embodiments, the anti-TIM-3 antibody is selected from the group consisting of:

[0704] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 118-120, respectively;

[0705] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 116;

[0706] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 116;

[0707] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 116 and SEQ ID NO: 374, 375, 376, or 377;

[0708] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 121-123, respectively;

[0709] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 117;

[0710] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 117;

[0711] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 117 and 378;

[0712] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequence of SEQ ID NOs: 118-123;

[0713] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 116 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 117;

[0714] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 116 and whose VL comprises the amino acid sequence of SEQ ID NO: 117; and

[0715] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 116 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377; and whose LC comprises the amino acid sequences of SEQ ID NO: 117 and 378.

[0716] In some embodiments, the anti-TIM-3 antibody is selected from the group consisting of:

[0717] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 128-130, respectively;

[0718] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 126;

[0719] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 126;

[0720] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 126 and SEQ ID NO: 374, 375, 376, or 377;

[0721] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 131-133, respectively;

[0722] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 127;

[0723] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 127;

[0724] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 127 and 378;

[0725] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequences of SEQ ID NOs: 128-133;

[0726] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 126 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 127;

[0727] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 126 and whose VL comprises the amino acid sequence of SEQ ID NO: 127; and

[0728] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 126 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377; and whose LC comprises the amino acid sequences of SEQ ID NO: 127 and 378.

[0729] In some embodiments, the anti-TIM-3 antibody is selected from the group consisting of:

[0730] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 138-140, respectively;

[0731] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 136;

[0732] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 136;

[0733] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 136 and SEQ ID NO: 374, 375, 376, or 377;

[0734] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 141-143, respectively;

[0735] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 137;

[0736] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 137;

[0737] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 137 and 378;

[0738] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequence of SEQ ID NOs: 138-143;

[0739] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 136 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 137;

[0740] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 136 and whose VL comprises the amino acid sequence of SEQ ID NO: 137; and

[0741] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 136 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377; and whose LC comprises the amino acid sequences of SEQ ID NO: 137 and 378.

[0742] In some embodiments, the anti-TIM-3 antibody is selected from the group consisting of:

[0743] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 148-150, respectively;

[0744] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 146;

[0745] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 146;

[0746] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 146 and SEQ ID NO: 374, 375, 376, or 377;

[0747] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 151-153, respectively;

[0748] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 147;

[0749] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 147;

[0750] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 147 and 378;

[0751] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequences of SEQ ID NOs: 148-53;

[0752] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 146 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 147;

[0753] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 146 and whose VL comprises the amino acid sequence of SEQ ID NO: 147; and

[0754] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 146 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377; and whose LC comprises the amino acid sequences of SEQ ID NO: 147 and 378.

[0755] In some embodiments, the anti-TIM-3 antibody is selected from the group consisting of:

[0756] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 158-160, respectively;

[0757] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 156;

[0758] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 156;

[0759] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 156 and SEQ ID NO: 374, 375, 376, or 377;

[0760] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 161-163, respectively;

[0761] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 157;

[0762] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 157;

[0763] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 157 and 378;

[0764] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequence of SEQ ID NOs: 158-163;

[0765] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 156 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 157;

[0766] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 156 and whose VL comprises the amino acid sequence of SEQ ID NO: 157; and

[0767] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 156 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377; and whose LC comprises the amino acid sequences of SEQ ID NO: 157 and 378.

[0768] In some embodiments, the anti-TIM-3 antibody is selected from the group consisting of:

[0769] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 168-170, respectively;

[0770] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 166;

[0771] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 166;

[0772] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 166 and SEQ ID NO: 374, 375, 376, or 377;

[0773] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 171-173, respectively;

[0774] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 167;

[0775] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 167;

[0776] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 167 and 378;

[0777] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequence of SEQ ID NOs: 168-173;

[0778] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 166 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 167;

[0779] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 166 and whose VL comprises the amino acid sequence of SEQ ID NO: 167; and

[0780] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 166 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377; and whose LC comprises the amino acid sequences of SEQ ID NO: 167 and 378.

[0781] In some embodiments, the anti-TIM-3 antibody is selected from the group consisting of:

[0782] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 178-80, respectively;

[0783] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 176;

[0784] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 176;

[0785] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 176 and SEQ ID NO: 374, 375, 376, or 377;

[0786] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 181-183, respectively;

[0787] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 177;

[0788] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 177;

[0789] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 177 and 378;

[0790] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequence of SEQ ID NOs: 178-183;

[0791] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 176 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 177;

[0792] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 176 and whose VL comprises the amino acid sequence of SEQ ID NO: 177; and

[0793] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 176 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377; and whose LC comprises the amino acid sequences of SEQ ID NO: 177 and 378.

[0794] In some embodiments, the anti-TIM-3 antibody is selected from the group consisting of:

[0795] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 188-190, respectively;

[0796] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 186;

[0797] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 186;

[0798] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 186 and SEQ ID NO: 374, 375, 376, or 377;

[0799] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 191-193, respectively;

[0800] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 187;

[0801] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 187;

[0802] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 187 and 378;

[0803] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequences of SEQ ID NOs: 188-193;

[0804] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 186 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 187;

[0805] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 186 and whose VL comprises the amino acid sequence of SEQ ID NO: 187; and

[0806] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 186 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377; and whose LC comprises the amino acid sequences of SEQ ID NO: 187 and 378.

[0807] In some embodiments, the anti-TIM-3 antibody is selected from the group consisting of:

[0808] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 198-200, respectively;

[0809] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 196;

[0810] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 196;

[0811] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 196 and SEQ ID NO: 374, 375, 376, or 377;

[0812] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 201-203, respectively;

[0813] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 197;

[0814] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 197;

[0815] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 197 and 378;

[0816] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequence of SEQ ID NOs: 198-203;

[0817] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 196 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 197;

[0818] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 196 and whose VL comprises the amino acid sequence of SEQ ID NO: 197; and

[0819] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 196 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377; and whose LC comprises the amino acid sequences of SEQ ID NO: 197 and 378.

[0820] In some embodiments, the anti-TIM-3 antibody is selected from the group consisting of:

[0821] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 208-210, respectively;

[0822] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 206;

[0823] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 206;

[0824] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 206 and SEQ ID NO: 374, 375, 376, or 377;

[0825] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 211-213, respectively;

[0826] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 207;

[0827] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 207;

[0828] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 207 and 378;

[0829] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequences of SEQ ID NOs: 208-213;

[0830] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 206 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 207;

[0831] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 206 and whose VL comprises the amino acid sequence of SEQ ID NO: 207; and

[0832] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 206 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377; and whose LC comprises the amino acid sequences of SEQ ID NO: 207 and 378.

[0833] In some embodiments, the anti-TIM-3 antibody is selected from the group consisting of:

[0834] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 218-220, respectively;

[0835] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 216;

[0836] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 216;

[0837] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 216 and SEQ ID NO: 374, 375, 376, or 377;

[0838] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 221-223, respectively;

[0839] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 217;

[0840] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 217;

[0841] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 217 and 378;

[0842] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequence of SEQ ID NOs: 218-223;

[0843] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 216 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 217;

[0844] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 216 and whose VL comprises the amino acid sequence of SEQ ID NO: 217; and

[0845] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NO: 216 and the amino acid sequence of SEQ ID NO: 374, 375, 376, or 377; and whose LC comprises the amino acid sequences of SEQ ID NO: 217 and 378.

[0846] In some embodiments, any of the anti-TIM-3 antibodies or antigen-binding portions described herein can inhibit the binding of ligands such as galectin-9, CEACAM1, HMGB-1, and phosphatidylserine to TIM-3.

[0847] In some embodiments, any anti-TIM-3 antibody or antigen-binding portion described herein can increase the activity of NK cells. In some embodiments, this activity can mediate ADCC.

[0848] In some embodiments, administration of an anti-TIM-3 antibody or antigen-binding portion thereof as described herein can activate dendritic cells, causing their maturation and thereby their ability to stimulate T cells. While not intending to be bound by any particular theory, it is believed that the anti-TIM-3 antibodies described herein act as TIM-3 dendritic cell activators, thereby acting on the efficacy of dendritic cells to stimulate T cells. In a tumor-associated setting, the anti-TIM-3 antibodies thus cause the maturation and activation of tumor-associated dendritic cells, leading to the activation of tumor-specific T cells.

[0849] In some embodiments, administration of an anti-TIM-3 antibody, or antigen-binding portion thereof, as described herein can directly activate T cells.

[0850] In some embodiments, the anti-TIM-3 antibodies or antigen-binding portions described herein have at least one of the following properties:

[0851] a) with a K of 23 nM or lower D Binding to human TIM-3, as measured by surface plasmon resonance;

[0852] b) with a K of 22 nM or lower D Binding to cynomolgus monkey TIM-3, as measured by surface plasmon resonance;

[0853] c) binds to human TIM-3 with an EC50 of 1.2 nM or less, as measured by ELISA;

[0854] d) binds to cynomolgus monkey TIM-3 with an EC50 of 46 nM or less, as measured by ELISA;

[0855] e) increased IFN-γ secretion in a one-way mixed lymphocyte reaction assay;

[0856] f) increased IFN-γ secretion in a two-way mixed lymphocyte reaction assay;

[0857] g) increased TNF-α secretion in a one-way mixed lymphocyte reaction assay;

[0858] h) increasing TNF-α secretion from dendritic cells; and

[0859] i) Inhibits the interaction between TIM-3 and phosphatidylserine.

[0860] 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 antibody 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 portion has all of the aforementioned properties. In some embodiments, the anti-TIM-3 antibody or antigen-binding portion has at least properties a, c, d, e, g, and h. In some embodiments, the anti-TIM-3 antibody or antigen-binding portion has at least properties a, c, d, e, g, h, and i. In some embodiments, the anti-TIM-3 antibody or antigen-binding portion has at least properties a, c, and d. In some embodiments, the anti-TIM-3 antibody or antigen-binding portion has at least properties a, b, c, d, e, f, and h. In some embodiments, the anti-TIM-3 antibody or antigen-binding portion has at least properties c, e, f, and h. In some embodiments, the anti-TIM-3 antibody or antigen-binding portion has at least properties c, d, e, f, and h.

[0861] In some embodiments, an anti-TIM-3 antibody, or antigen-binding portion thereof, as described herein binds to an epitope of TIM-3 that includes at least one (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) of the following residues of SEQ ID NO: 389: P50, V60, F61, E62, G64, R69, I117, M118, and D120. In certain embodiments, the antibody or antigen-binding portion binds to an epitope of TIM-3 that includes the following residues of SEQ ID NO: 389: P50, V60, F61, E62, G64, R69, I117, M118, and D120 (such as antibodies 15086.15086, 15086.16837, 15086.17145, or 15086.17144). In certain embodiments, the antibody or antigen binding portion binds to an epitope of TIM-3 comprising the following residues of SEQ ID NO: 389: F61, R69, and I117 (such as antibody 20293). In certain embodiments, the antibody or antigen binding portion binds to an epitope of TIM-3 comprising the following residues of SEQ ID NO: 389: P50, F61, E62, I117, M118, and D120 (such as antibody 20131).

[0862] In some embodiments, the anti-TIM-3 antibody, or antigen-binding portion thereof, binds to an epitope of TIM-3 comprising 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 comprises 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., antibody 15086.15086, 15086.16837, 15086.17145, 15086.17144, or 20131) and may additionally comprise amino acid residues V60 and G64 (e.g., antibody 15086.15086, 15086.16837, 15086.17145, or 15086.17144).

[0863] In some embodiments, an anti-TIM-3 antibody, or antigen-binding portion thereof, as described herein binds to an epitope of TIM-3 that includes at least one (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) of the following residues of SEQ ID NO: 236: P50, V60, F61, E62, G64, R69, I117, M118, and D120. Epitopes having any combination of the above residues are contemplated.

[0864] In some embodiments, an anti-TIM-3 antibody or antigen-binding portion thereof as 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 portion binds to residues 62-67 of SEQ ID NO: 389 (or a fragment thereof, such as a one, two, three, four, or five residue fragment) (e.g., antibodies 15086, 15086, 15086, 16837, 15086, 17145, 15086, 17144, and 20293). In some embodiments, the antibody or portion binds to residues 114-117 of SEQ ID NO: 389 (or a fragment thereof, such as a one, two, or three residue fragment) (e.g., antibody 20131). Epitopes having any combination of the above residues are also contemplated.

[0865] In some embodiments, the anti-TIM-3 antibody, or antigen-binding portion thereof, does not compete with ABTIM3 (from PCT Patent Publication WO 2015 / 117002) and / or mAb 15 (from PCT Patent Publication WO 2016 / 111947) for binding to TIM-3. In some embodiments, the anti-TIM-3 antibody, or antigen-binding portion thereof, does not bind to the same epitope as ABTIM3 and / or mAb 15; for example, the antibody or portion binds to one or more residues on TIM-3 that are not bound by ABTIM3 and / or mAb 15.

[0866] In some embodiments, the anti-TIM-3 antibody, or antigen-binding portion thereof, as described herein is an anti-TIM-3 antibody, or antigen-binding portion thereof, described in PCT Patent Publication WO 2017 / 178493, which is herein incorporated by reference in its entirety.

[0867] Anti-LAG-3 antibodies

[0868] In some embodiments, the anti-LAG-3 antibodies disclosed herein are human antibodies produced from transgenic rats capable of producing antibodies with a human idiotype. In another embodiment, the antibody is a chicken-derived chimeric antibody comprising chicken CDR sequences and human framework regions, wherein the framework regions have undergone humanization.

[0869] 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 not intending to be bound by any particular theory, it is believed that anti-LAG-3 antibodies are able to block the interaction of LAG-3 with its putative ligands, such as MHC II and LSECtin. The antibodies may accomplish this directly by blocking the ligand binding region or by inducing LAG-3 internalization. Another potential advantage of the anti-LAG-3 antibodies described herein is the low level of secondary effector function of antibodies with the "LALA" mutation (L234A / L235A), which prevents significant antibody binding to human FcgR (Fc gamma receptor) and thereby depletes effector T cells.

[0870] In some embodiments, the combination therapy or composition comprises an anti-LAG-3 antibody, or an antigen-binding portion thereof, wherein the LAG-3 antibody is an antibody referred to herein as antibody 15646, 15532, 15723, 15595, 15431, 15572, or 15011, or a variant of any of these, wherein the variant may, for example, contain certain minimal amino acid changes relative to the antibody (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes, which may, for example, be located in the framework regions without losing the antigen-binding specificity of the antibody.

[0871] In some embodiments, the anti-LAG-3 antibody (or antigen-binding portion thereof) competes or cross-competes for binding to human LAG-3 with, or binds to the same epitope of human LAG-3 as, antibodies 15646, 15532, 15723, 15595, 15431, 15572, or 15011. In some embodiments, the antibody has an IgG1 or IgG2 format. In certain embodiments, the antibody has an IgG2 format.

[0872] In some embodiments, the anti-LAG-3 antibody competes or cross-competes for binding to human LAG-3 with an antibody whose heavy chain (H) CDR1-3 and light chain (L) CDR1-3, respectively, comprise SEQ ID NO: 308-313, 318-323, 328-333, 338-343, 348-353, 358-363, or 368-373.

[0873] In some embodiments, the anti-LAG-3 antibody comprises an H-CDR3 comprising the following H-CDR3 amino acid sequence: SEQ ID NO: 310, 320, 330, 340, 350, 360, or 370.

[0874] In some embodiments, the anti-LAG-3 antibody contains H-CDR1-3 comprising the following H-CDR1-3 amino acid sequences, respectively: SEQ ID NO: 308-310, 318-320, 328-330, 338-340, 348-350, 358-360, or 368-370.

[0875] In some embodiments, the anti-LAG-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 NO: 306, 316, 326, 336, 346, 356, or 366.

[0876] In some embodiments, the anti-LAG-3 antibody has a VH comprising SEQ ID NO: 306, 316, 326, 336, 346, 356, or 366.

[0877] In some embodiments, the anti-LAG-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 sequence to SEQ ID NO: 306, 316, 326, 336, 346, 356, or 366; and a CH 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 sequence to SEQ ID NO: 375.

[0878] In some embodiments, the anti-LAG-3 antibody has a 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.

[0879] In some embodiments, the anti-LAG-3 antibody contains an L-CDR3 comprising the following L-CDR3 amino acid sequence: SEQ ID NO: 313, 323, 333, 343, 353, 363, or 373.

[0880] In some embodiments, the anti-LAG-3 antibody comprises L-CDR1-3 comprising the following L-CDR1-3 amino acid sequences, respectively: SEQ ID NO: 311-313, 321-323, 331-333, 341-343, 351-353, 361-363, or 371-373.

[0881] In some embodiments, the anti-LAG-3 antibody has a VL that is at least 90% identical in sequence (e.g., at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) to the VL amino acid sequence of SEQ ID NO: 307, 317, 327, 337, 347, 357, or 367.

[0882] In some embodiments, the anti-LAG-3 antibody has a VL comprising the VL amino acid sequence of SEQ ID NO: 307, 317, 327, 337, 347, 357, or 367.

[0883] In some embodiments, the anti-LAG-3 antibody has a VL 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 to the VL amino acid sequence of SEQ ID NO: 307, 317, 327, 337, 347, or 357; and a CL 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 to SEQ ID NO: 378.

[0884] In some embodiments, the anti-LAG-3 antibody has a LC comprising a VL amino acid sequence of SEQ ID NO: 307, 317, 327, 337, 347, or 357 and a CL amino acid sequence of SEQ ID NO: 378. In some embodiments, the anti-LAG-3 antibody has a LC comprising a VL amino acid sequence of SEQ ID NO: 367 and a CL amino acid sequence of SEQ ID NO: 379.

[0885] In some embodiments, the anti-LAG-3 antibody comprises any of the above heavy chain sequences and any of the above light chain sequences.

[0886] In some embodiments, the anti-LAG-3 antibody comprises H-CDR3 and L-CDR3 comprising the following H-CDR3 and L-CDR3 amino acid sequences, respectively: SEQ ID NO: 310 and 313, 320 and 323, 330 and 333, 340 and 343, 350 and 353, 360 and 363, or 370 and 373.

[0887] In some embodiments, the anti-LAG-3 antibody comprises H-CDR1-3 and L-CDR1-3 comprising the following H-CDR1-3 and L-CDR1-3 sequences, respectively: SEQ ID NO: 308-313, 318-323, 328-333, 338-343, 348-353, 358-363, or 368-373.

[0888] In some embodiments, the anti-LAG-3 antibody has a VH that is at least 90% (e.g., at least 92%, at least 95%, at least 98%, or at least 99%) identical in sequence to the amino acid sequence of SEQ ID NO: 306, 316, 326, 336, 346, 356, or 366, and a VL that is at least 90% (e.g., at least 92%, at least 95%, at least 98%, or at least 99%) identical in sequence to the amino acid sequence of SEQ ID NO: 307, 317, 327, 337, 347, 357, or 367.

[0889] In some embodiments, the anti-LAG-3 antibody has a VH comprising the amino acid sequence of SEQ ID NO: 306, 316, 326, 336, 346, 356, or 366, and a VL comprising the amino acid sequence of SEQ ID NO: 307, 317, 327, 337, 347, 357, or 367.

[0890] In some embodiments, the anti-LAG-3 antibody has a 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 a 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.

[0891] In some embodiments, the anti-LAG-3 antibody has a 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 a LC comprising the amino acid sequence of SEQ ID NO: 367 and the amino acid sequence of SEQ ID NO: 379.

[0892] In some embodiments, the anti-LAG-3 antibody comprises the following H-CDR1-3 and L-CDR1-3 amino acid sequences:

[0893] a) SEQ ID NOs: 308-313, respectively;

[0894] b) SEQ ID NOs: 318-323, respectively;

[0895] c) SEQ ID NOs: 328-333, respectively;

[0896] d) SEQ ID NOs: 338-343, respectively;

[0897] e) SEQ ID NOs: 348-353, respectively;

[0898] f) SEQ ID NO: 358-363, respectively; or

[0899] g) SEQ ID NOs: 368-373, respectively.

[0900] In some embodiments, the anti-LAG-3 antibody comprises a heavy chain variable domain and a light chain variable domain having the following amino acid sequence:

[0901] a) SEQ ID NOs: 306 and 307, respectively;

[0902] b) SEQ ID NOs: 316 and 317, respectively;

[0903] c) SEQ ID NOs: 326 and 327, respectively;

[0904] d) SEQ ID NOs: 336 and 337, respectively;

[0905] e) SEQ ID NOs: 346 and 347, respectively;

[0906] f) SEQ ID NOs: 356 and 357, respectively; or

[0907] g) SEQ ID NOs: 366 and 367, respectively.

[0908] In some embodiments, the anti-LAG-3 antibody comprises:

[0909] a) an HC comprising the amino acid sequence of SEQ ID NO: 306 and the amino acid sequence of SEQ ID NO: 375, and an LC comprising the amino acid sequence of SEQ ID NO: 307 and the amino acid sequence of SEQ ID NO: 378;

[0910] b) an HC comprising the amino acid sequence of SEQ ID NO: 316 and the amino acid sequence of SEQ ID NO: 375, and an LC comprising the amino acid sequence of SEQ ID NO: 317 and the amino acid sequence of SEQ ID NO: 378;

[0911] c) an HC comprising the amino acid sequence of SEQ ID NO: 326 and the amino acid sequence of SEQ ID NO: 375, and an LC comprising the amino acid sequence of SEQ ID NO: 327 and the amino acid sequence of SEQ ID NO: 378;

[0912] d) an HC comprising the amino acid sequence of SEQ ID NO: 336 and the amino acid sequence of SEQ ID NO: 375, and an LC comprising the amino acid sequence of SEQ ID NO: 337 and the amino acid sequence of SEQ ID NO: 378;

[0913] e) an HC comprising the amino acid sequence of SEQ ID NO: 346 and the amino acid sequence of SEQ ID NO: 375, and an LC comprising the amino acid sequence of SEQ ID NO: 347 and the amino acid sequence of SEQ ID NO: 378;

[0914] f) HC comprising the amino acid sequence of SEQ ID NO: 356 and the amino acid sequence of SEQ ID NO: 375, and LC comprising the amino acid sequence of SEQ ID NO: 357 and the amino acid sequence of SEQ ID NO: 378; or

[0915] g) HC comprising the amino acid sequence of SEQ ID NO: 366 and the amino acid sequence of SEQ ID NO: 375, and LC comprising the amino acid sequence of SEQ ID NO: 367 and the amino acid sequence of SEQ ID NO: 379.

[0916] In some embodiments, the anti-LAG-3 antibody is selected from the group consisting of:

[0917] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 308-310, respectively;

[0918] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 306;

[0919] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 306;

[0920] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NOs: 306 and 375;

[0921] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 311-313, respectively;

[0922] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 307;

[0923] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 307;

[0924] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 307 and 378;

[0925] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequences of SEQ ID NOs: 308-313;

[0926] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 306 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 307;

[0927] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 306 and whose VL comprises the amino acid sequence of SEQ ID NO: 307; and

[0928] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NOs: 306 and 375; and whose LC comprises the amino acid sequence of SEQ ID NOs: 307 and 378.

[0929] In some embodiments, the anti-LAG-3 antibody is selected from the group consisting of:

[0930] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 318-320, respectively;

[0931] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 316;

[0932] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 316;

[0933] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NOs: 316 and 375;

[0934] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 321-323, respectively;

[0935] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 317;

[0936] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 317;

[0937] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 317 and 378;

[0938] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequence of SEQ ID NOs: 318-323;

[0939] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 316 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 317;

[0940] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 316 and whose VL comprises the amino acid sequence of SEQ ID NO: 317; and

[0941] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NOs: 316 and 375; and whose LC comprises the amino acid sequence of SEQ ID NOs: 317 and 378.

[0942] In some embodiments, the anti-LAG-3 antibody is selected from the group consisting of:

[0943] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 328-330, respectively;

[0944] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 326;

[0945] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 326;

[0946] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NOs: 326 and 375;

[0947] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 331-333, respectively;

[0948] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 327;

[0949] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 327;

[0950] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 327 and 378;

[0951] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequences of SEQ ID NOs: 328-333;

[0952] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 326 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 327;

[0953] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 326 and whose VL comprises the amino acid sequence of SEQ ID NO: 327; and

[0954] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NOs: 326 and 375; and whose LC comprises the amino acid sequence of SEQ ID NOs: 327 and 378.

[0955] In some embodiments, the anti-LAG-3 antibody is selected from the group consisting of:

[0956] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 338-340, respectively;

[0957] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 336;

[0958] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 336;

[0959] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NOs: 336 and 375;

[0960] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 341-343, respectively;

[0961] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 337;

[0962] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 337;

[0963] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 337 and 378;

[0964] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequences of SEQ ID NOs: 338-343;

[0965] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 336 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 337;

[0966] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 336 and whose VL comprises the amino acid sequence of SEQ ID NO: 337; and

[0967] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NOs: 336 and 375; and whose LC comprises the amino acid sequence of SEQ ID NOs: 337 and 378.

[0968] In some embodiments, the anti-LAG-3 antibody is selected from the group consisting of:

[0969] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 348-350, respectively;

[0970] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 346;

[0971] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 346;

[0972] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NOs: 346 and 375;

[0973] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 351-353, respectively;

[0974] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 347;

[0975] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 347;

[0976] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 347 and 378;

[0977] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequences of SEQ ID NOs: 348-353;

[0978] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 346 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 347;

[0979] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 346 and whose VL comprises the amino acid sequence of SEQ ID NO: 347; and

[0980] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NOs: 346 and 375; and whose LC comprises the amino acid sequence of SEQ ID NOs: 347 and 378.

[0981] In some embodiments, the anti-LAG-3 antibody is selected from the group consisting of:

[0982] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 358-360, respectively;

[0983] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 356;

[0984] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 356;

[0985] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NOs: 356 and 375;

[0986] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 361-363, respectively;

[0987] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 357;

[0988] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 357;

[0989] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 357 and 378;

[0990] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequence of SEQ ID NOs: 358-363;

[0991] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 356 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 357;

[0992] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 356 and whose VL comprises the amino acid sequence of SEQ ID NO: 357; and

[0993] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NOs: 356 and 375; and whose LC comprises the amino acid sequence of SEQ ID NOs: 357 and 378.

[0994] In some embodiments, the anti-LAG-3 antibody is selected from the group consisting of:

[0995] a) an antibody whose H-CDR1-3 comprises the amino acid sequence of SEQ ID NOs: 368-370, respectively;

[0996] b) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 366;

[0997] c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 366;

[0998] d) an antibody whose HC comprises the amino acid sequence of SEQ ID NOs: 366 and 375;

[0999] e) an antibody whose L-CDR1-3 comprises the amino acid sequences of SEQ ID NOs: 371-373, respectively;

[1000] f) an antibody whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 367;

[1001] g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 367;

[1002] h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 367 and 379;

[1003] i) an antibody whose H-CDR1-3 and L-CDR1-3 respectively comprise the amino acid sequence of SEQ ID NOs: 368-373;

[1004] j) an antibody whose VH is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 366 and whose VL is at least 90% identical in sequence to the amino acid sequence of SEQ ID NO: 367;

[1005] k) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 366 and whose VL comprises the amino acid sequence of SEQ ID NO: 367; and

[1006] 1) an antibody whose HC comprises the amino acid sequence of SEQ ID NOs: 366 and 375; and whose LC comprises the amino acid sequence of SEQ ID NOs: 367 and 379.

[1007] In some embodiments, any of the anti-LAG-3 antibodies or antigen-binding portions described herein can bind to human LAG-3, e.g., with an EC50 of 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 portions described herein can bind to cynomolgus monkey LAG-3, e.g., with an EC50 of 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 specific embodiments, any of the anti-LAG-3 antibodies or antigen-binding portions described herein can bind to human LAG-3 with an EC50 of, e.g., 0.1 nM or less and to cynomolgus monkey LAG-3 with an EC50 of, e.g., 0.3 nM or less.

[1008] In some embodiments, any anti-LAG-3 antibody or antigen-binding portion described herein can bind to human LAG-3, for example, with an EC50 of 0.1 nM or less. In some embodiments, any anti-LAG-3 antibody or antigen-binding portion described herein can bind to cynomolgus monkey LAG-3, for example, with an EC50 of 0.3 nM or less. In specific embodiments, any anti-LAG-3 antibody or antigen-binding portion described herein can bind to human LAG-3, for example, with an EC50 of 0.1 nM or less and to cynomolgus monkey LAG-3, for example, with an EC50 of 0.3 nM or less.

[1009] In some embodiments, any of the anti-LAG-3 antibodies or antigen-binding portions described herein can inhibit the binding of a ligand, such as MHC class II (MHCII) or LSECtin, to LAG-3. For example, at 20 μg / mL, the anti-LAG-3 antibody or antigen-binding portion can reduce LAG-3 binding 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, the anti-LAG-3 antibody or antigen-binding protein can reduce LAG-3 binding to MHCII by greater than 85% compared to a negative control. In one embodiment, the anti-LAG-3 antibody or antigen-binding protein can reduce LAG-3 binding to MHC II by between about 25% and 95%, 30% and 90%, or 35% and 85% compared to a negative control.

[1010] In some embodiments, any of the anti-LAG-3 antibodies or antigen-binding portions described herein can block binding between LAG-3 and class II MHC (e.g., human LAG-3 expressed on Jurkat cells and human MHC class II expressed on Raji cells) (e.g., at a concentration 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).

[1011] In some embodiments, any of the anti-LAG-3 antibodies or antigen-binding portions described herein may have a K of D Binding to human LAG-3: 5.0 x 10 -8 or lower, 4.0x 10 -8 or lower, 3.0x 10-8 or lower, 2.0x 10 -8 or lower, 1.0x 10 -8 or lower, 9.0x 10 -9 or lower, 8.0x 10 -9 or lower, 7.0x 10 -9 or lower, 6.0x 10 -9 or lower, 5.0x 10 -9 or lower, 4.0x 10 -9 or lower, 3.0x 10 -9 or lower, 2.0x 10 -9 or lower, or 1.0x 10 -9 or lower, as measured by surface plasmon resonance.

[1012] In some embodiments, any of the anti-LAG-3 antibodies or antigen-binding portions described herein may have a K of D Binding to cynomolgus monkey LAG-3: 1.5 x 10 -7 or lower, 1.0x 10 -7 or lower, 9.0x 10 -8 or lower, 8.0x 10 -8 or lower, 7.0x 10 -8 or lower, 6.0x 10 -8 or lower, 5.0x 10 -8 or lower, 4.0x 10 -8 or lower, 3.0x 10 -8 or lower, 2.0x 10 -8 or lower, or 1.0x 10 -8 or lower, as measured by surface plasmon resonance.

[1013] In some embodiments, any of the anti-LAG-3 antibodies or antigen-binding portions described herein may have a K of D Binding to mouse LAG-3: 5.0 x 10 -8 or lower, 4.5x 10 -8 or lower, 4.0x 10 -8 or lower, 3.5x 10 -8 or lower, or 3.0x 10 -8 or lower, as measured by surface plasmon resonance.

[1014] In some embodiments, any anti-LAG-3 antibody or antigen-binding portion described herein can stimulate IL-2 production, e.g., from SEB-stimulated PBMCs.

[1015] In some embodiments, any of the anti-LAG-3 antibodies or antigen-binding portions described herein can reduce cellular and / or soluble levels of LAG-3, e.g., in a human T cell line, such as a human T cell line that overexpresses LAG-3.

[1016] In some embodiments, any of the anti-LAG-3 antibodies or antigen-binding portions described herein can induce tumor growth regression and / or delay tumor growth in vivo.

[1017] In some embodiments, any of the anti-LAG-3 antibodies or antigen-binding portions described herein may bind to a different epitope of human LAG-3 than antibody 25F7-Lag3.5.

[1018] In some embodiments, any of the anti-LAG-3 antibodies or antigen-binding portions described herein can activate T cells, resulting in increased anti-tumor activity.

[1019] In some embodiments, the anti-LAG-3 antibody, or antigen-binding portion, described herein has at least one of the following properties:

[1020] a) reduces the binding of human LAG-3 to human MHC class II on A375 cells by greater than 85% compared to a negative control antibody at a concentration of 20 μg / mL, as determined by a flow cytometry competition assay;

[1021] b) reduces human LAG-3 binding to human MHC class II on A375 cells by between 35% and 85% compared to a negative control antibody at a concentration of 20 μg / mL, as determined by a flow cytometry competition assay;

[1022] c) blocking the binding between human LAG-3 expressed on Jurkat cells and human MHC class II expressed on Raji cells;

[1023] d) binds to human LAG-3 with an EC50 of 0.1 nM or less, as measured by flow cytometry;

[1024] e) binds to cynomolgus monkey LAG-3 with an EC50 of 0.3 nM or less, as measured by flow cytometry;

[1025] f) binds to human LAG-3 with a KD of 3.0 x 10-8 or less as measured by surface plasmon resonance;

[1026] g) binds to cynomolgus monkey LAG-3 with a KD of 1.5 x 10-7 or less, as measured by surface plasmon resonance;

[1027] h) binds to mouse LAG-3 with a KD of 3.5 x 10-8 or less, as measured by surface plasmon resonance;

[1028] i) stimulates IL-2 production in human peripheral blood mononuclear cells (PBMCs) treated with Staphylococcal enterotoxin B (SEB);

[1029] j) reducing cellular levels of LAG-3 in human T cells;

[1030] k) reducing soluble levels of LAG-3 in cultures of human T cells;

[1031] 1) Inducing tumor growth regression in vivo;

[1032] m) slowing tumor growth in vivo; and

[1033] n) does not bind to the same epitope of human LAG-3 as antibody 25F7-Lag3.5.

[1034] Examples of such antibodies include, but are not limited to, antibody 15646 (having at least properties b, c, d, e, i, and n), antibody 15532 (having at least properties a, c, d, e, f, g, i, j, k, m, and n), antibody 15723 (having at least properties b, c, d, e, i, and n), antibody 15595 (having at least properties a, c, d, e, i, and n), antibody 15431 (having at least properties a, c, d, e, f, g, i, and n), antibody 15572 (having at least properties b, c, d, e, f, g, i, and n), and antibody 15011 (having at least properties a, c, d, e, f, g, h, i, j, k, l, m, and n). In some embodiments, the anti-TIM-3 antibodies or antigen-binding portions of the invention have at least 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 of the properties described. In some embodiments, the anti-TIM-3 antibodies or antigen-binding portions of the invention have 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; at least properties b, 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.

[1035] In some embodiments, the anti-LAG-3 antibodies, or antigen-binding portions, of the invention compete with antibodies 15011, 15572, and / or 15431 for binding to human LAG-3.

[1036] In some embodiments, the anti-LAG-3 antibodies, or antigen-binding portions, of the invention bind to an epitope of human LAG-3 having:

[1037] a) amino acid residues H85, P86, A87, P89, S91, W92, and G93 of SEQ ID NO:68;

[1038] 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;

[1039] 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

[1040] d) amino acid residues G107, L109, R110, and S111 of SEQ ID NO: 68.

[1041] In some embodiments, the anti-LAG-3 antibodies or antigen-binding portions of the invention bind 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.

[1042] In some embodiments, the anti-LAG-3 antibodies, or antigen-binding portions, of the invention bind to an epitope having:

[1043] a) amino acid residues 78-105 and 123-131 of SEQ ID NO: 68;

[1044] b) amino acid residues 23-30, 40-66, 88-105, 123-137, and 148-152 of SEQ ID NO: 68; or

[1045] c) amino acid residues 23-30, 40-66, 98-105, 118-137, and 148-161 of SEQ ID NO: 68

[1046] In some embodiments, an anti-LAG-3 antibody, or antigen-binding portion thereof, as described herein is an anti-LAG-3 antibody, or antigen-binding portion thereof, described in PCT patent application PCT / EP2017 / 076188, which is herein incorporated by reference in its entirety.

[1047] The class of the antibodies described herein can be changed or converted to another class or subclass. In one aspect, the nucleic acid molecules encoding VL or VH are isolated using methods known in the art so that they do not include nucleic acid sequences encoding CL or CH. The nucleic acid molecules encoding VL or VH are then operably linked to nucleic acid sequences encoding CL or CH from different types of immunoglobulin molecules, respectively. This can be achieved using vectors or nucleic acid molecules comprising CL or CH chains, as described above. For example, the class of an antibody that was originally IgM can be converted to IgG. In addition, class conversion can be used to convert one IgG subclass into another, such as from IgG1 to IgG2. The kappa light chain constant region can be changed (for example) to a lambda light chain constant region. A preferred method for producing an antibody as described herein having a desired Ig isotype comprises the following steps: isolating a nucleic acid molecule encoding the heavy chain of an antibody and a nucleic acid molecule encoding the light chain of an antibody, obtaining the heavy chain variable domain, connecting the heavy chain variable domain to a heavy chain constant region of the desired isotype, expressing the light chain and the connected heavy chain in cells, and collecting antibodies having the desired isotype.

[1048] The antibodies described herein can be IgG, IgM, IgE, IgA, or IgD molecules, but are typically of the IgG isotype, for example, IgG subclass IgG1, IgG2a or IgG2b, IgG3 or IgG4. In one embodiment, the antibody is IgG1. In another embodiment, the antibody is IgG2.

[1049] In one embodiment, the antibody may comprise at least one mutation in the Fc region. Many different Fc mutations are known, wherein these mutations provide altered effector function. For example, in many cases (e.g., where ligand / receptor interactions are undesirable or in the case of antibody-drug conjugates), it may be desirable to reduce or eliminate effector function.

[1050] In one embodiment, the antibody comprises at least one mutation in the Fc region that reduces effector function. Fc region amino acid positions that may be advantageous for mutation to reduce effector function include one or more of positions 228, 233, 234, and 235, wherein the amino acid positions are based on Numbering scheme number.

[1051] In some embodiments, one or both of the amino acid residues at positions 234 and 235 can be mutated, for example, from Leu to Ala (L234A / L235A). These mutations reduce the effector function of the Fc region of the IgG1 antibody. Additionally or alternatively, the amino acid residue at position 228 can be mutated, for example, to Pro. In some embodiments, the amino acid residue at position 233 can be mutated, for example, to Pro, the amino acid residue at position 234 can be mutated, for example, to Val, and / or the amino acid residue at position 235 can be mutated, for example, to Ala. The amino acid positions are determined according to Numbering scheme number.

[1052] In some embodiments, where the antibody is of the IgG4 subclass, it may comprise the mutation S228P, ie, a proline at position 228, wherein the amino acid position is according to Eu Numbering scheme No. This mutation is known to reduce undesired Fab arm exchange (Angal et al., Mol Immunol. 30:105-8 (1993)).

[1053] In certain embodiments, an antibody or antigen-binding portion thereof as described herein may be part of a larger immunoadhesion molecule formed by covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of a streptavidin core region to make tetrameric scFv molecules (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 make bivalent and biotinylated scFv molecules (Kipriyanov et al., Mol. Immunol. 31:1047-1058 (1994)). Other examples include cases where one or more CDRs from an antibody are covalently or non-covalently incorporated into a molecule to make it an immunoadhesin that specifically binds to an antigen of interest. In such embodiments, the CDRs may be incorporated as part of a larger polypeptide chain, may be covalently linked to another polypeptide chain, or may be incorporated non-covalently.

[1054] In another embodiment, a fusion antibody or immunoadhesin comprising all or part of an antibody described herein connected to another polypeptide can be made. In certain embodiments, only the variable domain of the antibody is connected to the polypeptide. In certain embodiments, the VH domain of the antibody is connected to a first polypeptide, and the VL domain of the antibody is connected to a second polypeptide, which is connected to the first polypeptide in a manner such 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., single-chain antibodies). The VH-linker-VL antibody is then connected to the polypeptide of interest. In addition, a fusion antibody can be created in which two (or more) single-chain antibodies are connected to each other. This is useful if you want to create a divalent or multivalent antibody on a single polypeptide chain or if you want to create a bispecific antibody.

[1055] To create a single-chain antibody (scFv), the VH- and VL-encoding DNA fragments are operably linked to another fragment encoding a flexible linker (e.g., encoding the amino acid sequence (Gly4-Ser)3 (SEQ ID NO: 396)) so that the VH and VL sequences can be expressed as a continuous single-chain protein in which the VL and VH domains are connected by the flexible linker. See, e.g., 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). The single-chain antibody can be monovalent, if only a single VH and VL are used; bivalent, if two VH and VL are used; or multivalent, if more than two VH and VL are used. For example, bispecific or multivalent antibodies can be generated that specifically bind to human PD-1, TIM-3, or LAG-3 and to another molecule. In some embodiments, the bispecific or multivalent antibody can 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.

[1056] In other embodiments, other modified antibodies can be prepared using antibody encoding nucleic acid molecules. For example, kappa bodies (Ill et al., Protein Eng. 10:949-57 (1997)), "minibodies" (Martin et al., EMBO J. 13:5303-9 (1994)), "dibodies" (Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)), or "Janusins" (Traunecker et al., EMBO J. 10:3655-3659 (1991) and Traunecker et al., Int. J. Cancer (Suppl.) 7:51-52 (1992)) can be prepared using standard molecular biology techniques according to the instructions of the specification.

[1057] As described herein, antibodies or antigen-binding portions thereof can be derivatized or connected to another molecule (e.g., another peptide or protein). In general, antibodies or portions thereof are derivatized so that antigen binding is not negatively affected by the derivatization or labeling. Therefore, the antibodies and antibody portions that can be used in the combination therapies and compositions of the present invention are intended to include both the complete form and the modified form of the antibodies described herein. For example, antibodies or antibody portions as described herein can be functionally connected (by chemical coupling, gene fusion, non-covalent connection or other) to one or more other molecular entities, such as another antibody (e.g., bispecific antibody or diabody), a detection agent, a pharmaceutical agent, and / or a protein or peptide (such as a streptavidin core region or a polyhistidine tag) that can mediate the connection of the antibody or antibody portion to another molecule.

[1058] One type of derivatized antibody is generated by cross-linking two or more antibodies (of the same type or different types, e.g., to create bispecific antibodies). Suitable cross-linkers include heterobifunctional linkers (which have two different reactive groups separated by an appropriate spacer, e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional linkers, e.g., disuccinimidyl suberate). Such linkers are available, for example, from Pierce Chemical Company, Rockford, IL.

[1059] Antibodies can also be derivatized with chemical groups such as polyethylene glycol (PEG), methyl or ethyl groups, or carbohydrate groups. These groups can be used to improve the biological characteristics of antibodies, for example, to increase serum half-life.

[1060] The antibodies described herein may also be labeled. As used herein, the term "label" or "labeled" refers to the incorporation of another molecule into an antibody. In one embodiment, the label is a detectable marker, such as the incorporation of a radiolabeled amino acid or the attachment of a polypeptide to a biotinyl moiety that can be detected by labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatically active streptavidin that can be detected by optical or colorimetric methods). In another embodiment, the label or marker can 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 labels for polypeptides include, but are not limited to, radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent labels, biotinyl groups, predetermined epitopes of the polypeptide recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), magnetic agents such as gadolinium chelates, toxins, In some embodiments, the label is attached via spacer arms of various lengths to reduce potential steric hindrance.

[1061] In certain embodiments, the antibodies described herein may exist in neutral form (including zwitterionic form) or as positively or negatively charged species. In some embodiments, the antibodies may be complexed with counterions to form pharmaceutically acceptable salts.

[1062] The term "pharmaceutically acceptable salt" refers to a complex comprising one or more antibodies and one or more counterions, wherein the counterions are derived from pharmaceutically acceptable inorganic and organic acids and bases.

[1063] Combination therapy

[1064] The present invention provides combination therapies (e.g., compositions) comprising any (e.g., any) anti-PD-1 antibody or antigen-binding portion thereof described herein and any (e.g., any) anti-TIM-3 antibody or antigen-binding portion thereof described herein. In some embodiments, the combination therapy comprises any (e.g., any) anti-PD-1 antibody or antigen-binding portion thereof described herein and any (e.g., any) anti-LAG-3 antibody or antigen-binding portion thereof described herein. In some embodiments, the combination therapy comprises any (e.g., any) anti-TIM-3 antibody or antigen-binding portion thereof described herein and any (e.g., any) anti-LAG-3 antibody or antigen-binding portion thereof described herein. In specific embodiments, the combination therapy comprises any (e.g., any) anti-PD-1 antibody or antigen-binding portion thereof described herein, any (e.g., any) anti-TIM-3 antibody or antigen-binding portion thereof described herein, and any (e.g., any) anti-LAG-3 antibody or antigen-binding portion thereof described herein. The combination therapy can take the form of, for example, a method for treatment using the antibody or antigen-binding portion or a pharmaceutical composition comprising the antibody or antigen-binding portion.

[1065] 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.

[1066] In certain embodiments, the combination therapy or composition of the invention comprises:

[1067] - an anti-PD-1 antibody, or an antigen-binding portion thereof, comprising the H-CDR1-3 and L-CDR1-3 amino acid sequences of SEQ ID NOs: 228-233, respectively; and an anti-TIM-3 antibody, or an antigen-binding portion thereof, comprising the H-CDR1-3 and L-CDR1-3 amino acid sequences of SEQ ID NOs: 8-13, respectively;

[1068] - an anti-PD-1 antibody, or an antigen-binding portion thereof, comprising the VH and VL amino acid sequences of SEQ ID NOs: 226 and 227, respectively; and an anti-TIM-3 antibody, or an antigen-binding portion thereof, comprising the VH and VL amino acid sequences of SEQ ID NOs: 7 and 4, respectively; or

[1069] - an anti-PD-1 antibody comprising a HC comprising the amino acid sequence of SEQ ID NOs: 226 and 375 and a LC comprising the amino acid sequence of SEQ ID NOs: 227 and 379; and an anti-TIM-3 antibody comprising a HC comprising the amino acid sequence of SEQ ID NOs: 7 and 377 and a LC comprising the amino acid sequence of SEQ ID NOs: 4 and 378.

[1070] In certain embodiments, the combination therapy or composition of the invention comprises:

[1071] - an anti-PD-1 antibody, or an antigen-binding portion thereof, comprising the H-CDR1-3 and L-CDR1-3 amino acid sequences of SEQ ID NOs: 228-233, respectively; and an anti-LAG-3 antibody, or an antigen-binding portion thereof, comprising the H-CDR1-3 and L-CDR1-3 amino acid sequences of SEQ ID NOs: 318-323, respectively;

[1072] - an anti-PD-1 antibody, or an antigen-binding portion thereof, comprising the VH and VL amino acid sequences of SEQ ID NOs: 226 and 227, respectively; and an anti-LAG-3 antibody, or an antigen-binding portion thereof, comprising the VH and VL amino acid sequences of SEQ ID NOs: 316 and 317, respectively; or

[1073] - an anti-PD-1 antibody comprising a HC comprising the amino acid sequence of SEQ ID NOs: 226 and 375 and a LC comprising the amino acid sequence of SEQ ID NOs: 227 and 379; and an anti-LAG-3 antibody comprising a HC comprising the amino acid sequence of SEQ ID NOs: 316 and 375 and a LC comprising the amino acid sequence of SEQ ID NOs: 317 and 378.

[1074] In certain embodiments, the combination therapy or composition of the invention comprises:

[1075] An anti-TIM-3 antibody, or an antigen-binding portion thereof, comprising the H-CDR1-3 and L-CDR1-3 amino acid sequences of SEQ ID NOs: 8-13, respectively; and an anti-LAG-3 antibody, or an antigen-binding portion thereof, comprising the H-CDR1-3 and L-CDR1-3 amino acid sequences of SEQ ID NOs: 318-323, respectively;

[1076] - an anti-TIM-3 antibody, or an antigen-binding portion thereof, comprising the VH and VL amino acid sequences of SEQ ID NOs: 7 and 4, respectively; and an anti-LAG-3 antibody, or an antigen-binding portion thereof, comprising the VH and VL amino acid sequences of SEQ ID NOs: 316 and 317, respectively; or

[1077] - an anti-TIM-3 antibody comprising a HC comprising the amino acid sequence of SEQ ID NOs: 7 and 377 and a LC comprising the amino acid sequence of SEQ ID NOs: 4 and 378; and an anti-LAG-3 antibody comprising a HC comprising the amino acid sequence of SEQ ID NOs: 316 and 375 and a LC comprising the amino acid sequence of SEQ ID NOs: 317 and 378.

[1078] In certain embodiments, the combination therapy or composition of the invention comprises:

[1079] - an anti-PD-1 antibody, or an antigen-binding portion thereof, comprising the H-CDR1-3 and L-CDR1-3 amino acid sequences of SEQ ID NOs: 228-233, respectively; an anti-TIM-3 antibody, or an antigen-binding portion thereof, comprising the H-CDR1-3 and L-CDR1-3 amino acid sequences of SEQ ID NOs: 8-13, respectively; and an anti-TIM-3 antibody, or an antigen-binding portion thereof, comprising the H-CDR1-3 and L-CDR1-3 amino acid sequences of SEQ ID NOs: 318-323, respectively;

[1080] - an anti-PD-1 antibody, or an antigen-binding portion thereof, comprising the VH and VL amino acid sequences of SEQ ID NOs: 226 and 227, respectively; an anti-TIM-3 antibody, or an antigen-binding portion thereof, comprising the VH and VL amino acid sequences of SEQ ID NOs: 7 and 4, respectively; and an anti-LAG-3 antibody, or an antigen-binding portion thereof, comprising the VH and VL amino acid sequences of SEQ ID NOs: 316 and 317, respectively; or

[1081] -An anti-PD-1 antibody comprising a HC comprising the amino acid sequence of SEQ ID NOs: 226 and 375 and a LC comprising the amino acid sequence of SEQ ID NOs: 227 and 379; an anti-TIM-3 antibody comprising a HC comprising the amino acid sequence of SEQ ID NOs: 7 and 377 and a LC comprising the amino acid sequence of SEQ ID NOs: 4 and 378; and an anti-LAG-3 antibody comprising a HC comprising the amino acid sequence of SEQ ID NOs: 316 and 375 and a LC comprising the amino acid sequence of SEQ ID NOs: 317 and 378.

[1082] In some embodiments, any or all antibodies in the combination therapy or composition can be IgG, eg, IgG1 or IgG2.

[1083] Multispecific binding molecules

[1084] In further aspects, the invention provides multispecific binding molecules with the following binding specificities (e.g., an antigen binding portion comprising the following, such as an antigen binding portion comprising the following six CDRs):

[1085] - an anti-PD-1 antibody as described herein and an anti-TIM-3 antibody as described herein;

[1086] - an anti-PD-1 antibody as described herein and an anti-LAG-3 antibody as described herein; or

[1087] - an anti-TIM-3 antibody as described herein and an anti-LAG-3 antibody as described herein.

[1088] 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 multispecific binding molecule has the binding specificity of the anti-PD-1 antibody described herein, the anti-TIM-3 antibody described herein, and the anti-LAG-3 antibody described herein. Multispecific binding molecules are known in the art, and examples of different types of multispecific binding molecules are given elsewhere herein. Such multispecific (e.g., bispecific or trispecific) binding molecules are encompassed by the combination therapies of the present invention.

[1089] In another aspect, the present invention provides a combination therapy using two or more of the bispecific binding molecules targeting PD-1, the bispecific binding molecules targeting TIM-3, and the bispecific binding molecules targeting LAG-3. The bispecific binding molecules targeting PD-1, TIM-3, or LAG-3 may have the binding specificity of an antibody targeting an antigen as described herein and 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, cancer antigen, or another cell surface molecule thereof that mediates a disease state (such as cancer)). Such bispecific binding molecules are known in the art, and examples of different types of bispecific binding molecules are given in other parts of this article.

[1090] Nucleic acid molecules and vectors

[1091] Also described are nucleic acid molecules and sequences encoding the anti-PD-1, anti-TIM-3, and / or anti-LAG-3 antibodies, or antigen-binding portions thereof, described herein. In some embodiments, different nucleic acid molecules encode the heavy and light chain amino acid sequences of an anti-PD-1 antibody, or antigen-binding portion thereof, an anti-TIM-3 antibody, or antigen-binding portion thereof, or an anti-LAG-3 antibody, or antigen-binding portion thereof. In other embodiments, the same nucleic acid molecule encodes the heavy and light chain amino acid sequences of the anti-PD-1 antibody, or antigen-binding portion thereof, an anti-TIM-3 antibody, or antigen-binding portion thereof, or an anti-LAG-3 antibody, or antigen-binding portion thereof.

[1092] Reference to a nucleotide sequence encompasses its complement, unless otherwise specifically indicated. Thus, reference to a nucleic acid having a particular sequence should be understood to encompass its complementary strand having its complementary sequence. As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides (ribonucleotides or deoxynucleotides or modified forms of either) of at least 10 bases in length. This term includes both single-stranded and double-stranded forms.

[1093] In some embodiments, the nucleotide sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to one or more nucleotide sequences described herein, for example, a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 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, 196, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215 95, 204, 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. The term "percent sequence identity" before a nucleic acid sequence and hereinafter refers to the residues that are the same in two sequences when aligned for maximum correspondence. The length of the sequence identity comparison can extend across at least about nine nucleotides, usually at least about 18 nucleotides, more usually at least about 24 nucleotides, typically 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, which are programs in the Wisconsin Package version 10.0, Genetics Computer Group (GCG), Madison, Wisconsin. FASTA (which includes, for example, the programs FASTA2 and FASTA3) provides alignment and percent sequence identity for query and search regions of optimal overlap between sequences (see, e.g., 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). Unless otherwise specifically stated, 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 (a word size of 6 and a NOPAM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG version 6.1, which is incorporated herein by reference.

[1094] In some embodiments, a nucleic acid molecule comprising one or more nucleotide sequences selected from the group consisting of SEQ ID NO: 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, 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.

[1095] In any of the above embodiments, the nucleic acid molecule can be isolated. Nucleic acid molecules encoding the heavy chain and / or light chain of the antibody described herein or its antigen-binding portion thereof can be isolated from any source that generates such antibodies or portions. In various embodiments, nucleic acid molecules are isolated from B cells expressing antibodies isolated from animals immunized with PD-1, TIM-3, or LAG-3 antigens or from immortalized cells generated from such B cells. Methods for isolating nucleic acids encoding antibodies are well known in the art. MRNA can be isolated and used to generate cDNA for polymerase chain reaction (PCR) or cDNA cloning of antibody genes. In certain embodiments, nucleic acid molecules as described herein can be synthesized rather than isolated.

[1096] In some embodiments, a nucleic acid molecule as described herein may comprise a nucleotide sequence encoding a VH domain from an antibody or antigen-binding portion described herein, linked in frame to a nucleotide sequence encoding a heavy chain constant region from any source. Similarly, a nucleic acid molecule as described herein may comprise a nucleotide sequence encoding a VL domain from an antibody or antigen-binding portion described herein, linked in frame to a nucleotide sequence encoding a light chain constant region from any source.

[1097] In other aspects, nucleic acid molecules encoding heavy chain variable domains (VH) and / or light chain variable domains (VL) can be "converted" into full-length antibody genes. In one embodiment, nucleic acid molecules encoding VH or VL domains are converted into full-length antibody genes by the following: respectively inserted into an expression vector encoding heavy chain constant (CH) or light chain constant (CL) region so that the VH segment is operably connected to the CH segment in the vector, and / or the VL segment is operably connected to the CL segment in the vector. In another embodiment, nucleic acid molecules encoding VH and / or VL domains are converted into full-length antibody genes by the following: nucleic acid molecules encoding VH and / or VL domains are linked (e.g., connected) to nucleic acid molecules encoding CH and / or CL regions using standard molecular biology techniques. Nucleic acid molecules encoding full-length heavy and / or light chains can then be expressed from the cells into which the nucleic acid molecules have been introduced and antibodies can be isolated.

[1098] Nucleic acid molecules can be used to recombinantly express large quantities of antibodies. Nucleic acid molecules can also be used to generate chimeric antibodies, bispecific antibodies, single-chain antibodies, immunoadhesins, diabodies, mutated antibodies, and antibody derivatives, as described herein.

[1099] Also described herein are vectors suitable for expressing one or both of the chains of the anti-PD-1 antibodies, or antigen-binding portions thereof, anti-TIM-3 antibodies, or antigen-binding portions thereof, and / or anti-LAG-3 antibodies, or antigen-binding portions thereof, as described herein. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is attached. In some embodiments, the vector is a plasmid, i.e., a circular, double-stranded fragment of DNA to which additional DNA segments can be attached. In some embodiments, the vector is a viral vector, wherein additional DNA segments can be attached to its viral genome. In some embodiments, the vector is capable of autonomously replicating in the host cell into which it is introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). In other embodiments, the vector (e.g., non-episomal mammalian vectors) can be incorporated into the host cell's genome immediately after introduction into the host cell and thereby replicate as the host genome replicates. In addition, certain vectors are capable of directing the expression of genes to which they are operably attached. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

[1100] In some embodiments, the vector comprises a nucleic acid molecule encoding a heavy chain, a light chain, or both a heavy chain and a light chain of an antibody or antigen-binding portion thereof described herein. In some embodiments, the vector comprises a nucleic acid molecule encoding a fusion protein, a modified antibody, an antibody fragment, and a probe thereof.

[1101] In some embodiments, anti-PD-1, anti-TIM-3, or anti-LAG-3 antibodies or antigen-binding portions thereof are expressed by inserting DNA encoding partial or full-length light and heavy chains obtained as described above into expression vectors so that the genes are operably linked to necessary expression control sequences, such as transcription and translation control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses, such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV-derived episomes, and the like. The antibody coding sequence can be linked to a vector so that the transcription and translation control sequences within the vector perform their intended functions of regulating the transcription and translation of the antibody coding sequence. The expression vector and expression control sequence can be selected to be compatible with the expression host cell used. The antibody light chain coding sequence and the antibody heavy chain coding sequence can be inserted into separate vectors and can be operably linked to the same or different expression control sequences (e.g., promoters). In one embodiment, the two coding sequences are inserted into the same expression vector and can be operably linked to the same expression control sequence (e.g., a common promoter), to separate identical expression control sequences (e.g., promoters), or to different expression control sequences (e.g., promoters). The antibody coding sequence can be inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and the vector, or blunt end ligation if no restriction sites are present).

[1102] Convenient vectors are those that encode functionally fully human CH or CL immunoglobulin sequences and have appropriate restriction sites engineered to allow for easy insertion and expression of any VH or VL sequence (as described above). The HC- and LC-encoding genes in such vectors may contain intronic sequences that increase the overall yield of the antibody protein by stabilizing the associated mRNA. The intronic sequences are flanked by splice donor and splice acceptor sites, which determine where RNA splicing occurs. The intronic sequence location may be in the variable or constant region of the antibody chain, or in both the variable and constant regions when multiple introns are used. Polyadenylation and transcription termination may occur at the native chromosomal location downstream of the coding region. The recombinant expression vector may also encode a signal peptide that promotes secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into the vector so that the signal peptide is linked in frame 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).

[1103] In addition to the antibody chain genes, the recombinant expression vector may carry regulatory sequences that control the expression of the antibody chain genes in the host cell. Those skilled in the art will appreciate that expression vector design (including regulatory sequence selection) may depend on factors such as the choice of host cells to be transformed, the level of expression of the desired protein, and the like. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high-level protein expression in mammalian cells, such as promoters and / or enhancers derived from retroviral LTRs, promoters and / or enhancers derived from cytomegalovirus (CMV) (such as CMV promoter / enhancer), promoters and / or enhancers derived from simian virus 40 (SV40) (such as SV40 promoter / enhancer), promoters and / or enhancers derived from adenovirus (e.g., adenovirus major late promoter (AdMLP)), polyoma, and strong mammalian promoters, such as natural immunoglobulin and actin promoters. For further 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 transformation, are known in the art. See, for example, U.S. Patent 6,517,529. Methods for expressing polypeptides in bacterial cells or fungal cells (e.g., yeast cells) are also known in the art.

[1104] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vector may carry additional sequences, such as sequences that regulate replication of the vector in the host cell (e.g., an origin of replication) and a selectable marker gene. The selectable marker gene facilitates 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, typically, the selectable marker gene confers resistance to drugs (such as G418, hygromycin, or methotrexate) to the host cells into which the vector has been introduced. For example, selectable marker genes include the dihydrofolate reductase (DHFR) gene (for selecting / amplifying dhfr- host cells with methotrexate), the neo gene (for G418 selection), and the glutamate synthetase gene.

[1105] The term "expression control sequence" as used herein refers to polynucleotide sequences that are necessary for the expression and processing of the coding sequences to which they are linked. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that increase translation efficiency (i.e., Kozak consensus sequences); sequences that increase protein stability; and, when desired, sequences that increase protein secretion. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences generally include promoters, ribosome binding sites, and transcription termination sequences; in eukaryotes, such control sequences generally include promoters and transcription termination sequences. The term "control sequence" is intended to include, at a minimum, all components whose presence is necessary for expression and processing, and may also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

[1106] Host cells and methods for producing antibodies and antibody compositions

[1107] Methods for producing the combination therapies (e.g., compositions) 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 the antibody, culturing the host cell under conditions suitable for expression of the antibody, and isolating the resulting antibody. Antibodies produced by such expression in such recombinant host cells are referred to herein as "recombinant antibodies." Progeny cells of such host cells, and antibodies produced thereby, are also described.

[1108] As used herein, the term "recombinant host cell" (or simply "host cell") refers to a cell into which a recombinant expression vector has been introduced. A host cell may comprise, for example, one or more vectors as described herein. A host cell may comprise, for example, a nucleotide sequence encoding a heavy chain or antigen-binding portion thereof of an anti-PD-1, anti-TIM-3, and / or anti-LAG-3 antibody or antigen-binding portion thereof as described herein, a nucleotide sequence encoding a light chain or antigen-binding portion thereof of an anti-PD-1, anti-TIM-3, and / or anti-LAG-3 antibody or antigen-binding portion thereof as described herein, or both. It should be understood that "recombinant host cell" and "host cell" refer not only to the particular target cell but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not be (in fact) completely identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[1109] Nucleic acid molecules encoding anti-PD-1, anti-TIM-3, and / or anti-LAG-3 antibodies or antigen-binding portions thereof and vectors comprising these nucleic acid molecules can be used to transfect suitable mammalian, plant, bacterial or yeast host cells. Transformation can be 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-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in lipid particles, and direct microinjection of DNA into the nucleus. In addition, nucleic acid molecules can 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 are well known in the art and include, for example, Agrobacterium-mediated transformation, biolistic transformation, direct injection, electroporation, and viral transformation. Methods for transforming bacterial and yeast cells are also well known in the art.

[1110] Mammalian cells that can be obtained as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These particularly include Chinese hamster ovary (CHO) cells, NSO cells, SP2 cells, HEK-293T cells, 293Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), A549 cells, and many other cell lines. Particularly preferred cell lines are selected by determining which cell line has a high expression level. Other cell lines that can be used are insect cell lines, such as Sf9 or Sf21 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell or (more preferably) secretion of the antibody into the culture medium of the host cell. The antibody can be recovered from the culture medium using standard protein purification methods. Plant host cells include, for example, Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, etc. Bacterial host cells include Escherichia coli and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Pichia pastoris.

[1111] In addition, the expression of the antibodies or antigen-binding portions thereof described herein from production cell lines can be increased using a number of known techniques. For example, the glutamine synthetase gene expression system (GS system) is a common method for increasing expression under certain conditions. The GS system is discussed in conjunction with all or part of EP Patents 0 216 846, 0 256 055, 0 323 997, and 0 338 841.

[1112] It is likely that antibodies expressed by different cell lines or in transgenic animals will have different glycosylation patterns from one another. However, all antibodies encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein are part of the present invention, regardless of the glycosylation state of the antibodies, and more generally, regardless of the presence or absence of post-translational modifications.

[1113] 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, the method comprising:

[1114] - providing a first and a second host cell, wherein the first host cell is capable of expressing an anti-PD-1 antibody as described herein and the second host cell is capable of expressing an anti-TIM-3 antibody as described herein,

[1115] - culturing the first and second host cells under conditions suitable for the expression of anti-PD-1 antibody and anti-TIM-3 antibody, and

[1116] - Isolating the resulting antibodies.

[1117] In some embodiments, the present invention relates to a method for generating an antibody composition comprising an anti-PD-1 antibody and an anti-LAG-3 antibody, the method comprising:

[1118] - providing a first and a second host cell, wherein the first host cell is capable of expressing an anti-PD-1 antibody as described herein and the second host cell is capable of expressing an anti-LAG-3 antibody as described herein,

[1119] - culturing the first and second host cells under conditions suitable for expression of the anti-PD-1 antibody and the anti-LAG-3 antibody, and

[1120] - Isolating the resulting antibodies.

[1121] 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, the method comprising:

[1122] - providing a first and a second host cell, wherein the first host cell is capable of expressing an anti-TIM-3 antibody as described herein and the second host cell is capable of expressing an anti-LAG-3 antibody as described herein,

[1123] - culturing the first and second host cells under conditions suitable for expression of the anti-TIM-3 antibody and the anti-LAG-3 antibody, and

[1124] - Isolating the resulting antibodies.

[1125] In some embodiments, the present invention relates to a method for generating an antibody composition comprising an anti-PD-1 antibody, an anti-TIM-3 antibody, and an anti-LAG-3 antibody, the method comprising:

[1126] - providing first, second, and third host cells, wherein the first host cell is capable of expressing an anti-PD-1 antibody as described herein, the second host cell is capable of expressing an anti-TIM-3 antibody as described herein, and the third host cell is capable of expressing an anti-LAG-3 antibody as described herein,

[1127] - culturing the first, second, and third host cells under conditions suitable for expression of the anti-PD-1 antibody, the anti-TIM-3 antibody, and the anti-LAG-3 antibody, and

[1128] - Isolating the resulting antibodies.

[1129] For the generation of the antibody composition of the present invention, antibodies against different targets can be generated separately, i.e., each antibody is generated in a separate bioreactor, or individual antibodies can be generated together in a single bioreactor. If the antibody composition is generated in more than one bioreactor, a purified antibody composition can be obtained by combining the antibodies obtained from the individually purified supernatants of each bioreactor. Various methods for the generation of polyclonal antibody compositions in multiple bioreactors (wherein cell lines or antibody preparations are combined at a later time point upstream or before downstream processing or during downstream processing) are described in PCT Publication WO 2009 / 129814.

[1130] In the case of individual antibodies produced in a single bioreactor, this can be performed, for example, as described in PCT publications WO 2004 / 061104 or WO 2008 / 145133. The method described in WO 2004 / 061104 is based on site-specific incorporation of antibody coding sequences into the genome of individual host cells, while the method of WO 2008 / 145133 involves an alternative method of using random incorporation to produce antibodies in a single bioreactor.

[1131] Further information regarding methods suitable for generating the antibody compositions of the invention can be found in PCT publications WO 2012 / 059857 and WO 2013 / 164689.

[1132] The present invention also provides polyclonal cell lines that produce:

[1133] - at least one anti-PD-1 antibody, or an antigen-binding portion thereof, as described herein and at least one anti-TIM-3 antibody, or an antigen-binding portion thereof, as described herein;

[1134] - at least one anti-PD-1 antibody, or antigen-binding portion thereof, as described herein and at least one anti-LAG-3 antibody, or antigen-binding portion thereof, as described herein;

[1135] - at least one anti-TIM-3 antibody, or antigen-binding portion thereof, as described herein and at least one anti-LAG-3 antibody, or antigen-binding portion thereof, as described herein; or

[1136] - at least one anti-PD-1 antibody, or an antigen-binding portion thereof, as described herein, at least one anti-TIM-3 antibody, or an antigen-binding portion thereof, as described herein, and at least one anti-LAG-3 antibody, or an antigen-binding portion thereof, as described herein.

[1137] The present invention also provides a method for generating the above polyclonal cell line, comprising providing host cells each comprising a nucleotide sequence encoding a heavy chain, or antigen-binding portion thereof, of at least one of the antibodies or moieties produced by the polyclonal cell line and a nucleotide sequence encoding a light chain, or antigen-binding portion thereof, of at least one of the antibodies or moieties produced by the polyclonal cell line.

[1138] The present invention also provides a host cell comprising:

[1139] - a nucleotide sequence encoding the heavy chain or antigen-binding portion thereof of an anti-PD-1 antibody as described herein, a nucleotide sequence encoding the light chain or antigen-binding portion thereof of an anti-PD-1 antibody as described herein, or both, and a nucleotide sequence encoding the heavy chain or antigen-binding portion thereof of an anti-TIM-3 antibody as described herein, a nucleotide sequence encoding the light chain or antigen-binding portion thereof of an anti-TIM-3 antibody as described herein, or both;

[1140] - a nucleotide sequence encoding the heavy chain or antigen-binding portion thereof of an anti-PD-1 antibody as described herein, a nucleotide sequence encoding the light chain or antigen-binding portion thereof of an anti-PD-1 antibody as described herein, or both; and a nucleotide sequence encoding the heavy chain or antigen-binding portion thereof of an anti-LAG-3 antibody as described herein, a nucleotide sequence encoding the light chain or antigen-binding portion thereof of an anti-LAG-3 antibody as described herein, or both;

[1141] - a nucleotide sequence encoding the heavy chain or antigen-binding portion thereof of an anti-TIM-3 antibody as described herein, a nucleotide sequence encoding the light chain or antigen-binding portion thereof of an anti-TIM-3 antibody as described herein, or both; and a nucleotide sequence encoding the heavy chain or antigen-binding portion thereof of an anti-LAG-3 antibody as described herein, a nucleotide sequence encoding the light chain or antigen-binding portion thereof of an anti-LAG-3 antibody as described herein, or both; or

[1142] - a nucleotide sequence encoding the heavy chain or antigen binding portion thereof of an anti-PD-1 antibody as described herein, a nucleotide sequence encoding the light chain or antigen binding portion thereof of an anti-PD-1 antibody as described herein, or both; a nucleotide sequence encoding the heavy chain or antigen binding portion thereof of an anti-TIM-3 antibody as described herein, a nucleotide sequence encoding the light chain or antigen binding portion thereof of an anti-TIM-3 antibody as described herein, or both; and a nucleotide sequence encoding the heavy chain or antigen binding portion thereof of an anti-LAG-3 antibody as described herein, a nucleotide sequence encoding the light chain or antigen binding portion thereof of an anti-LAG-3 antibody as described herein, or both.

[1143] Pharmaceutical composition

[1144] Another aspect of the invention is a pharmaceutical composition comprising as an active ingredient (e.g. as the only active ingredient):

[1145] at least one anti-PD-1 antibody, or an antigen-binding portion thereof, as described herein and at least one anti-TIM-3 antibody, or an antigen-binding portion thereof, as described herein;

[1146] - at least one anti-PD-1 antibody, or antigen-binding portion thereof, as described herein and at least one anti-LAG-3 antibody, or antigen-binding portion thereof, as described herein;

[1147] - at least one anti-TIM-3 antibody, or antigen-binding portion thereof, as described herein and at least one anti-LAG-3 antibody, or antigen-binding portion thereof, as described herein; or

[1148] - At least one anti-PD-1 antibody, or an antigen-binding portion thereof, as described herein, at least one anti-TIM-3 antibody, or an antigen-binding portion thereof, as described herein, and at least one anti-LAG-3 antibody, or an antigen-binding portion thereof, as described herein.

[1149] In some aspects, the pharmaceutical composition comprises a multispecific binding molecule (e.g., a multispecific binding molecule having the binding specificities of an anti-PD-1 antibody as described herein and an anti-TIM-3 or anti-LAG-3 antibody as described herein; or an anti-PD-1 antibody, an anti-TIM-3 antibody, and an anti-LAG-3 antibody as described herein).

[1150] In some embodiments, the pharmaceutical composition may further comprise one or more additional antibodies that target one or more relevant cell surface receptors (eg, one or more cancer-associated receptors).

[1151] In some embodiments, the pharmaceutical composition is intended to improve, prevent, and / or treat a disorder, disease, or condition by modulating PD-1, TIM-3, and / or LAG-3 to improve or slow the progression of the disorder, disease, or condition. In some embodiments, the pharmaceutical composition is intended to improve, prevent, and / or treat cancer. In some embodiments, the pharmaceutical composition is intended to activate the immune system.

[1152] The ratios between the antibodies of their antigen-binding portions in the pharmaceutical compositions of the present invention (or the ratios of the individual antibodies or portions described herein, administered simultaneously, sequentially, or separately) tend to result in the antibodies being administered in the same amount, but this is not necessarily the case. Thus, a composition of the present invention comprising an anti-PD-1 antibody and an anti-TIM-3 antibody, an anti-PD-1 antibody and an anti-LAG-3 antibody, or an anti-TIM-3 antibody and an anti-LAG-3 antibody may contain the antibodies in a ratio of approximately 1:1. A composition of the present invention comprising an anti-PD-1 antibody, an anti-TIM-3 antibody, and an anti-LAG-3 antibody may contain the antibodies in a ratio of approximately 1:1:1 (i.e., in the same amount). However, depending on the characteristics of the individual antibodies, it may be desirable to use different antibodies in different amounts. Suitable ratios for the different antibodies in the compositions of the present invention can be determined as described in PCT Publication WO2010 / 040356, which describes methods for identifying chemical entities in a combination drug product (e.g., a polyclonal antibody composition) and selecting the optimal stoichiometric ratio between them to obtain a combination drug with optimal efficacy and potency.

[1153] In general, the pharmaceutical compositions described herein are suitable for administration in a formulation in combination with one or more pharmaceutically acceptable excipients (eg, as described below).

[1154] The term "excipient" is used herein to describe any ingredient other than the compounds of the present invention. The selection of an excipient will depend largely on factors such as the particular mode of administration, the excipient's effect on solubility and stability, and the characteristics of the dosage form. As used herein, "pharmaceutically acceptable excipients" include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption-retarding agents, and the like. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many instances, it is preferred to include an isotonic agent (e.g., a sugar, a polyol such as mannitol, sorbitol, or sodium chloride) in the composition. Other examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances, such as wetting agents or emulsifiers, preservatives, or buffers, that enhance the shelf life or effectiveness of the antibody.

[1155] The pharmaceutical compositions of the present invention and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation can be found, for example, in Remington's Pharmaceutical Sciences, 19th edition (Mack Publishing Company, 1995). The pharmaceutical compositions are preferably produced under GMP (Good Manufacturing Practice) conditions.

[1156] The pharmaceutical compositions of the present invention can be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition that contains a predetermined amount of an active ingredient. The amount of active ingredient is generally equal to the dose that the active ingredient would be administered to a subject or a convenient fraction of such a dose, such as, for example, half or one-third of such a dose.

[1157] Any art-accepted method for administering peptides, proteins, or antibodies can be suitably employed with the antibodies and antigen-binding portions described herein.

[1158] Typically, the pharmaceutical compositions of the present invention are suitable for parenteral administration. As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physically opening a subject's tissue and administering the pharmaceutical composition through the opening in the tissue, thereby generally resulting in direct administration into the bloodstream, muscle, or internal organs. Thus, parenteral administration includes, but is not limited to, administering the composition by injection, administering the composition through a surgical incision, administering the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, ​​intracranial, intratumoral, and intrasynovial injection or infusion; and renal dialysis infusion techniques. Regional perfusion is also contemplated. Particular embodiments include intravenous and subcutaneous routes.

[1159] Typically, formulations suitable for parenteral administration of pharmaceutical compositions comprise active ingredients in combination with a pharmaceutically acceptable carrier (such as sterile water or sterile isotonic saline). Such formulations can be prepared, packaged, or sold in a form suitable for bolus injection or for continuous administration. Injectable formulations can be prepared, packaged, or sold in unit dosage form (such as in ampoules) or in multi-dose containers containing preservatives. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further comprise one or more additional ingredients, including, but not limited to, suspending agents, stabilizers, or dispersants. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) before parenteral administration of the reconstituted composition. Parenteral formulations also include aqueous solutions that may contain excipients such as salts, carbohydrates, and buffers (preferably to a pH of 3 to 9), but in some applications, they may be more suitably formulated into sterile non-aqueous solutions or dry forms to be used in conjunction with a suitable vehicle (such as sterile pyrogen-free water). Exemplary parenteral administration forms include solutions or suspensions in sterile aqueous solutions (e.g., propylene glycol or dextrose aqueous solutions). If desired, such dosage forms may be appropriately buffered. Other useful parenteral formulations include those comprising an active ingredient in microcrystalline form or in a liposomal formulation. Parenteral formulations can be formulated to be direct and / or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted, and programmed release.

[1160] For example, in one aspect, sterile injectable solutions can be prepared by incorporating the desired amount of the composition and one or more of the listed ingredients or combinations (if necessary) into an appropriate solvent followed by filtration sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the desired other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze drying, which produce a powder of the active ingredient from its previously sterile-filtered solution plus any other desired ingredients. This can be achieved, for example, by using a coating such as lecithin, by maintaining the desired particle size (in the case of dispersions), and by using a surfactant to maintain the appropriate fluidity of the solution. Long-term absorption of the injectable composition can be achieved by including an agent that delays absorption (e.g., monostearate and gelatin) in the composition and / or by using a modified release coating (e.g., sustained-release coating).

[1161] Therapeutic uses of the combination therapy and compositions of the present invention

[1162] In one aspect, the combination therapy and composition of the present invention are used to enhance or activate the immune system in patients (e.g., humans) in need. In some embodiments, the patient is immunosuppressed. In some embodiments, the physician can promote the anti-cancer activity of the patient's own immune system by administering the combination therapy or composition of the present invention alone or in combination with other therapeutic agents (sequentially or simultaneously). The combination therapy or composition regulates the activity of PD-1, TIM-3, and / or LAG-3 in immune cells, resulting in an increase in anti-cancer immunity. In certain embodiments, the combination therapy and composition of the present invention are used to treat cancer, for example, cancers derived from tissues such as skin, lungs, small intestine, colon, ovaries, brain, prostate, kidneys, soft tissues, hematopoietic system, head and neck, liver, bladder, breast, stomach, uterus and pancreas, and any cancer or other condition that relies on PD-1, TIM-3, and / or LAG-3 activity and / or wherein the patient expresses or overexpresses any of these ligands.

[1163] In some embodiments, cancers treated by the combination therapies and compositions of the invention can include, for example, melanoma (e.g., advanced or metastatic melanoma), non-small cell lung cancer, head and neck squamous cell carcinoma, renal cell carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, glioblastoma, glioma, squamous cell lung cancer, small cell lung cancer, hepatocellular carcinoma, bladder cancer, upper urinary tract cancer, esophageal cancer, gastroesophageal junction cancer, gastric cancer, liver cancer, colon cancer, colorectal cancer, multiple myeloma, sarcoma, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, nasopharyngeal carcinoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, small lymphocytic lymphoma, ovarian cancer, gastrointestinal cancer, Primary peritoneal cancer, fallopian tube cancer, urothelial cancer, HTLV-associated T-cell leukemia / lymphoma, prostate cancer, genitourinary tract cancer, meningioma, adrenocortical cancer, gliosarcoma, fibrosarcoma, renal cancer, breast cancer, pancreatic cancer, endometrial cancer, basal cell carcinoma of the skin, appendix cancer, biliary tract cancer, salivary gland cancer, advanced Merkel cell carcinoma, diffuse large B-cell lymphoma, follicular lymphoma, mesothelioma, neuroendocrine tumors, urological cancer, bone cancer, breast cancer, respiratory cancer, adenoid cystic carcinoma, cervical cancer, astrocytoma, chordoma, neuroblastoma, oral cancer, squamous cell carcinoma of the skin, thyroid cancer, Kaposi's sarcoma, anal cancer, gallbladder cancer, thymic cancer, uterine cancer, and solid tumors. Cancer can be, for example, early, intermediate, advanced, or metastatic.

[1164] In specific embodiments, cancers treated by the combination therapies and compositions of the invention can 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's lymphoma, non-Hodgkin's lymphoma, leukemia (e.g., acute myeloid leukemia), hematological malignancies, solid tumors (e.g., advanced or metastatic solid tumors), MSI-high tumors, HPV- and HIV-associated malignancies, and tumors with BRAC1 and BRAC2 mutations.

[1165] 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, and / or ulcerative colitis.

[1166] In some embodiments, the combination therapy or composition is used to treat viral and / or parasitic infections, for example, wherein the pathogen suppresses the host immune response. For example, the pathogen can be, for example, HIV, hepatitis (A, B, or C), human papillomavirus (HPV), lymphocytic choriomeningitis virus (LCMV), adenovirus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus (cornovirus), respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, poxvirus, human T cell lymphotropic virus (HTLV), human cytomegalovirus (HCMV), dengue virus, molluscum virus, poliovirus, rabies virus, John Cunningham (JC) virus, arboviral encephalitis virus, simian immunodeficiency virus (SIV), influenza, herpes, Giardia, malaria, Leishmania, Staphylococcus aureus, or Pseudomonas aeruginosa.

[1167] In some embodiments, the combination therapies and compositions of the invention are useful for treating patients who are immunocompromised (eg, due to chemotherapeutic agents or radiation therapy) or who are at risk of being immunocompromised.

[1168] In some embodiments, the combination therapies and compositions of the invention can be used to activate and expand antigen-specific T cells ex vivo.

[1169] In some embodiments, the patient may have previously been treated for a condition characterized by overexpression or overactivity of any of PD-1, TIM-3, and / or LAG-3 or their ligands (e.g., cancer or an immune disorder). 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 the drug.

[1170] "Treat," "treating," and "treatment" refer to a method of alleviating or stopping a biological disorder and / or at least one symptom associated therewith. As used herein, "alleviating" a disease, disorder, or condition means reducing the severity and / or frequency of symptoms of the disease, disorder, or condition. Furthermore, references to "treating" herein include references to curative, palliative, and prophylactic treatments.

[1171] A "therapeutically effective amount" refers to an administered therapeutic dose that will alleviate to some extent one or more of the symptoms of the disorder being treated. A therapeutically effective amount of an anti-cancer therapeutic can, for example, result in tumor shrinkage, increased survival, elimination of cancer cells, decreased disease progression, reversal of metastasis, or other clinical endpoints desired by a healthcare provider.

[1172] In some embodiments, the antibodies, antigen-binding portions, or multispecific binding molecules in the combination therapies of the present invention are administered as a single composition. In other embodiments, the antibodies, antigen-binding portions, or multispecific binding molecules are administered as more than one composition. For example, a combination therapy comprising an anti-PD-1 antibody, an anti-TIM-3 antibody, and an anti-LAG-3 antibody may involve administration of a single composition comprising all three antibodies, a composition comprising two of the antibodies, and a composition comprising one of the antibodies, or separate compositions for each antibody. In the case of more than one composition, the compositions may be administered simultaneously, sequentially, separately, or any combination thereof.

[1173] The combination therapies and compositions of the present invention can be administered alone or in combination with one or more other drugs or antibodies (or as any combination thereof). The pharmaceutical compositions, methods and uses of the present invention therefore also encompass embodiments of combinations with other active agents (co-administration), as described in detail below.

[1174] As used herein, the terms "co-administration," "co-administered," and "in combination with" in reference to a combination therapy or composition of the present invention and one or more other therapeutic agents are intended to mean and do mean and include the following:

[1175] - administering such combination therapy (or components thereof) or composition and therapeutic agent simultaneously to a patient in need of treatment when such components are formulated together in a single dosage form that releases said components to said patient at substantially the same time,

[1176] - administering such combination therapy (or components thereof) or composition and therapeutic agent substantially simultaneously to a patient in need of treatment when such components are formulated separately from one another into separate dosage forms that are ingested by said patient at substantially the same time, whereupon said components are released to said patient at substantially the same time,

[1177] - sequential administration of such combination therapy (or components thereof) or composition and therapeutic agent to a patient in need of treatment, when such components are formulated separately from one another in separate dosage forms to be ingested by the patient at sequential times (with significant time intervals between administrations), whereupon said components are released to said patient at substantially different times; and

[1178] - administering such combination therapy (or components thereof) or composition and therapeutic agent sequentially to a patient in need of treatment, when such components are formulated together into a single dosage form that releases said components in a controlled manner, and then they are released to said patient simultaneously, sequentially, and / or overlappingly at the same and / or different times, wherein each part can be administered by the same or different routes.

[1179] The combination therapy and composition of the present invention can be administered without additional therapeutic treatment, i.e., as a separate therapy (i.e., monotherapy). Alternatively, the treatment utilizing the combination therapy or composition of the present invention may include at least one additional therapeutic treatment, such as another immunostimulant, anticancer agent, antiviral agent, or vaccine (e.g., tumor vaccine). In some embodiments, the combination therapy or composition can be co-administered or formulated with another medication / drug for the treatment of cancer. Additional therapeutic treatments may include, for example, chemotherapeutic agents, anti-neoplastic agents, or anti-angiogenic agents, different anti-cancer antibodies, and / or radiotherapy.

[1180] Efficacy can be further improved by combining the combination therapies and compositions of the present invention with agents known to induce terminal differentiation of cancer cells. Such compounds can be, for example, selected from the group consisting of retinoic acid, trans-retinoic acid, cis-retinoic acid, phenyl butyrate, nerve growth factor, dimethyl sulfoxide, active form of vitamin D3, peroxisome proliferator-activated receptor gamma, 12-O-tetradecanoylphorbol 13-acetate, hexamethylene-bis-acetamide, transforming growth factor beta, butyric acid, cyclic AMP, and vesnarinone. In some embodiments, the compound is selected from retinoic acid, phenyl butyrate, all-trans-retinoic acid, and active form of vitamin D.

[1181] A pharmaceutical product comprising a 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 therapy for simultaneous, separate, or sequential administration in the treatment of cancer. The other agent can be any agent suitable for treating the particular cancer under consideration, for example, an agent selected from the group consisting of an alkylating agent, e.g., a platinum derivative, such as cisplatin, carboplatin, and / or oxaliplatin; a plant alkaloid, e.g., paclitaxel, docetaxel, and / or irinotecan; an antitumor antibiotic, e.g., doxorubicin (adriamycin), daunomycin, epirubicin, idarubicin, mitoxantrone, dactinomycin, bleomycin, actinomycin, luteomycin, and / or mitomycin; a topoisomerase inhibitor, such as topotecan; and / or an antimetabolite, e.g., fluorouracil and / or other fluoropyrimidines. In some embodiments, the additional agent is dacarbazine or gemcitabine.

[1182] The combination therapy or composition of the present invention can also be used in combination with other anti-cancer therapies such as vaccines, cytokines, enzyme inhibitors, immunostimulatory compounds, and T cell therapies. In the case of a vaccine, it can be, for example, a protein, peptide or DNA vaccine containing one or more antigens associated with the cancer being treated, or a vaccine comprising dendritic cells and antigens. Suitable cytokines include, for example, IL-2, IFN-gamma and GM-CSF. An example of a class of enzyme inhibitors with anti-cancer activity is indoleamine-2,3-dioxygenase (IDO) inhibitors, such as 1-methyl-D-tryptophan (1-D-MT). Adoptive T cell therapy refers to various immunotherapy techniques involving the expansion or engineering of a patient's own T cells to recognize and attack their tumors.

[1183] It is also contemplated that the combination therapies or compositions of the present invention can be used in adjunctive therapy with tyrosine kinase inhibitors. These are synthetic (primarily quinazoline-derived) low molecular weight molecules that interact with the intracellular tyrosine kinase domain of the receptor and inhibit ligand-induced receptor phosphorylation by competing for the intracellular Mg-ATP binding site.

[1184] In some embodiments, the combination therapy or composition can be used in combination with another agent / drug that mediates immune system activation, 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 antigen-binding fragment thereof that binds to one of the above molecules. In specific embodiments, the antibodies, or antigen-binding portions thereof, compositions, or bispecific binding molecules of the invention may be administered in combination with a CTLA-4 inhibitor (e.g., an anti-CTLA-4 antibody such as tremelimumab or ipilimumab). In one embodiment, the antibodies, or antigen-binding portions thereof, compositions, or bispecific binding molecules of the invention may be administered in combination with ipilimumab. It is also contemplated that the combination therapies or compositions of the invention may be used in combination with cytokines (e.g., IL-1, IL-2, IL-12, IL-15, or IL-21), EGFR inhibitors, VEGF inhibitors, and the like.

[1185] In certain aspects, the combination therapies and compositions of the present invention may be administered in combination with another inhibitor of the PD-1, TIM-3, or LAG-3 pathway that targets one or more of PD-1, TIM-3, LAG-3, or a ligand for any of these targets. Examples of such inhibitors include:

[1186] - Other anti-PD-1 antibodies and antibodies targeting PD-1 ligands and / or co-receptors (such as PD-L1 or PD-L2) (e.g., pembrolizumab and / or nivolumab);

[1187] - Other anti-TIM-3 antibodies and antibodies targeting TIM-3 ligands and / or co-receptors (such as galectin-9, HMGB-1, phosphatidylserine lipids, CEACAM1, LILRA1-6, or LILRB1-5) (e.g., MGB453, TSR-022, and / or LY3321367); and

[1188] - Other anti-LAG-3 antibodies and 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).

[1189] It should be understood that the combination therapies and compositions of the present invention can be used in the methods of treatment as described herein, can be used in the treatment as described herein, and / or can be used to generate medicaments for the treatment as described herein. The present invention also provides kits and articles of manufacture comprising the combination therapies or compositions of the present invention as described herein.

[1190] Dosage and route of administration

[1191] The combination therapies and compositions of the present invention will be administered in an effective amount for the treatment of the condition in question (i.e., at dosages and for periods of time necessary to achieve the desired result). The therapeutically effective amount may vary based on factors such as the particular condition being treated, the age, sex, and weight of the patient, and whether the antibody is administered as a monotherapy or in combination with one or more additional anti-cancer therapies.

[1192] The dosage regimen may 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 as indicated by the exigencies of the therapeutic situation. Formulating parenteral compositions in unit dosage form is particularly advantageous for ease of administration and uniformity of dosage. As used herein, unit dosage form refers to physically discrete units suitable as single doses for the patient / subject to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect, in combination with the required pharmaceutical carrier. The specifications of the unit dosage forms of the present invention are generally determined by and directly depend on: (a) the unique characteristics of the chemotherapeutic agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such active compounds to treat sensitivity in individuals.

[1193] Therefore, based on the disclosure provided herein, those skilled in the art will appreciate that dosages and administration regimens can be adjusted according to methods known in the art of therapeutics. That is, a maximum tolerable dose can be easily established, and an effective amount that provides a detectable therapeutic benefit to a patient can also be determined, and the time required for administering each agent to provide a detectable therapeutic benefit to a patient can be determined. Therefore, although certain dosages and administration regimens are exemplified herein, these examples are not in any way intended to be the dosages and administration regimens that may be provided to a patient when practicing the present invention.

[1194] It should be noted that the dosage value may vary with the type and severity of the condition to be alleviated, and may include a single dose or multiple doses. It should be further understood that for any particular subject, a particular dosage regimen should be adjusted over time based on individual needs and the professional judgment of the person administering or supervising the composition, and the dosage ranges proposed herein are only exemplary and are not intended to limit the scope or implementation of the combination therapy embodied. In addition, the dosage regimen using the combination therapy and composition of the present invention can 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 particular antibody utilized. Therefore, the dosage regimen can vary widely, but can be routinely determined using standard methods. For example, the dosage can be adjusted based on pharmacokinetic or pharmacodynamic parameters (which may include clinical efficacy, such as toxic effects and / or laboratory values). Therefore, the present invention encompasses intra-patient dose increases, as determined by those skilled in the art. Determining appropriate dosage and regimen is well known in the relevant art and it should be understood that it will be included by those skilled in the art once the teachings disclosed herein are provided.

[1195] It is envisioned that a suitable dosage of the antibody in the combination therapy or composition of the present invention will be in the range of 0.1-100 mg / kg, such as about 0.5-50 mg / kg, for example, about 1-20 mg / kg. The antibody can be administered at a dose of at least 0.25 mg / kg, for example, 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, such as at least 4 mg / kg, for example, at least 5 mg / kg; and for example, up to a maximum of 50 mg / kg, such as up to a maximum of 30 mg / kg, for example, up to a maximum of 20 mg / kg, such as up to a maximum of 15 mg / kg. Administration will generally be repeated at a suitable interval of, for example, once a week, once every two weeks, once every three weeks, or once every four weeks, and for a time deemed appropriate by the physician in charge (who may optionally increase or decrease the dosage if necessary).

[1196] An effective amount for tumor therapy can be measured by its ability to stabilize disease progression and / or improve symptoms in a patient, and preferably to reverse disease progression (e.g., by reducing tumor size). The ability of the combination therapies of the present invention to inhibit cancer can be assessed by in vitro assays (e.g., as described in the Examples) and in suitable animal models predictive of efficacy in human tumors (see, e.g., the Examples). An appropriate dosage regimen will be selected to provide the optimal therapeutic response in each particular case, for example, administration as a single bolus or as a continuous infusion, with possible adjustments in dosage as indicated by the exigencies of each case.

[1197] Products and kits

[1198] The present invention also provides a preparation comprising an anti-PD-1 antibody and an anti-TIM-3 antibody or an anti-LAG-3 antibody that competes for binding to human PD-1 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.

[1199] In some embodiments, the article of manufacture comprises:

[1200] - at least one anti-PD-1 antibody, or an antigen-binding portion thereof, as described herein and at least one anti-TIM-3 antibody, or an antigen-binding portion thereof, as described herein;

[1201] - at least one anti-PD-1 antibody, or antigen-binding portion thereof, as described herein and at least one anti-LAG-3 antibody, or antigen-binding portion thereof, as described herein;

[1202] - at least one anti-TIM-3 antibody, or antigen-binding portion thereof, as described herein and at least one anti-LAG-3 antibody, or antigen-binding portion thereof, as described herein; or

[1203] - at least one anti-PD-1 antibody, or an antigen-binding portion thereof, as described herein, at least one anti-TIM-3 antibody, or an antigen-binding portion thereof, as described herein, and at least one anti-LAG-3 antibody, or an antigen-binding portion thereof, as described herein;

[1204] and methods for producing the articles.

[1205] The present invention also provides a kit comprising an anti-PD-1 antibody that competes for binding to human PD-1 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, and an anti-TIM-3 antibody or an anti-LAG-3 antibody.

[1206] In some embodiments, the kit comprises:

[1207] - at least one anti-PD-1 antibody, or an antigen-binding portion thereof, as described herein and at least one anti-TIM-3 antibody, or an antigen-binding portion thereof, as described herein;

[1208] - at least one anti-PD-1 antibody, or antigen-binding portion thereof, as described herein and at least one anti-LAG-3 antibody, or antigen-binding portion thereof, as described herein;

[1209] - at least one anti-TIM-3 antibody, or antigen-binding portion thereof, as described herein and at least one anti-LAG-3 antibody, or antigen-binding portion thereof, as described herein; or

[1210] - at least one anti-PD-1 antibody, or an antigen-binding portion thereof, as described herein, at least one anti-TIM-3 antibody, or an antigen-binding portion thereof, as described herein, and at least one anti-LAG-3 antibody, or an antigen-binding portion thereof, as described herein.

[1211] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described below. In the event of a conflict, the present specification, including definitions, shall prevail.

[1212] Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Enzyme reactions and purification techniques are according to the manufacturer's specifications, as commonly accomplished in the art or performed as described herein.

[1213] Furthermore, unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and embodiments, the words "have" and "comprise" or variations such as "has," "having," "comprises," or "comprising" shall be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[1214] All publications and other references mentioned herein are incorporated by reference in their entirety.Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[1215] In order to make the present invention more clearly understood, the following examples are presented. These examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention in any way.

[1216] Example

[1217] Example 1: Improved in vitro activity of combined PD-1 / TIM-3 targeting in a one-way MLR assay

[1218] This example demonstrates that combined targeting of PD-1 and TIM-3 using anti-PD-1 antibody 12819 and anti-TIM-3 antibody 15086.17145 increases IFN-γ secretion in a one-way mixed lymphocyte reaction (MLR) assay.

[1219] In a one-way MLR assay, dendritic cells (DCs) isolated from two different healthy human donors and CD4 + T cells were used to induce heterotypic antigen-specific responses that resulted in cytokine production and T cell activation / proliferation. Dendritic cells (DCs) were cultured for 6 days using 20 ng / ml granulocyte-macrophage colony-stimulating factor (GM-CSF) and 20 ng / ml interleukin-4 (IL-4) from CD14 + Monocyte differentiation and comparison with CD4 +T cells were mixed at a 1:10 ratio. The indicated antibodies or antibody mixtures were added to a final total concentration of 10 μg / mL. The antibody mixture contained anti-PD-1 and anti-TIM-3 antibodies at a 1:1 ratio. After 5 days of culture, supernatants were harvested and IFN-γ levels were measured using the Meso Scale electrochemiluminescent cytokine assay. The Student's unpaired t-test was used for statistical analysis and the Bonferroni correction was used to adjust for multiple comparisons. Corrected p-values ​​of <0.05 were considered statistically significant.

[1220] Figure 1 The efficacy of anti-PD-1 and anti-TIM-3 antibodies on IFN-γ secretion in three independent donor pairs in a one-way MLR assay was shown. Both anti-PD-1 (12819) and anti-TIM-3 (15086) antibodies were functional in the one-way MLR assay and increased IFN-γ secretion in all three donor pairs. In addition, the combination of anti-PD-1 and anti-TIM-3 antibodies showed improved activity by inducing a statistically significant increase in IFN-γ compared to the individual antibodies.

[1221] Example 2: Improved in vitro activity of combined PD-1 / TIM-3 targeting in a two-way MLR assay

[1222] This example demonstrates that combined targeting of PD-1 and TIM-3 using anti-PD-1 antibody 12819 and anti-TIM-3 antibody 15086.17145 in a two-way MLR assay increases IFN-γ secretion.

[1223] In the two-way MLR assay, PBMCs from two different healthy human donors were co-cultured to induce an alloantigen-specific response that results in cytokine production and T cell activation / proliferation. PBMCs from 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, supernatants were harvested and IFN-γ levels were measured using the MesoScale electrochemiluminescent cytokine assay. Statistical analysis was performed using the Student's unpaired t-test with a Bonferroni correction to adjust for multiple comparisons. Corrected p-values ​​< 0.05 were considered statistically significant.

[1224] Figure 2 The efficacy of anti-PD-1 and anti-TIM-3 antibodies on IFN-γ secretion in a two-way MLR assay was demonstrated in three independent human donor pairs. Both anti-PD-1 and anti-TIM-3 antibodies increased IFN-γ secretion in the two-way MLR assay. Furthermore, the combination of anti-PD-1 and anti-TIM-3 antibodies demonstrated enhanced activity compared to the individual antibodies by inducing increased IFN-γ secretion.

[1225] Example 3: Anti-TIM-3 antibodies induce T cell proliferation in vitro in a one-way MLR assay

[1226] The ability of the anti-TIM-3 antibody 15086.17145 to induce T cell proliferation was investigated in a one-way MLR assay. Anti-TIM-3 antibody, a positive control antibody against anti-PD-1 (12819), or a negative control IgG2 antibody were added to a final concentration of 25 μg / mL and incubated for 5 days. Afterwards, 1 μCi / well of 3H-thymidine was added for an additional 18 hours. Cells were harvested, and 3H-thymidine incorporation was measured by liquid scintillation counting (MicroBeta2).

[1227] like Figure 3 As shown in Figure 2, anti-TIM-3 antibody 15086.17145 induced the proliferation of both CD4+ and CD8+ T cells to a similar extent as the positive control antibody for anti-PD-1 (panel A). The addition of soluble anti-CD3 enhanced the proliferation induced by anti-TIM-3 antibody (panel B).

[1228] Example 4: Anti-TIM-3 Antibody Induces IL-12p40 Secretion from Dendritic Cells

[1229] Monocyte-derived dendritic cells were generated from healthy donor material as previously described in Example 1. Dendritic cells were incubated for 5 days with 10 μg / mL of anti-TIM-3 antibody 15086.17145 or a negative control IgG2 antibody or without treatment, and IL-12p40 levels in the supernatant were measured using a standard ELISA cytokine assay.

[1230] like Figure 4 As shown in , TIM-3 ligation by anti-TIM-3 antibody 15086.17145 resulted in increased IL-12p40 secretion from dendritic cells.

[1231] Example 5: Anti-TIM-3 Antibody Induces Expression of Activation Markers in Dendritic Cells

[1232] To further investigate the functional effects of targeting TIM-3, the expression levels of selected activation markers on monocyte-derived dendritic cells were determined after treatment with the anti-TIM-3 antibody 15086.17145.

[1233] Dendritic cell lines were treated with 25 μg / mL anti-TIM-3 antibody for 24 hours and gene expression of costimulatory molecules was measured using NanoString Technologies. Gene expression levels were normalized to 30 uniformly expressed housekeeping genes. Data for selected genes of interest are presented as fold change relative to untreated control cells.

[1234] Gene expression analysis showed upregulation of several activation markers and co-stimulatory molecules including MHC-II (HLA-DQB1 and HLA-DQA1), CD80 and CD86 ( Figure 5 , panel A). FACS analysis was used to verify that the cell surface expression of CD80 and CD86 increased after treatment with anti-TIM-3 antibody 15086.17145 ( Figure 5 , Panel B). The histogram overlay shown represents CD11c+ dendritic cells, and the numbers adjacent to the histograms indicate the MFI values.

[1235] Example 6: In vivo efficacy of anti-TIM-3 antibody 15086 on human lung PDX tumor growth

[1236] This example shows that the anti-TIM-3 antibody 15086.17145 can be expressed in CD34 + In vivo efficacy in a human lung patient-derived xenograft (PDX) tumor model in humanized NSG-SGM3 mice.

[1237] NSG-SGM3 mice use umbilical cord blood-derived CD34 + The cells were humanized and implanted with patient-derived lung tumor fragments (LG1306) on the right flank. 3 Mice were randomized and treatment was initiated based on tumor size between treatments. Mice received either vehicle or anti-TIM-3 antibody 15086 intraperitoneally at an initial dose of 10 mg / kg, followed by 5 mg / kg 5x Q5D. Tumors were measured three times weekly in two dimensions using calipers and in mm. 3 The tumor volume was calculated according to the following formula: (width) 2 x length x 0.5. Gray areas represent treatment periods. Two-way ANOVA with Bonferroni multiple comparisons was used to compare tumor volumes between treatment groups at each time point. Data are presented as mean ± SEM, **p < 0.01.

[1238] Treatment with anti-TIM-3 antibody 15086 in CD34 + Humanized NSG-SGM3 mice resulted in significant tumor growth inhibition in a human lung PDX model ( Figure 6 ).

[1239] Example 7: Improved efficacy of combined PD-1 / LAG-3, PD-1 / TIM-3, or PD-1 / TIM-3 / LAG-3 targeting in the PBMC+SEB assay

[1240] This example describes the improved efficacy of combining anti-PD-1 antibody 12819 with anti-LAG-3 antibody 15532 or anti-TIM-3 antibody 15086.17145 in the PBMC+SEB (Staphylococcal Enterotoxin B) assay.

[1241] SEB is a superantigen that binds to the specific Vβ region of class II MHC molecules and T cell receptors (TCRs) and drives nonspecific stimulation of T cells. This results in polyclonal T cell activation / proliferation and cytokine release (including IL-2). Human PBMCs isolated from buffy coats from healthy donors were seeded in 384-well plates and kept untreated or treated with 10 ng / mL SEB and 10 μg / mL of the indicated single antibody or antibody mixture. The combination of antibodies was a 1:1 or 1:1:1 mixture of the indicated antibodies. After 48 hours in a humidified incubator at 37°C, the supernatant was removed and analyzed for IL-2 levels using an IL-2 ELISA kit (Life Technologies). Data are presented as mean ± SEM. Significant differences were tested using Student's t-test with Bonferroni correction.

[1242] Figure 7 Increased IL-2 secretion was shown following treatment with a single anti-PD-1, anti-LAG-3, or anti-TIM-3 antibody, a mixture of two of these antibodies, or a mixture of all three antibodies. The data demonstrate that IL-2 levels were increased by monotherapy with anti-PD-1, anti-LAG-3, or anti-TIM-3 antibodies, and IL-2 secretion was further increased by treatment with a combination of anti-PD-1 and anti-LAG-3 antibodies, or anti-PD-1 and anti-TIM-3 antibodies. The triple combination of anti-PD-1, anti-LAG-3, and anti-TIM-3 antibodies increased IL-2 secretion more than any combination of the two antibodies.

[1243] Example 8: Improved in vivo efficacy of combined anti-PD-1 and anti-LAG-3 antibodies in two syngeneic mouse tumor models

[1244] This example demonstrates the in vivo efficacy of combining the anti-PD-1 antibody 12819 with the anti-LAG-3 antibody C9B7W (which is reactive to mouse LAG-3; BioXcell) or the anti-LAG-3 antibody 15011 in two syngeneic mouse tumor models.

[1245] 0.5x10 6 MC38 (colon cancer) or 5x10 6ASB-XIV (lung cancer) cells were subcutaneously inoculated into the flanks of 6-8 week old female BALB / cAnNRj (ASB-XIV) or C57BL / 6 (MC38) mice. Tumors were measured three times a week in two dimensions using calipers and in mm. 3 The tumor volume was calculated according to the following formula: (width) 2 x length x 0.5. On the 5th day after inoculation (ASB-XIV) or 13th day after inoculation (MC38), the 3 Based on the mean tumor size, mice were randomly divided into four groups of ten animals and treatment was initiated. Mice were treated three times weekly with intraperitoneal injections of vehicle 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 for a total of six treatments. Antibody treatment was administered at a dose of 10 mg / kg / target. Tumor volumes were compared between treatment groups at each time point using a two-way ANOVA with Bonferroni multiple comparisons test. Statistical analysis was performed using GraphPad Prism version 5.0 (GraphPad Software, Inc.).

[1246] MC38 syngeneic tumors treated with the monoclonal anti-PD-1 antibody 12819 exhibited continued tumor growth, although the tumors were significantly different from those treated with vehicle ( Figure 8 ) had slower growth kinetics. No efficacy was observed with treatment with the anti-LAG-3 antibody C9B7W alone. The combination of anti-PD-1 and anti-LAG-3 antibodies showed improved tumor growth inhibition compared to anti-PD-1 treatment and significant tumor growth inhibition compared to vehicle-treated tumors (p < 0.001) ( Figure 8 ).

[1247] ASB-XIV syngeneic tumors treated with the anti-PD-1 antibody 12819 showed delayed tumor growth compared to vehicle-treated tumors ( Figure 8 and 9 No efficacy was observed with treatment using the anti-LAG-3 antibody C9B7W alone ( Figure 8 ), while anti-LAG-3 antibody 15011 significantly inhibited tumor growth compared with vehicle treatment (p<0.05) ( Figure 9 The combination of anti-PD-1 antibody and anti-LAG-3 antibody C9B7W significantly improved the anti-tumor efficacy compared to vehicle treatment ( Figure 8 ). A significantly improved tumor inhibitory efficacy was observed in the combination of anti-PD-1 antibody and anti-LAG-3 antibody 15011 compared to monotherapy with the anti-PD-1 antibody (p<0.0001) or monotherapy with the anti-LAG-3 antibody (p<0.05). Figure 9 ).

[1248] Example 9: Improved in vivo efficacy of combined anti-PD-1 and anti-TIM-3 antibodies in two syngeneic mouse tumor models

[1249] This example demonstrates the in vivo efficacy of combining the anti-PD-1 antibody 12819 and the anti-TIM-3 antibody 5D12 (which is reactive to mouse TIM-3; Anderson et al., Science 318: 1141-43 (2007)) in two syngeneic mouse tumor models.

[1250] 0.2x10 6 Sa1N (fibrosarcoma) or 5x10 6 ASB-XIV (lung cancer) cells were subcutaneously inoculated into the flanks of 6-8 week old female A / J (Sa1N) and BALB / cAnNRj (ASB-XIV) mice. Tumors were measured three times a week in two dimensions using calipers and in mm. 3 The tumor volume was calculated according to the following formula: (width) 2 x length x 0.5. 60-110mm 3 Mice were randomized and treatment was initiated based on the mean tumor size. Mice were treated with intraperitoneal injections of vehicle buffer, anti-PD-1 antibody 12819, and / or anti-TIM-3 antibody 5D12 as a single dose (Sa1N) or three times weekly for a total of six treatments (ASB-XIV). Antibody treatment was administered at a dose of 10 mg / kg / dose in mice bearing ASB-XIV tumors. Mice bearing Sal1N tumors were administered with 1 mg / kg and 10 mg / kg of anti-PD-1 and anti-TIM-3 antibodies, respectively. Tumor volumes were compared between treatment groups at each time point using a two-way ANOVA with Bonferroni multiple comparison tests. Statistical analysis was performed using GraphPad Prism version 5.0 (GraphPad Software, Inc.).

[1251] On the 6th day after inoculation of ASB-XIV tumor cells, 3 The mice were randomly divided into four groups of ten animals and treatment was initiated. Compared with vehicle treatment, treatment with anti-PD-1 antibody 12819 delayed tumor growth, while treatment with anti-TIM-3 antibody 5D12 had no effect on tumor growth. Compared with single treatment with either antibody, significant tumor inhibitory efficacy was seen by combining anti-PD-1 antibody and anti-TIM-3 antibody (p < 0.001) ( Figure 10 ).

[1252] On the 13th day after inoculation of Sa1N tumor cells,3 The mice were randomly divided into four groups of ten animals and administered with a single antibody treatment based on the average tumor size. The results showed that single antibody treatment with either the anti-PD-1 antibody 12819 or the anti-TIM-3 antibody 5D12 delayed initial tumor growth. Compared to single antibody treatment, improved anti-tumor efficacy was observed by combining the anti-PD-1 antibody with the anti-TIM-3 antibody. Combination treatment also significantly inhibited tumor growth compared to vehicle treatment (p<0.0001)( Figure 10 ).

[1253] Example 10: In vivo efficacy of combined anti-PD-1 and anti-TIM-3 antibodies in a human xenograft tumor model

[1254] This example demonstrates the improved in vivo efficacy of combining anti-PD-1 antibody 12819 with anti-TIM-3 antibody 15086.17145 in a human xenograft tumor model in which A375 cells (human melanoma) were implanted into mice reconstituted with human PBMCs.

[1255] Human PBMCs were injected intraperitoneally into NOG (donor 1 and donor 2) or NOG-EXL (hGM-CSF / hIL-3-NOG) (donor 3) mice one day prior to subcutaneous implantation of human A375 melanoma cells. Treatment began on the day of PBMC injection and mice were treated three times weekly with intraperitoneal injections of vehicle buffer, anti-PD-1 antibody 12819, anti-TIM-3 antibody 15086, 17145, or a combination of anti-PD-1 and anti-TIM-3 antibodies for a total of six treatments. Antibody treatment was administered at a dose of 10 mg / kg / target. Tumors were measured three times weekly in two dimensions and in mm using a caliper. 3 The tumor volume was calculated according to the following formula: (width) 2 x length x 0.5. Two-way ANOVA plus Bonferroni multiple comparison test was applied to compare tumor volumes between treatment groups at various time points. Statistical analysis was performed using GraphPad Prism version 5.0 (GraphPad Software, Inc.).

[1256] like Figure 11 As shown in , treatment with a combination of anti-PD-1 and anti-TIM-3 antibodies resulted in significant (and synergistic, in at least donor 3) tumor growth delay compared to the vehicle-treated group (p < 0.05 and p < 0.01), while single antibody treatment (anti-PD-1 or anti-TIM-3) showed limited efficacy on tumor growth.

[1257] Example 11: Combined PD-1 and TIM-3 targeting leads to increased survival in mice co-implanted with human immune and tumor cells

[1258] This example demonstrates the in vivo efficacy of combining anti-PD-1 antibody 12819 with anti-TIM-3 antibody 15086.17145 in a mouse model of human tumors.

[1259] 2x10 6 A375 (melanoma) cells and 2x10 6 Individual human PBMCs were mixed and inoculated subcutaneously into the flanks of 6-8 week old female NOD-scid mice. Treatment was initiated at the time of cell inoculation. Mice were treated intraperitoneally with vehicle buffer, anti-PD-1 antibody 12819, anti-TIM-3 antibody 15086.17145, or a combination of anti-PD-1 and anti-TIM-3 three times a week for a total of six treatments. For each antibody, antibody treatment was administered at a dose of 10 mg / kg. Tumors were measured three times a week in two dimensions and in mm using a caliper. 3 The tumor volume was calculated according to the following formula: (width) 2 x length x 0.5.

[1260] Survival is defined as having <400mm 3 The results showed that the survival of mice treated with the dual combination of anti-PD-1 and anti-TIM-3 antibodies was increased compared to any single antibody treatment ( Figure 12 , sub-figure AE)).

[1261] Example 12: Combined PD-1, LAG-3, and TIM-3 targeting results in increased survival in a syngeneic mouse tumor model

[1262] This example demonstrates the in vivo efficacy of combining the anti-PD-1 antibody 12819 with the anti-LAG-3 antibody C9B7 and the anti-TIM-3 antibody 5D12 in a syngeneic mouse tumor model.

[1263] 5x10 6 ASB-XIV (lung cancer) cells were inoculated subcutaneously into the flanks of 6-8 week old female BALB / cAnNRj mice. Tumors were measured three times a week in two dimensions using calipers and in mm. 3 The tumor volume was calculated according to the following formula: (width) 2 x length x 0.5. On the 5th day after inoculation, 3At an average tumor size of 100, mice were randomly divided into seven groups of ten animals and treatment was initiated. Mice were treated three times weekly with intraperitoneal injections of vehicle 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, anti-PD-1 and anti-TIM-3, or anti-PD-1, anti-LAG-3, and anti-TIM-3 antibodies for a total of six treatments. Antibody treatment was administered at a dose of 10 mg / kg for each antibody.

[1264] Survival is defined as having ≤400mm 3 The results showed that the survival of mice treated with the triple combination of anti-PD-1, anti-LAG-3, and anti-TIM-3 antibodies was increased compared to any single or dual antibody treatment ( Figure 13 , sub-figures AH).

[1265] Table 1: Antibody sequence identification codes

[1266]

[1267]

[1268]

[1269]

[1270]

[1271]

[1272] Table 3: Target protein sequences

[1273]

[1274] Table 4: Nucleotide sequences of heavy and light chain variable domains of anti-TIM-3 antibodies

[1275]

[1276]

[1277]

[1278]

[1279]

[1280]

[1281] Table 5: Anti-TIM3 antibody heavy chain and light chain variable domain amino acid sequences

[1282] (CDRs are in bold and italics)

[1283]

[1284]

[1285]

[1286]

[1287] Table 6: Nucleotide sequences of heavy and light chain variable domains of anti-PD-1 antibodies

[1288]

[1289]

[1290]

[1291] Table 7: Anti-PD-1 Antibody Heavy and Light Chain Variable Domain Amino Acid Sequences

[1292] (CDRs are in bold and italics)

[1293]

[1294]

[1295] Table 8: Nucleotide sequences of heavy and light chain variable domains of anti-LAG-3 antibodies

[1296]

[1297]

[1298]

[1299] Table 9: Anti-LAG-3 Antibody Heavy and Light Chain Variable Domain Amino Acid Sequences

[1300] (CDRs are in bold and italics)

[1301]

[1302]

[1303] The present invention provides:

[1304] 1. A method for improving immunity in a human patient in need thereof, comprising administering to said patient

[1305] an anti-PD-1 antibody, or an antigen-binding portion thereof, that competes for binding to human PD-1 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; and

[1306] An anti-TIM-3 antibody, or an antigen-binding portion thereof, or an anti-LAG-3 antibody, or an antigen-binding portion thereof.

[1307] 2. The method of claim 1 , wherein the method comprises administering the anti-PD-1 antibody or antigen-binding portion thereof, the anti-TIM-3 antibody or antigen-binding portion thereof, and the anti-LAG-3 antibody or antigen-binding portion thereof.

[1308] 3. The method of claim 1 or 2, wherein the anti-PD-1 antibody binds to an epitope of human PD-1 comprising:

[1309] a) residues V64, L128, P130, K131, and A132 of SEQ ID NO: 388;

[1310] b) residues V44 and T145 of SEQ ID NO: 388;

[1311] c) residues K131 and E136 of SEQ ID NO: 388; or

[1312] d) residues V44 and T145 of SEQ ID NO: 388.

[1313] 4. The method of claim 1 or 2, wherein the anti-PD-1 antibody binds to an epitope of human PD-1 comprising:

[1314] a) residues 56-64, 69-90, and 122-140 of SEQ ID NO:388;

[1315] b) residues 69-90 and 122-140 of SEQ ID NO:388;

[1316] c) residues 69-75 of SEQ ID NO:388;

[1317] d) residues 136-140 of SEQ ID NO: 388; or

[1318] e) residues 69-75 and 136-140 of SEQ ID NO:388.

[1319] 5. The method of any one of items 1 to 4, wherein the anti-PD-1 antibody has at least one of the following properties:

[1320] a) With a K of 750 pM or lower D Binds to human PD-1;

[1321] b) with a K of 7 nM or lower D Binds to cynomolgus monkey PD-1;

[1322] c) with a K of 1 nM or lower D Binds to mouse PD-1;

[1323] d) does not bind to rat PD-1;

[1324] e) increased IL-2 secret...

Claims

1. A composition comprising: a) an anti-LAG-3 antibody, or an antigen-binding portion thereof; and b) anti-PD-1 antibodies or antigen-binding portions thereof; wherein the anti-LAG-3 antibody, or antigen-binding portion thereof, comprises H-CDR1-3 and L-CDR1-3 with amino acid sequences of SEQ ID NOs: 318-323, respectively.

2. The composition of claim 1, wherein the anti-LAG-3 antibody, or antigen-binding portion thereof, has at least one of the following properties: a) reduces human LAG-3 binding to human MHC class II on A375 cells by greater than 85% compared to a negative control antibody at a concentration of 20 μg / mL, as measured by flow cytometry competition assay; b) reduced human LAG-3 binding to human MHC class II on A375 cells by between 35% and 85% compared to a negative control antibody at a concentration of 20 μg / mL, as measured by flow cytometry competition assay; c) blocking the binding between human LAG-3 expressed on Jurkat cells and human MHC class II expressed on Raji cells; d) binds to human LAG-3 with an EC50 of 0.1 nM or less as measured by flow cytometry; e) binds to cynomolgus monkey LAG-3 with an EC50 of 0.3 nM or less as measured by flow cytometry; f) Measured by surface plasmon resonance, 3.0x10 -8 or lower K D Binds to human LAG-3; g) Measured by surface plasmon resonance, with a value of 1.5x10 -7 or lower K D binds to cynomolgus monkey LAG-3; h) Measured by surface plasmon resonance, 3.5x10 -8 or lower K D Binds to mouse LAG-3; i) stimulate IL-2 production in human peripheral blood mononuclear cells (PBMCs) treated with Staphylococcal enterotoxin B (SEB); j) reduce cellular levels of LAG-3 in human T cells; k) reducing soluble levels of LAG-3 in cultures of human T cells; l) inducing tumor growth regression in vivo; m) slowing tumor growth in vivo; and n) Does not bind to the same epitope of human LAG-3 as antibody 25F7-Lag3.

5.

3. The composition of claim 1, wherein the anti-LAG-3 antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain and a light chain variable domain comprising the amino acid sequences of SEQ ID NOs: 316 and 317, respectively.

4. The composition of any one of claims 1 to 3, wherein the anti-LAG-3 antibody, or antigen-binding portion thereof, binds to an epitope of human LAG-3 comprising: a) residues H85, P86, A87, P89, S91, W92, and G93 of SEQ ID NO: 68; or b) residues 78-105 and 123-131 of SEQ ID NO:

68.

5. The composition of any one of claims 1-3, wherein the anti-LAG-3 antibody is of the IgG subclass IgGl isotype.

6. The composition of claim 5, wherein the anti-LAG-3 antibody comprises a mutation from Leu to Ala at one or both of heavy chain amino acid positions 234 and 235, according to Eu numbering.

7. The composition of any one of claims 1 to 3, wherein the antibody is of the IgG subclass IgG4 isotype, wherein the amino acid residue at position 228 by Eu numbering is mutated from Ser to Pro.

8. A composition comprising: a) anti-LAG-3 antibodies; and b) anti-PD-1 antibodies or antigen-binding portions thereof; The anti-LAG-3 antibody comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable domain represented by SEQ ID NO: 316 and a heavy chain constant region represented by SEQ ID NO: 375, and the light chain comprising a light chain variable domain represented by SEQ ID NO: 317 and a light chain constant region represented by SEQ ID NO:

378.

9. A pharmaceutical composition comprising: (a) an anti-LAG-3 antibody, or an antigen-binding portion thereof, and an anti-PD-1 antibody, or an antigen-binding portion thereof; and (b) pharmaceutically acceptable excipients; wherein the anti-LAG-3 antibody, or antigen-binding portion thereof, comprises H-CDR1-3 and L-CDR1-3 with amino acid sequences of SEQ ID NOs: 318-323, respectively.

10. The pharmaceutical composition of claim 9, wherein the anti-LAG-3 antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain and a light chain variable domain comprising the amino acid sequences of SEQ ID NOs: 316 and 317, respectively.

11. The pharmaceutical composition of claim 9 or 10, wherein the anti-LAG-3 antibody, or antigen-binding portion thereof, binds to an epitope of human LAG-3 comprising: a) residues H85, P86, A87, P89, S91, W92, and G93 of SEQ ID NO: 68; or b) residues 78-105 and 123-131 of SEQ ID NO:

68.

12. The pharmaceutical composition of claim 9 or 10, wherein the anti-LAG-3 antibody is of the IgG subclass IgGl isotype.

13. The pharmaceutical composition of claim 12, wherein the anti-LAG-3 antibody comprises a mutation from Leu to Ala at one or both of heavy chain amino acid positions 234 and 235, according to Eu numbering.

14. The pharmaceutical composition of claim 9 or 10, wherein the antibody is of the IgG subclass IgG4 isotype, wherein the amino acid residue at position 228 according to Eu numbering is mutated from Ser to Pro.

15. The pharmaceutical composition of claim 9 or 10, wherein the anti-LAG-3 antibody comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable domain set forth in SEQ ID NO: 316 and a heavy chain constant region set forth in SEQ ID NO: 375, and the light chain comprising a light chain variable domain set forth in SEQ ID NO: 317 and a light chain constant region set forth in SEQ ID NO:

378.

16. The pharmaceutical composition of claim 9 or 10, wherein the pharmaceutical composition is suitable for parenteral administration.

17. The pharmaceutical composition of claim 15, wherein the pharmaceutical composition is suitable for parenteral administration.

18. The pharmaceutical composition of claim 16, wherein the parenteral administration is intravenous or subcutaneous administration.

19. The pharmaceutical composition of claim 17, wherein the parenteral administration is intravenous or subcutaneous administration.

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