LAG-3 and PD-1 / LAG-3 antibodies

Abstract

The present disclosure relates to antibodies that bind to and stimulate human LAG-3, as well as bispecific antibodies that bind to and stimulate each of human PD-1 and human LAG-3.

Description

[Technical Field]

[0001] The present disclosure is in the field of medicine. In particular, the present disclosure relates to novel antibodies that stimulate human LAG-3 and novel bispecific antibodies that stimulate both human LAG-3 and human PD-1, compositions comprising such antibodies, and methods of using such antibodies for the treatment of autoimmune diseases. [Background technology]

[0002] Immune checkpoint pathways regulate both autoimmune and anti-cancer immune responses (Isakov, N., J. Autoimmune Disorders 2016;2(2):17). In the treatment of autoimmune diseases, it is desirable to promote, i.e., stimulate, the effects of immunoinhibitory pathways so that the immune response is suppressed. Conversely, in the treatment of cancer, it is desirable to inhibit, i.e., antagonize, the effects of immunoinhibitory pathways so that the immune response is stimulated.

[0003] PD-1 is a type I cell membrane protein. Activation of the PD-1 pathway leads to inhibition of immune cell activation, including reduced cell proliferation, reduced cell survival, and inhibition of inflammatory cytokines (e.g., IFNγ, TNFα, and IL-2). PD-1 is expressed on recently activated immune cells, such as T cells, B cells, NKT cells, monocytes, and dendritic cells, and its expression is tightly regulated. The importance of PD-1-mediated signaling in regulating human immune responses has been demonstrated by the successful treatment of cancer patients with antagonistic PD-1 antibodies. Furthermore, there are other known correlations between autoimmune disease activity and the PD-1 pathway (Mozaffarian, N., et al., 2008) and autoantibodies against PD-1 (Shi, H., et al., 2017). Human PD-1 agonist antibodies are known in the art (see, e.g., WO 2019 / 168745 and WO 2020 / 247648).

[0004] LAG-3 (CD223) is a cell surface inhibitory receptor. LAG-3 regulates T cell effector function, and LAG-3 antagonist therapy for cancer is being evaluated (Lecocq Q, et al., Int. J. Molec. Sci. 2021;22(1):75; Chocarro L, et al., Immuno-Oncology and Technology 2022;14(C):100079.doi:10.1016 / j.iotech.2022.100079). PD-1 / LAG-3 bispecific antagonist antibodies for cancer treatment have been disclosed (Angin M, et al., J. Immunol. 2020;204(4):810-818). LAG-3 agonist antibodies have been described (Angin M, et al., J. Immunol. 2020;204(4):810-818). However, it is believed that PD-1 / LAG-3 dual agonist bispecific antibodies have not been previously reported.

[0005] Binds to human LAG-3 and does not deplete T cells; stimulates the LAG-3 signaling pathway; binds to human PD-1 and human LAG-3 with desirable association and dissociation rates for optimal agonist activity; simultaneously stimulates each of the PD-1 and LAG-3 signaling pathways; reduces T cell proliferation by promoting downregulation of T cell receptor signaling rather than by T cell depletion; stimulates human PD-1 and human LAG-3 in an immunologically relevant context to achieve in vivo efficacy; and combination of a human LAG-3 agonist antibody with a human PD-1 agonist antibody. There are no known treatments for treating autoimmune diseases involving dual agonist antibody constructs that demonstrate greater in vitro and / or in vivo efficacy than the combined efficacy of a single antibody, demonstrate sufficient potency as a bispecific molecule for the treatment and / or prevention of autoimmune disorders, and / or provide an effective alternative to drug switching, where treatment is discontinued due to at least one adverse event and / or ineffectiveness (particularly anti-drug antibody (ADA)-mediated reduced efficacy) during treatment of an autoimmune disorder with another human PD-1 agonist antibody or a human LAG-3 agonist antibody. Summary of the Invention

[0006] Thus, the present disclosure provides novel human LAG-3 agonist antibodies and novel human LAG-3 / human PD-1 dual agonist bispecific antibodies.

[0007] The present disclosure represents an advancement in the art by providing compositions and methods useful for preventing, down-regulating, or ameliorating autoimmune and / or immune tolerance-related disorders through immune checkpoint stimulation using engineered human LAG-3 / human PD-1 dual agonist bispecific antibodies. The human LAG-3 / human PD-1 dual agonist bispecific antibodies of the present disclosure may be capable of ameliorating or reversing immunopathology, preferably through inhibiting the adaptive arm of the immune response, abrogating antigen-specific immune processes, and thereby directly addressing the underlying disease pathology. Clinically, the use of such antibodies may result in long-term persistence or remission of the disease being treated. DETAILED DESCRIPTION OF THE INVENTION

[0008] The preparation of bispecific molecules is generally known to be an unpredictable endeavor. For example, coexpression of two heavy chains and two light chains to generate an IgG bispecific antibody can result in several erroneous constructs and unwanted by-products (Lewis SM et al., Nature Biotechnology 2014;32:191-202; Leaver-Fay A, et al., Structure 2016;24:641-651). Therefore, the present disclosure provides a human LAG-3 / human PD-1 bispecific molecule that reduces Fc receptor binding, minimizes complement fixation, minimizes oxidation, facilitates heteromab construction, is cross-reactive with human LAG-3 and human PD-1, and exhibits in vivo efficacy in at least one preclinical model of autoimmune disorder.

[0009] The present disclosure provides an antibody that binds to human LAG-3, wherein the antibody comprises an antigen-binding domain comprising a heavy chain variable region (VH) and a light chain variable region (VL); VH comprises heavy chain complementarity determining regions (HCDRs): HCDR1 comprising SEQ ID NO:1, HCDR2 comprising SEQ ID NO:2, and HCDR3 comprising SEQ ID NO:3; The VL provides an antibody comprising light chain complementarity determining regions (LCDRs): LCDR1 comprising SEQ ID NO:4, LCDR2 comprising SEQ ID NO:5, and LCDR3 comprising SEQ ID NO:6.

[0010] In one preferred embodiment, the VH comprises SEQ ID NO:13 and the VL comprises SEQ ID NO:14.

[0011] In one preferred embodiment, the human LAG-3 antibody comprises a constant heavy (CH) region comprising SEQ ID NO:15 and a constant light (CL) region comprising SEQ ID NO:16.

[0012] In another preferred embodiment, the human LAG-3 antibody comprises a light chain (LC) comprising SEQ ID NO:22 and a heavy chain (HC) comprising SEQ ID NO:37, wherein X at position 328 of SEQ ID NO:37 is a lysine residue or an alanine residue. In another preferred embodiment, X at position 328 of SEQ ID NO:37 is a lysine residue. In another preferred embodiment, X at position 328 of SEQ ID NO:37 is an alanine residue.

[0013] In another preferred embodiment, the human LAG-3 antibody comprises (a) an LC comprising SEQ ID NO: 22, and (b) an HC comprising SEQ ID NO: 21. In another preferred embodiment, the antibody comprises (a) an LC comprising SEQ ID NO: 22, and (b) an HC comprising SEQ ID NO: 31. In another preferred embodiment, the human LAG-3 antibody comprises an HC comprising SEQ ID NO: 21, and an LC comprising SEQ ID NO: 22.

[0014] The present disclosure also provides an antibody that binds to human LAG-3, the antibody comprising an antigen-binding domain comprising a VH and a VL, VH comprises heavy chain complementarity determining regions: HCDR1 comprising SEQ ID NO:44, HCDR2 comprising SEQ ID NO:45, and HCDR3 comprising SEQ ID NO:47; The VL provides an antibody comprising light chain complementarity determining regions: LCDR1 comprising SEQ ID NO:48, LCDR2 comprising SEQ ID NO:49, and LCDR3 comprising SEQ ID NO:50.

[0015] In one preferred embodiment, the VH comprises SEQ ID NO:53 and the VL comprises SEQ ID NO:56.

[0016] In another preferred embodiment, the human LAG-3 antibody comprises a constant heavy CH region comprising SEQ ID NO:58 and a CL region comprising SEQ ID NO:60.

[0017] In another preferred embodiment, the human LAG-3 antibody comprises an LC comprising SEQ ID NO:64 and an HC comprising SEQ ID NO:61.

[0018] The present disclosure also provides an antibody that binds to human LAG-3, the antibody comprising an antigen-binding domain comprising a VH and a VL, VH comprises heavy chain complementarity determining regions: HCDR1 comprising SEQ ID NO:51, HCDR2 comprising SEQ ID NO:52, and HCDR3 comprising SEQ ID NO:47; The VL provides an antibody comprising light chain complementarity determining regions: LCDR1 comprising SEQ ID NO:48, LCDR2 comprising SEQ ID NO:49, and LCDR3 comprising SEQ ID NO:50.

[0019] In one preferred embodiment, the VH comprises SEQ ID NO:54 and the VL comprises SEQ ID NO:56.

[0020] In another preferred embodiment, the human LAG-3 antibody comprises a CH region comprising SEQ ID NO:58 and a CL region comprising SEQ ID NO:60.

[0021] In another preferred embodiment, the human LAG-3 antibody comprises an LC comprising SEQ ID NO:64 and an HC comprising SEQ ID NO:62.

[0022] In another preferred embodiment, the human LAG-3 antibody is of the human IgG1 or IgG4 isotype. In another preferred embodiment, the antibody is of the human IgG1 isotype.

[0023] In another preferred embodiment, the antigen-binding domain of human LAG-3 is a single-chain variable fragment (scFv).

[0024] Lysine-to-alanine substitutions in the Fc region of an antibody can be useful in reducing ADCC and CDC. The present disclosure also provides a nucleic acid comprising a sequence encoding one or both of SEQ ID NO:22 or SEQ ID NO:37, wherein X at position 328 in SEQ ID NO:37 is a lysine residue or an alanine residue. In a preferred embodiment, X at position 328 in SEQ ID NO:37 is a lysine residue. In another preferred embodiment, X at position 328 in SEQ ID NO:37 is an alanine residue. In another preferred embodiment, the present disclosure provides a vector comprising the nucleic acid.

[0025] In another preferred embodiment, the present disclosure provides a composition comprising a first vector comprising a nucleic acid sequence encoding SEQ ID NO:22 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:37, wherein X at position 328 of SEQ ID NO:37 is a lysine residue or an alanine residue.

[0026] The present disclosure also provides a nucleic acid comprising a sequence encoding one or both of SEQ ID NO: 64 or SEQ ID NO: 61. The present disclosure also provides a nucleic acid comprising a sequence encoding one or both of SEQ ID NO: 64 or SEQ ID NO: 62.

[0027] In another preferred embodiment, the present disclosure provides a composition comprising a first vector comprising a nucleic acid sequence encoding SEQ ID NO:64 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:61.

[0028] In another preferred embodiment, the present disclosure provides a composition comprising a first vector comprising a nucleic acid sequence encoding SEQ ID NO:64 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:62.

[0029] In a preferred embodiment, the present disclosure provides a cell comprising the vector. In another preferred embodiment, the present disclosure provides a cell comprising the composition. In another preferred embodiment, the cell is a mammalian cell. In another preferred embodiment, the cell or mammalian cell is isolated. In another preferred embodiment, the present disclosure provides a method for producing an antibody, comprising culturing cells under conditions in which the antibody is expressed and then recovering the expressed antibody from the culture medium. In another preferred embodiment, the present disclosure provides an antibody produced by culturing cells under conditions in which the antibody is expressed and recovering the expressed antibody from the culture medium. In another preferred embodiment, the present disclosure provides a composition comprising the human LAG-3 antibody of the present invention.

[0030] The present disclosure also provides pharmaceutical compositions comprising a human LAG-3 antibody of the present invention and a pharmaceutically acceptable excipient, diluent, or carrier. In a preferred embodiment, the pharmaceutical composition comprises arginine.

[0031] The present disclosure also provides a pre-filled syringe of a pharmaceutical composition comprising a human LAG-3 antibody of the present invention and a pharmaceutically acceptable excipient, diluent, or carrier. In a preferred embodiment, the pharmaceutical composition comprises arginine.

[0032] The present disclosure also provides a method for treating an autoimmune disease in a subject in need thereof, comprising administering a therapeutically effective amount of a human LAG-3 antibody of the present invention to the subject. In a preferred embodiment, the autoimmune disease is rheumatoid arthritis, ulcerative colitis, type 1 diabetes, or systemic lupus erythematosus. In a preferred embodiment, the autoimmune disease is an active disease. In another preferred embodiment, the autoimmune disease is in remission.

[0033] The present disclosure also provides the human LAG-3 antibodies of the invention for use in therapy.

[0034] The present disclosure also provides the human LAG-3 antibody of the present invention for use in treating autoimmune disease. In a preferred embodiment, the autoimmune disease is rheumatoid arthritis, ulcerative colitis, type 1 diabetes, or systemic lupus erythematosus. In a preferred embodiment, the autoimmune disease is an active disease. In another preferred embodiment, the autoimmune disease is in remission.

[0035] The present disclosure also provides a pharmaceutical composition comprising the human LAG-3 antibody of the present invention for use in treating rheumatoid arthritis, ulcerative colitis, type 1 diabetes, or systemic lupus erythematosus. In a preferred embodiment, the immune disease is active. In another preferred embodiment, the immune disease is in remission.

[0036] The present disclosure also provides the use of a human LAG-3 antibody of the invention in the manufacture of a medicament for treating rheumatoid arthritis, ulcerative colitis, type 1 diabetes, or systemic lupus erythematosus.

[0037] The present disclosure also provides an antibody that binds to human PD-1, the antibody comprising an antigen-binding domain comprising a heavy chain variable region (VH) and a light chain variable region (VL), VH comprises heavy chain complementarity determining regions (HCDRs): HCDR1 comprising SEQ ID NO:7, HCDR2 comprising SEQ ID NO:8, and HCDR3 comprising SEQ ID NO:9; The VL provides an antibody comprising light chain complementarity determining regions (LCDRs): LCDR1 comprising SEQ ID NO:10, LCDR2 comprising SEQ ID NO:11, and LCDR3 comprising SEQ ID NO:12.

[0038] In one preferred embodiment, the human PD-1 antibody VH comprises SEQ ID NO:17 and the VL comprises SEQ ID NO:20.

[0039] In another preferred embodiment, the human PD-1 antibody comprises a constant heavy (CH) region comprising SEQ ID NO:19 and a constant light region comprising SEQ ID NO:20.

[0040] In another preferred embodiment, the human PD-1 antibody comprises a light chain (LC) comprising SEQ ID NO:24 and a heavy chain (HC) comprising SEQ ID NO:38, wherein X at position 328 of SEQ ID NO:38 is a lysine residue or an alanine residue. In another preferred embodiment, X at position 328 of SEQ ID NO:38 is a lysine residue. In another preferred embodiment, X at position 328 of SEQ ID NO:38 is an alanine residue.

[0041] In another preferred embodiment, the human PD-1 antibody comprises (a) an LC comprising SEQ ID NO: 24, and (b) an HC comprising SEQ ID NO: 23. In another preferred embodiment, the antibody comprises (a) an LC comprising SEQ ID NO: 24, and (b) an HC comprising SEQ ID NO: 32. In another preferred embodiment, the human PD-1 antibody comprises a heavy chain comprising SEQ ID NO: 23, and a light chain comprising SEQ ID NO: 24.

[0042] In another preferred embodiment, the human PD-1 antibody VH comprises SEQ ID NO: 55 and the VL comprises SEQ ID NO: 57. In another preferred embodiment, the human PD-1 antibody comprises a constant heavy (CH) region comprising SEQ ID NO: 59 and a constant light region comprising SEQ ID NO: 20. In another preferred embodiment, the human PD-1 antibody comprises a light chain comprising SEQ ID NO: 65 and a heavy chain comprising SEQ ID NO: 63.

[0043] In another preferred embodiment, the human PD-1 antibody is of the human IgG1 or IgG4 isotype. In another preferred embodiment, the antibody is of the human IgG1 isotype.

[0044] In another preferred embodiment, the antigen-binding domain of human PD-1 is a single-chain variable fragment (scFv).

[0045] The present disclosure also provides a nucleic acid comprising a sequence encoding SEQ ID NO:24 or SEQ ID NO:38, wherein X at position 328 in SEQ ID NO:38 is a lysine residue or an alanine residue. In a preferred embodiment, X at position 328 in SEQ ID NO:38 is a lysine residue. In another preferred embodiment, X at position 328 in SEQ ID NO:38 is an alanine residue. In another preferred embodiment, the present disclosure provides a vector comprising the nucleic acid.

[0046] In another preferred embodiment, the present disclosure provides a composition comprising a first vector comprising a nucleic acid sequence encoding SEQ ID NO:24 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:38, wherein X at position 328 of SEQ ID NO:38 is a lysine residue or an alanine residue.

[0047] The present disclosure also provides a nucleic acid comprising a sequence encoding SEQ ID NO:64 or SEQ ID NO:62. In another preferred embodiment, the present disclosure provides a vector comprising the nucleic acid. In another preferred embodiment, the present disclosure provides a composition comprising a first vector comprising a nucleic acid sequence encoding SEQ ID NO:64 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:62.

[0048] In a preferred embodiment, the present disclosure provides a cell comprising the vector. In another preferred embodiment, the present disclosure provides a cell comprising the composition. In another preferred embodiment, the cell is a mammalian cell. In another preferred embodiment, the cell or mammalian cell is isolated. In another preferred embodiment, the present disclosure provides a method for producing an antibody, comprising culturing cells under conditions in which the antibody is expressed, and then recovering the expressed antibody from the culture medium. In another preferred embodiment, the present disclosure provides an antibody produced by culturing cells under conditions in which the antibody is expressed, and recovering the expressed antibody from the culture medium. In another preferred embodiment, the present disclosure provides a composition comprising a human PD-1 antibody of the present invention.

[0049] The present disclosure also provides a human LAG-3 / human PD-1 dual agonist bispecific antibody, in which the dual agonist antibody binds to each of human LAG-3 and human PD-1. In a preferred embodiment, the dual agonist simultaneously binds to human LAG-3 and human PD-1. In another preferred embodiment, the dual agonist simultaneously binds to human LAG-3 and human PD-1 on the same T cell.

[0050] The present disclosure also provides human LAG-3 agonist antibodies that bind to an epitope on human LAG-3 comprising amino acid residues 183-202 of SEQ ID NO: 41 (human LAG-3). The present disclosure also provides human LAG-3 agonist antibodies that bind to an epitope on human LAG-3 comprising amino acid residues 203-225 of SEQ ID NO: 41. The present disclosure also provides human LAG-3 agonist antibodies that bind to an epitope on human LAG-3 comprising amino acid residues 183-202 of SEQ ID NO: 41 and amino acid residues 203-225 of SEQ ID NO: 41.

[0051] The present disclosure also provides a human LAG-3 / human PD-1 dual agonist bispecific antibody, wherein the LAG-3 binding portion of the bispecific antibody binds to an epitope on human LAG-3 comprising amino acid residues 183-202 of SEQ ID NO: 41 (human LAG-3). The present disclosure also provides a human Lag- / human PD-1 dual agonist bispecific antibody, wherein the LAG-3 binding portion of the bispecific antibody binds to an epitope on human LAG-3 comprising amino acid residues 203-225 of SEQ ID NO: 41 (human LAG-3). The present disclosure also provides a human LAG-3 / human PD-1 dual agonist bispecific antibody, wherein the LAG-3 binding portion of the bispecific antibody binds to an epitope on human LAG-3 comprising amino acid residues 183-202 of SEQ ID NO:41 (human LAG-3) and amino acid residues 203-225 of SEQ ID NO:41.

[0052] The present disclosure also provides pharmaceutical compositions comprising a human PD-1 antibody of the invention and a pharmaceutically acceptable excipient, diluent, or carrier.

[0053] The present disclosure also provides a method for treating an autoimmune disease in a subject in need thereof, comprising administering a therapeutically effective amount of a human LAG-3 / human PD-1 dual agonist bispecific antibody of the present invention to the subject. In a preferred embodiment, the autoimmune disease is rheumatoid arthritis, ulcerative colitis, type 1 diabetes, or systemic lupus erythematosus. In a preferred embodiment, the autoimmune disease is active. In another preferred embodiment, the autoimmune disease is in remission. In another preferred embodiment, the subject has been screened for the presence of LAG-3 and PD-1 on the same T cell or population of T cells. In another preferred embodiment, the subject has been identified as having both LAG-3 and PD-1 present on the same T cell or population of T cells.

[0054] The present disclosure also provides the human LAG-3 / human PD-1 dual agonist bispecific antibodies of the invention for use in therapy.

[0055] The present disclosure also provides a human LAG-3 / human PD-1 dual agonist bispecific antibody of the present invention for use in treating an autoimmune disease. In a preferred embodiment, the autoimmune disease is rheumatoid arthritis, ulcerative colitis, type 1 diabetes, or systemic lupus erythematosus. In a preferred embodiment, the autoimmune disease is an active disease. In another preferred embodiment, the autoimmune disease is in remission.

[0056] The present disclosure also provides a pharmaceutical composition comprising the human LAG-3 / human PD-1 dual agonist bispecific antibody of the present invention for use in treating rheumatoid arthritis, ulcerative colitis, type 1 diabetes, or systemic lupus erythematosus. In a preferred embodiment, the immune disease is active. In another preferred embodiment, the immune disease is in remission.

[0057] The present disclosure also provides the use of a human LAG-3 / human PD-1 dual agonist bispecific antibody of the invention in the manufacture of a medicament for treating rheumatoid arthritis, ulcerative colitis, type 1 diabetes, or systemic lupus erythematosus.

[0058] Mutations that promote desired qualities are known to those skilled in the art. However, it is not possible to predict which, if any, or how many such mutations will promote the desired qualities of any particular monospecific or bispecific antibody. The present invention provides preferred bispecific antibody constructs, antibodies that contain amino acid residue mutations that promote one or more of: reducing or eliminating C1q binding, reducing or eliminating nonspecific and self-interactions, reducing viscosity of the composition or formulation, eliminating deamidated residues, and reducing immunogenicity.

[0059] In a preferred embodiment, the LAG-3 / human PD-1 dual agonist bispecific antibody comprises the CDRs of a human LAG-3 agonist antibody of the invention (SEQ ID NOS: 1-6) and the CDRs of a human PD-1 agonist antibody. In another preferred embodiment, the LAG-3 / human PD-1 dual agonist bispecific antibody comprises the CDRs of a human LAG-3 agonist antibody of the invention (SEQ ID NOS: 1-6) and the CDR sequences of the human PD-1 agonist antibodies of SEQ ID NOS: 7-12.

[0060] In another preferred embodiment, the human LAG-3 / human PD-1 dual agonist bispecific antibody (a) a first antigen-binding domain that binds to human LAG-3, comprising a first heavy chain variable region (VH1) and a first light chain variable region (VL1), wherein VH1 comprises an HCDR1 comprising SEQ ID NO: 1, an HCDR2 comprising SEQ ID NO: 2, and an HCDR3 comprising SEQ ID NO: 3; and VL1 comprises an LCDR1 comprising SEQ ID NO: 4, an LCDR2 comprising SEQ ID NO: 5, and an LCDR3 comprising SEQ ID NO: 6; and (b) a second antigen-binding domain that binds to human PD-1, comprising a second heavy chain variable region (VH2) and a second light chain variable region (VL2), wherein VH2 comprises an HCDR1 comprising SEQ ID NO:7, an HCDR2 comprising SEQ ID NO:8, and an HCDR3 comprising SEQ ID NO:9; and VL2 comprises an LCDR1 comprising SEQ ID NO:10, an LCDR2 comprising SEQ ID NO:11, and an LCDR3 comprising SEQ ID NO:12. Includes.

[0061] In one preferred embodiment, VH1 comprises SEQ ID NO: 13 and VL1 comprises SEQ ID NO: 14. In another preferred embodiment, VH2 comprises SEQ ID NO: 17 and VL2 comprises SEQ ID NO: 18.

[0062] In another preferred embodiment, the human LAG-3 / human PD-1 dual agonist bispecific antibody comprises two heavy chains and two light chains, wherein the first heavy chain (HC1) comprises a first VH1 and a first heavy chain constant region, the first light chain (LC1) comprises a VL1 and a first light chain constant region, the second heavy chain (HC2) comprises a second VH2 and a second heavy chain constant region, and the second light chain (LC2) comprises a second VL2 and a second light chain constant region. In another preferred embodiment, the second light chain (LC2) comprises a VL2 and a second light chain constant region, wherein HC1 is linked to LC1 by an interchain disulfide bond, HC2 is linked to LC2 by an interchain disulfide bond, and HC1 is linked to HC2 by an interchain disulfide bond.

[0063] In another preferred embodiment, HC1 comprises SEQ ID NO: 37, wherein X at position 328 of SEQ ID NO: 37 is lysine or alanine; LC1 comprises SEQ ID NO: 22; HC2 comprises SEQ ID NO: 38, wherein X at position 324 of SEQ ID NO: 38 is lysine or alanine; and LC2 comprises SEQ ID NO: 24.

[0064] In another preferred embodiment, the X at position 328 of SEQ ID NO: 37 is lysine and the X at position 324 of SEQ ID NO: 38 is lysine. In another preferred embodiment, the X at position 328 of SEQ ID NO: 37 is alanine and the X at position 324 of SEQ ID NO: 38 is alanine.

[0065] In another preferred embodiment, the human LAG-3 / human PD-1 dual agonist bispecific antibody comprises a first HC1 comprising SEQ ID NO:21, a first LC1 comprising SEQ ID NO:22, a second HC2 comprising SEQ ID NO:23, and a second LC2 comprising SEQ ID NO:24.

[0066] In another preferred embodiment, the human LAG-3 / human PD-1 dual agonist bispecific antibody comprises a first HC1 comprising SEQ ID NO:21, a first LC1 comprising SEQ ID NO:22, a second HC2 comprising SEQ ID NO:23, and a second LC2 comprising SEQ ID NO:24.

[0067] In another preferred embodiment, the human LAG-3 / human PD-1 dual agonist bispecific antibody comprises a first HC1 comprising SEQ ID NO: 31, a first LC1 comprising SEQ ID NO: 22, a second HC2 comprising SEQ ID NO: 32, and a second LC2 comprising SEQ ID NO: 24.

[0068] In another preferred embodiment, the human LAG-3 / human PD-1 dual agonist bispecific antibody of the invention comprises a first constant heavy region (CH1) comprising SEQ ID NO: 15, a first constant light region (CL1) comprising SEQ ID NO: 16, a second constant heavy region (CH2) comprising SEQ ID NO: 19, and a second constant light region (CL2) comprising SEQ ID NO: 20.

[0069] In another preferred embodiment, the human LAG-3 / human PD-1 dual agonist bispecific antibody of the invention comprises a CH1 comprising SEQ ID NO:29, a CL1 comprising SEQ ID NO:16, a CH2 comprising SEQ ID NO:30, and a second constant light region (CL2) comprising SEQ ID NO:20.

[0070] In another preferred embodiment, the first antigen-binding domain of the bispecific antibody is an scFv and the second antigen-binding domain is an scFv.

[0071] The disclosure also provides nucleic acids comprising a sequence encoding one or more of SEQ ID NOs:22, 24, 37, and 38, wherein the X at position 328 of SEQ ID NO:37 is a lysine or an alanine residue, and the X at position 324 of SEQ ID NO:38 is a lysine or an alanine residue, such that if amino acid residue 328 of SEQ ID NO:37 is a lysine residue, then the X at position 324 of SEQ ID NO:38 is a lysine residue, and if the X at position 328 of SEQ ID NO:37 is an alanine residue, then the X at position 324 of SEQ ID NO:38 is an alanine residue.

[0072] The disclosure also provides a cell comprising a nucleic acid comprising a sequence encoding one or more of the polypeptides of SEQ ID NOs:22, 24, 37, and 38, wherein the X at position 328 of SEQ ID NO:37 is a lysine or an alanine residue, and amino acid residue 324 of SEQ ID NO:38 is a lysine or an alanine residue, and wherein if the X at position 328 of SEQ ID NO:37 is a lysine residue, then the X at position 324 of SEQ ID NO:38 is a lysine residue, and if the X at position 328 of SEQ ID NO:37 is an alanine residue, then the X at position 324 of SEQ ID NO:38 is an alanine residue, and wherein the cell is capable of expressing the one or more polypeptides.

[0073] In another preferred embodiment of the LAG-3 / human PD-1 dual agonist bispecific antibody of the invention, the LAG-3 / human PD-1 dual agonist bispecific antibody comprises the CDRs of a human LAG-3 agonist antibody of the invention (SEQ ID NOs: 44, 45, and 47-50) and the CDRs of a human PD-1 agonist antibody. In another preferred embodiment, the LAG-3 / human PD-1 dual agonist bispecific antibody comprises the CDRs of a human LAG-3 agonist antibody of the invention (SEQ ID NOs: 44, 45, and 47-50) and the CDR sequences of the human PD-1 agonist antibodies of SEQ ID NOs: 7, 46, and 9-12.

[0074] In another preferred embodiment, the human LAG-3 / human PD-1 dual agonist bispecific antibody (a) a first antigen-binding domain that binds to human LAG-3, comprising a first VH1 and a first VL1, wherein the VH1 comprises an HCDR1 comprising SEQ ID NO: 44, an HCDR2 comprising SEQ ID NO: 45, and an HCDR3 comprising SEQ ID NO: 47; and the VL1 comprises an LCDR1 comprising SEQ ID NO: 48, an LCDR2 comprising SEQ ID NO: 49, and an LCDR3 comprising SEQ ID NO: 50; and (b) a second antigen-binding domain that binds to human PD-1, comprising a second VH2 and a second VL2, wherein the VH2 comprises an HCDR1 comprising SEQ ID NO:7, an HCDR2 comprising SEQ ID NO:46, and an HCDR3 comprising SEQ ID NO:9; and the VL2 comprises an LCDR1 comprising SEQ ID NO:10, an LCDR2 comprising SEQ ID NO:11, and an LCDR3 comprising SEQ ID NO:12. Includes.

[0075] In another preferred embodiment, the human LAG-3 / human PD-1 dual agonist bispecific antibody comprises a first HC1 comprising SEQ ID NO: 61, a first LC1 comprising SEQ ID NO: 64, a second HC1 comprising SEQ ID NO: 63, and a second LC1 comprising SEQ ID NO: 65.

[0076] In another preferred embodiment, the human LAG-3 / human PD-1 dual agonist bispecific antibody comprises a first HC1 comprising SEQ ID NO: 62, a first LC1 comprising SEQ ID NO: 64, a second HC1 comprising SEQ ID NO: 63, and a second LC1 comprising SEQ ID NO: 65.

[0077] In another preferred embodiment, the first antigen-binding domain of the bispecific antibody is an scFv and the second antigen-binding domain is an scFv.

[0078] The present disclosure also provides a nucleic acid comprising a sequence encoding one or more of SEQ ID NOs: 66, 68, 69, and 70. The present disclosure also provides a cell comprising a nucleic acid comprising a sequence encoding one or more of the polypeptides of SEQ ID NOs: 61, 63, 64, and 65.

[0079] The present disclosure also provides a nucleic acid comprising a sequence encoding one or more of SEQ ID NOs: 67-70. The present disclosure also provides a cell comprising a nucleic acid comprising a sequence encoding one or more of the polypeptides of SEQ ID NOs: 62-65.

[0080] In a preferred embodiment of the cell, the cell is a mammalian cell. In another preferred embodiment, the cell or mammalian cell is isolated. In another preferred embodiment, the present disclosure provides a method for producing an antibody, comprising culturing cells under conditions in which a human LAG-3 / human PD-1 dual agonist antibody is expressed, and then recovering the expressed antibody from the culture medium. In another preferred embodiment, the present disclosure provides an antibody produced by culturing cells under conditions in which a human LAG-3 / human PD-1 dual agonist antibody is expressed, and recovering the expressed antibody from the culture medium.

[0081] The present disclosure also provides pharmaceutical compositions comprising a human LAG-3 / human PD-1 dual agonist bispecific antibody of the invention and a pharmaceutically acceptable excipient, diluent, or carrier.

[0082] The present disclosure also provides a method for treating an autoimmune disease in a subject in need thereof, comprising administering a therapeutically effective amount of a bispecific antibody of the invention to the subject. In a preferred embodiment, the autoimmune disease is rheumatoid arthritis, ulcerative colitis, type 1 diabetes, or systemic lupus erythematosus. In another preferred embodiment, the autoimmune disease is active. In another preferred embodiment, the autoimmune disease is in remission. In another preferred embodiment, the human LAG-3 / human PD-1 dual agonist bispecific antibody exhibits greater efficacy in vitro and / or in vivo than the efficacy of a combination of a corresponding monospecific human LAG-3 agonist antibody and a corresponding monospecific human PD-1 antibody.

[0083] The present disclosure also provides the human LAG-3 / human PD-1 dual agonist bispecific antibodies of the invention for use in therapy.

[0084] The present disclosure also provides a human LAG-3 / human PD-1 dual agonist bispecific antibody of the present invention for use in treating an autoimmune disease. In a preferred embodiment, the autoimmune disease is rheumatoid arthritis, ulcerative colitis, type 1 diabetes, or systemic lupus erythematosus. In a preferred embodiment, the autoimmune disease is an active disease. In another preferred embodiment, the autoimmune disease is in remission.

[0085] The present disclosure also provides a pharmaceutical composition comprising the human LAG-3 / human PD-1 dual agonist bispecific antibody of the present invention for use in treating an autoimmune disease. In a preferred embodiment, the autoimmune disease is rheumatoid arthritis, ulcerative colitis, type 1 diabetes, or systemic lupus erythematosus. In another preferred embodiment, the autoimmune disease is an active disease. In another preferred embodiment, the autoimmune disease is in remission.

[0086] The present disclosure also provides use of a human LAG-3 / human PD-1 dual agonist bispecific antibody of the present invention in the manufacture of a medicament for treating rheumatoid arthritis, ulcerative colitis, type 1 diabetes, or systemic lupus erythematosus. In a preferred embodiment, the immune disease is active. In another preferred embodiment, the immune disease is in remission.

[0087] In embodiments that refer to methods of treatment described herein, such embodiments are also further embodiments for use in that treatment, or alternatively for use in the manufacture of a medicament for use in that treatment.

[0088] In a preferred embodiment, the human LAG-3 antibody or human LAG-3 / human PD-1 dual agonist bispecific antibody of the invention is substantially pure. In another preferred embodiment, the human LAG-3 antibody or human LAG-3 / human PD-1 dual agonist bispecific antibody of the invention is sterile.

[0089] As used herein, the term "autoimmune disease" refers to an undesirable condition resulting from an inappropriate or unwanted immune response against one's own cells and / or tissues or transplanted cells and / or tissues. The term "autoimmune disease" is meant to include such conditions, whether mediated by a humoral or cellular immune response. Exemplary autoimmune diseases or disorders include, but are not limited to, graft-versus-host disease (GVHD), solid organ transplant rejection, vasculitis, systemic lupus erythematosus (SLE), type 1 diabetes mellitus (T1DM), multiple sclerosis (MS), giant cell arteritis (GCA), psoriasis (PsO), psoriatic arthritis (PsA), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), ulcerative colitis (UC), ankylosing spondylitis (AS), Sjögren's syndrome (SjS), autoimmune hepatitis, scleroderma, celiac disease, adenocarcinoma of the rectum, and thyroid cancer. These conditions include: Crohn's disease, Hashimoto's thyroiditis, Graves' disease, atrophic gastritis / pernicious anemia, acquired hypogonadism / infertility, hypoparathyroidism, Coombs-positive hemolytic anemia, chronic allergic diseases (such as asthma, hay fever, or allergic rhinitis), Crohn's disease, male or female infertility, Behçet's disease, Wegener's granulomatosis, myocarditis, myositis, polymyalgia rheumatica (PMR), spontaneous abortion, vitiligo, atherosclerosis, autoimmune pancreatitis, bullous pemphigoid, chronic viral infections, and myasthenia gravis. For purposes of this disclosure, preferred autoimmune diseases are graft-versus-host disease (GVHD), solid organ transplant rejection, vasculitis, systemic lupus erythematosus (SLE), type 1 diabetes mellitus (T1DM), multiple sclerosis (MS), giant cell arteritis (GCA), psoriasis (PsO), psoriatic arthritis (PsA), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), ulcerative colitis (UC), ankylosing spondylitis (AS), Sjögren's syndrome (SjS), autoimmune hepatitis, and scleroderma.

[0090] In a preferred embodiment, the immune disease is rheumatoid arthritis, ulcerative colitis, type 1 diabetes, or systemic lupus erythematosus. In another preferred embodiment, the immune disease is rheumatoid arthritis. In another preferred embodiment, the immune disease is ulcerative colitis. In another preferred embodiment, the immune disease is type 1 diabetes. In another preferred embodiment, the immune disease is systemic lupus erythematosus. In another preferred embodiment, the immune disease is an active disease. In another preferred embodiment, the immune disease is in remission.

[0091] In another preferred embodiment, the human LAG-3 / human PD-1 dual agonist bispecific antibody of the invention binds to human LAG-3 but does not deplete T cells.

[0092] In another preferred embodiment, the human LAG-3 / human PD-1 dual agonist bispecific antibody of the invention stimulates the LAG-3 signaling pathway.

[0093] In another preferred embodiment, the human LAG-3 / human PD-1 dual agonist bispecific antibodies of the invention bind to human PD-1 and human LAG-3 with desirable association and dissociation rates for optimal agonist activity.

[0094] In another preferred embodiment, the human LAG-3 / human PD-1 dual agonist bispecific antibody of the invention simultaneously stimulates each of the PD-1 and LAG-3 signaling pathways.

[0095] In another preferred embodiment, the human LAG-3 / human PD-1 dual agonist bispecific antibodies of the invention reduce T cell proliferation by promoting downregulation of T cell receptor signaling, rather than by T cell depletion.

[0096] In another preferred embodiment, the human LAG-3 / human PD-1 dual agonist bispecific antibodies of the invention stimulate human PD-1 and human LAG-3 in an immunologically relevant context to achieve in vivo efficacy.

[0097] In another preferred embodiment, the human LAG-3 / human PD-1 dual agonist bispecific antibodies of the invention exhibit an effect in vitro and / or in vivo that is greater than the effect of a combination of a human LAG-3 agonist antibody and a human PD-1 agonist antibody.

[0098] The present disclosure also provides a method comprising: (a) contacting a human LAG-3 / human PD-1 dual agonist bispecific antibody of the present invention with cells expressing the extracellular domain of human PD-1 fused to an enzyme acceptor subunit of a reporter gene and the extracellular domain of human LAG-3 fused to an enzyme donor subunit; (b) incubating the contacted cells under conditions suitable for binding of the bispecific antibody to the extracellular domain of human PD-1 and the extracellular domain of human LAG-3; (c) contacting the cells with a reporter compound; (d) assaying the amount of reporter compound; and (e) determining the IC50 for the bispecific antibody binding to the cells. In a preferred embodiment, the enzyme acceptor subunit of the reporter gene is β-galactosidase and the enzyme donor subunit is the PK1 enzyme donor subunit of β-galactosidase. In another preferred embodiment, the bispecific antibody comprises 1) a heavy chain (HC) of SEQ ID NO: 21 and a light chain (LC) of SEQ ID NO: 22, and 2) a HC of SEQ ID NO: 23 and a LC of SEQ ID NO: 24. In another preferred embodiment, the bispecific antibody comprises 1) a HC of SEQ ID NO: 31 and a LC of SEQ ID NO: 22, and 2) a HC of SEQ ID NO: 32 and a LC of SEQ ID NO: 24. In another preferred embodiment, the bispecific antibody comprises 1) a HC of SEQ ID NO: 61 and a LC of SEQ ID NO: 64, and 2) a HC of SEQ ID NO: 63 and a LC of SEQ ID NO: 65. In another preferred embodiment, the bispecific antibody comprises 1) a HC of SEQ ID NO: 62 and a LC of SEQ ID NO: 64, and 2) a HC of SEQ ID NO: 63 and a LC of SEQ ID NO: 65.

[0099] As used herein, "PD-1" refers to programmed cell death 1, also known as programmed death 1 (PD-1; CD279), a type I cell membrane protein belonging to the extended CD28 / CTLA-4 family that contains an extracellular IgV domain followed by transmembrane and intracellular domains.

[0100] As used herein, "hPD-1" or "human PD-1" refers to wild-type human PD-1, e.g., wild-type human PD-1 having the amino acid sequence set forth in SEQ ID NO: 39 (i.e., NCBI Reference Sequence NP_005009.2).

[0101] PD-1 polypeptide "extracellular domain" or "ECD" refers to a form of PD-1 polypeptide that is essentially free of transmembrane and cytoplasmic domains. Preferably, the PD-1 ECD has less than 1% of the transmembrane and cytoplasmic domains, and more preferably, the PD-1 ECD has less than 0.5% of such domains. Even more preferably, the human PD-1 ECD polypeptide is as set forth in SEQ ID NO: 40. PD-1 polypeptide ECD can be prepared using methods known in the art. Alternatively, human PD-1 polypeptide ECD can be commercially purchased from various vendors, such as Sino Biological (Beijing, China; reference number 10377-H08) and R&D Systems (Minneapolis, MN, USA; cat. #8986-PD).

[0102] As used herein, "LAG-3" refers to lymphocyte activation gene 3, also known as CD223, which belongs to the immunoglobulin superfamily. The terms LAG-3, LAG3, Lag-3 and Lag3 are synonymous.

[0103] As used herein, "hLAG-3" or "human LAG-3" refers to wild-type human LAG-3, preferably wild-type human LAG-3 having the amino acid sequence set forth in SEQ ID NO: 41 (i.e., NCBI reference sequence NP_002277).

[0104] The LAG-3 polypeptide "extracellular domain" or "ECD" refers to a form of the LAG-3 polypeptide that is essentially free of transmembrane and cytoplasmic domains. Preferably, the LAG-3 ECD has less than 1% of the transmembrane and cytoplasmic domains, and more preferably, the LAG-3 ECD has less than 0.5% of such domains. Even more preferably, the human LAG-3' ECD polypeptide is as set forth in SEQ ID NO: 42. The LAG-3 polypeptide ECD can be prepared using methods known in the art. Alternatively, the human LAG-3 polypeptide ECD can be commercially purchased from various vendors, such as Bertyn Bioreagent (Rockville, MD, USA, Cat. No. 32083).

[0105] As used herein, a "human PD-1 agonist antibody" refers to an antibody that binds to human PD-1 and, when administered in vivo, results in significantly reduced autoimmune activity, such as at least one of reduced anti-double-stranded DNA (ds-DNA) titers, reduced disease scores, or reduced inflammatory cytokines.

[0106] As used herein, "human LAG-3 agonist antibody" refers to an antibody that binds to human LAG-3 and, when administered in vivo, results in at least one significantly reduced autoimmune activity, such as reduced anti-double-stranded DNA (ds-DNA) titers, reduced disease scores, or reduced inflammatory cytokines. In a preferred embodiment, the LAG-3 agonist antibody is "Antibody E," which refers to a human LAG-3 binding agonist antibody having an HC of SEQ ID NO: 21 and an LC of SEQ ID NO: 22.

[0107] In another preferred embodiment, the LAG-3 agonist antibody is a variant of Antibody E, an antibody having an HC of SEQ ID NO:31 and an LC of SEQ ID NO:22.

[0108] In another preferred embodiment, the LAG-3 agonist antibody is "Antibody F," which refers to a human LAG-3 binding agonist antibody having an HC of SEQ ID NO:61 and an LC of SEQ ID NO:64.

[0109] In another preferred embodiment, the LAG-3 agonist antibody is a variant of antibody F, an antibody having an HC of SEQ ID NO:62 and an LC of SEQ ID NO:64.

[0110] In another preferred embodiment, the LAG-3 agonist antibody is a variant of Antibody F, referred to herein as "Antibody G," an antibody having an HC of SEQ ID NO: 61 and an LC of SEQ ID NO: 64, with a serine to histidine substitution at amino acid residue 30 and a threonine to serine substitution at amino acid residue 61.

[0111] As used herein, "human LAG-3 / human PD-1 dual agonist antibody" refers to an antibody comprising (a) the CDR sequences of SEQ ID NOs: 1-12, or (b) SEQ ID NOs: 44, 45, and 47-50, and 7, 46, and 9-12. In a preferred embodiment, the dual agonist antibody comprises 1) the variable heavy chain (VH) sequence of SEQ ID NO: 13 and the variable light chain (VL) sequence of SEQ ID NO: 14, and 2) the VH sequence of SEQ ID NO: 17 and the VL sequence of SEQ ID NO: 18. In another preferred embodiment, the dual agonist antibody is "Antibody A," which refers to a human PD-1-binding and human LAG-3-binding dual agonist antibody having 1) an HC of SEQ ID NO: 21 and an LC of SEQ ID NO: 22, and 2) an HC of SEQ ID NO: 23 and an LC of SEQ ID NO: 24. In another preferred embodiment, the dual agonist antibody is "Antibody B," which refers to a human PD-1-binding and human LAG-3-binding antibody having 1) an HC of SEQ ID NO: 31 and an LC of SEQ ID NO: 22, and 2) an HC of SEQ ID NO: 32 and an LC of SEQ ID NO: 24. Antibodies A and B bind to the same epitope on human LAG-3.

[0112] In another preferred embodiment, the dual agonist antibody comprises 1) the variable heavy chain VH sequence of SEQ ID NO: 53 and the variable light chain VL sequence of SEQ ID NO: 56, and 2) the VH sequence of SEQ ID NO: 55 and the VL sequence of SEQ ID NO: 57. In another preferred embodiment, the dual agonist antibody is "Antibody C," which refers to a human PD-1-binding and human LAG-3-binding dual agonist antibody having 1) an HC of SEQ ID NO: 61 and an LC of SEQ ID NO: 64, and 2) an HC of SEQ ID NO: 63 and an LC of SEQ ID NO: 65. Antibodies C and D bind to the same epitope on human LAG-3 that is different from the epitope on human LAG-3 bound by antibodies A and B.

[0113] In a preferred embodiment, the dual agonist antibody comprises 1) the variable heavy chain VH sequence of SEQ ID NO: 54 and the variable light chain VL sequence of SEQ ID NO: 56, and 2) the VH sequence of SEQ ID NO: 55 and the VL sequence of SEQ ID NO: 57. In another preferred embodiment, the dual agonist antibody is "Antibody D," which refers to a human PD-1-binding and human LAG-3-binding dual agonist antibody having 1) an HC of SEQ ID NO: 62 and an LC of SEQ ID NO: 64, and 2) an HC of SEQ ID NO: 63 and an LC of SEQ ID NO: 65.

[0114] The anti-human LAG-3 / anti-human PD-1 dual agonist molecules, e.g., dual agonist bispecific antibodies, disclosed herein are believed to be the first such dual agonist molecules disclosed.

[0115] The disclosure herein is believed to be the first report of a T cell population containing both human LAG-3 target protein and PD-1 target protein on the same T cell. The disclosure herein is also believed to be the first report that human LAG-3 is more highly expressed on CD8+ T cells than on CD4+ T cells, and that human PD-1 is more highly expressed on CD4+ T cells than on CD8+ T cells. The disclosure herein is also believed to be the first report of a T cell population containing a human PD-1 target protein at a concentration higher than the concentration of human LAG-3 target protein on the same T cell. In a preferred embodiment, the ratio of PD-1 protein to LAG-3 protein on the surface of the T cell is about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, about 5.5:1, or about 6:1. In another preferred embodiment, the ratio of PD-1 protein to LAG-3 protein on the surface of the T cell is about 3:1 to about 5:1.

[0116] As used herein, the term "antibody" refers to an immunoglobulin molecule that binds to an antigen. Antibody embodiments include monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific or multispecific antibodies, or conjugated antibodies. The antibody may be of any class (e.g., IgG, IgE, IgM, IgD, IgA) and any subclass (e.g., IgG1, IgG2, IgG3, IgG4). In a preferred embodiment, the antibody or bispecific antibody of the invention is an IgG1.

[0117] An exemplary antibody of the present disclosure is an immunoglobulin G (IgG) antibody composed of four polypeptide chains: two heavy chains (HC) and two light chains (LC) cross-linked via interchain disulfide bonds. The amino-terminal portion of each of the four polypeptide chains contains a variable region of approximately 100 to 125 amino acids or more, which is primarily responsible for antigen recognition. The carboxy-terminal portion of each of the four polypeptide chains contains a constant region, which is primarily responsible for effector function. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain is composed of a light chain variable region (VL) and a light chain constant region. IgG isotypes can be further divided into subclasses (e.g., IgG1, IgG2, IgG3, and IgG4).

[0118] The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), among which more conserved regions called framework regions (FRs) are located. CDRs are exposed on the surface of the protein and are important regions of the antibody for antigen-binding specificity. Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Herein, the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2, and HCDR3," and the three CDRs of the light chain are referred to as "LCDR1, LCDR2, and LCDR3." CDRs contain most of the residues that form specific interactions with the antigen.The assignment of amino acid residues to CDRs is based on the Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991)), Chothia (Chothia et al., "Canonical structures for the hypervariable regions of immunoglobulins", Journal of Molecular Biology, 196, 901-917 (1987), Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), North (North et al., "A New Clustering of Antibody CDR Loop Conformations", Journal of Molecular Biology, 406, 228-256 (2011)), or IMGT (the international ImMunoGeneTics This can be done by well-known schemes, including those described in the Imgt database, available at www.imgt.org (see Lefranc et al., Nucleic Acids Res. 1999;27:209-212). In the present disclosure, the Kabat method is used to assign amino acid residues to CDRs.

[0119] As used herein, embodiments of the present disclosure also include antibody fragments or antigen-binding fragments that comprise at least a portion of an antibody that retains the ability to specifically interact with an antigen or an epitope of an antigen, such as Fab, Fab', F(ab')2, Fv fragment, scFv antibody fragment, scFab, disulfide-linked Fv (sdFv), Fd fragment, etc.

[0120] The term "antigen-binding domain," as used herein, refers to a portion that binds to an antigen or an epitope of an antigen. For example, the antigen-binding domain in a human LAG-3 antibody of the present invention binds to human LAG-3 (SEQ ID NO: 41) or a fragment thereof, such as LAG-3 ECD (SEQ ID NO: 42). A bispecific antibody of the present invention comprises two antigen-binding domains, one of which binds to human LAG-3 (SEQ ID NO: 41) or a fragment thereof, such as LAG-3 ECD (SEQ ID NO: 42), and the other of which binds to human PD-1 (SEQ ID NO: 39) or a fragment thereof, such as PD-1 ECD (SEQ ID NO: 40).

[0121] As used herein, the term "bispecific" refers to a molecule that contains two different antigen-binding domains. A bispecific binding molecule can bind to two different antigens or two different epitopes of the same antigen. Exemplary embodiments of bispecific molecules include the bispecific antibodies disclosed herein.

[0122] The term "agonize," as used herein, refers to the ability of an antibody, antibody fragment, or binding molecule to induce or increase one or more activities or functions associated with an antigen.

[0123] Preferably, the antibodies of the present disclosure contain an Fc portion that is a human IgG1 subtype. It is well known that human IgG1 binds to the Fc-gamma receptor family (FcγR) and C1q. Interaction with these receptors can induce antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In a preferred embodiment, the monoclonal anti-human LAG-3 antibody of the present invention does not exhibit substantial ADCC activity. In another preferred embodiment, the monoclonal anti-human LAG-3 antibody of the present invention does not exhibit ADCC activity. In another preferred embodiment, the anti-human LAG-3 / anti-human PD-1 dual agonist bispecific antibody of the present invention does not exhibit substantial ADCC activity. In another preferred embodiment, the anti-human LAG-3 / anti-human PD-1 dual agonist bispecific antibody of the present invention does not exhibit ADCC activity.

[0124] In a preferred embodiment, the monoclonal anti-human LAG-3 antibodies of the invention do not substantially deplete T cells. In another preferred embodiment, the monoclonal anti-human LAG-3 antibodies of the invention do not substantially deplete T cells. In another preferred embodiment, the anti-human LAG-3 / anti-human PD-1 dual agonist bispecific antibodies of the invention do not substantially deplete T cells. In another preferred embodiment, the anti-human LAG-3 / anti-human PD-1 dual agonist bispecific antibodies of the invention do not deplete T cells.

[0125] The isolated DNA encoding the CH region can be converted into a full-length heavy chain gene by operably linking the DNA encoding the VH to another DNA molecule encoding a heavy chain constant region. The sequences of human and other mammalian heavy chain constant region genes are known in the art. DNA fragments encompassing these regions can be obtained, for example, by standard PCR amplification.

[0126] The isolated DNA encoding the VL region can be converted into a full-length light chain gene by operably linking the VL-encoding DNA to another DNA molecule encoding a light chain constant region. The sequences of light chain constant region genes from humans and other mammals are known in the art. DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region. In a preferred embodiment, the light chain constant region of the anti-LAG-3 antibody of the present invention is a kappa constant region. In another preferred embodiment, for the anti-LAG-3 / anti-PD-1 dual agonist antibody of the present invention, the light chain constant region of the anti-LAG-3 arm is a kappa constant region, and the light chain constant region of the anti-PD-1 arm is a lambda constant region.

[0127] The terms "nucleic acid" or "polynucleotide," as used interchangeably herein, refer to a polymer of nucleotides, including single- and / or double-stranded nucleotide-containing molecules, such as DNA, cDNA, and RNA molecules, that incorporate naturally occurring nucleotides, modified nucleotides, and / or nucleotide analogs. A polynucleotide of the present disclosure may also include substrates incorporated therein, for example, by a DNA or RNA polymerase or a synthetic reaction.

[0128] The polynucleotide of the present disclosure can be expressed in host cells after the sequence is operably linked to expression control sequence.Expression vectors are typically replicable in host organisms, either as episomes or as an integrated part of host chromosomal DNA.Generally, expression vectors contain selectable markers, such as tetracycline, neomycin, and dihydrofolate reductase, to allow detection of cells transformed with desired DNA sequence.

[0129] The antibodies of the present disclosure can be readily produced in mammalian cells, non-limiting examples of which include CHO, NS0, HEK293, or COS cells. Host cells are cultured using techniques well known in the art.

[0130] Vectors containing polynucleotide sequences of interest (eg, a polynucleotide encoding an antibody polypeptide and an expression control sequence) can be introduced into host cells by well-known methods, which vary depending on the type of cellular host.

[0131] Various methods of protein purification can be used to purify proteins, including but not limited to antibodies, and such methods are known in the art.

[0132] In another embodiment of the present disclosure, the cells, antibodies, or nucleic acids encoding the antibodies are provided in isolated form. As used herein, the term "isolated" refers to a cell, protein, peptide, or nucleic acid that is free or substantially free of any other macromolecular species found in the cellular environment. "Substantially free," as used herein, means that the protein, peptide, or nucleic acid of interest contains more than 80%, preferably more than 90%, and more preferably more than 95% (on a molar basis) of the macromolecular species of interest.

[0133] The antibody of the present disclosure or a pharmaceutical composition comprising the same can be administered parenterally, non-limiting examples of which include subcutaneous administration and intravenous administration. The antibody of the present disclosure can be administered alone to a patient in a single dose or multiple doses together with a pharmaceutically acceptable carrier, diluent, or excipient. The pharmaceutical composition of the present disclosure can be prepared by methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy, 22nd ed. (2012), A. Loyd et al., Pharmaceutical Press), and can include the antibody disclosed herein and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0134] As used herein with respect to the affinity of a human PD-1 agonist antibody for human PD-1 (SEQ ID NO: 39) or a human PD-1 ECD, preferably the human PD-1 ECD set forth in SEQ ID NO: 40, "binds to human PD-1," unless otherwise indicated, means that the antibody binds to or binds to human PD-1 at a density of about 1 x 10, as determined by methods known in the art, including, but not limited to, the use of a surface plasmon resonance (SPR) biosensor at 25°C or 37°C, and / or by methods essentially as described herein. -7 M, about 1x10 -8 M, about 1x10 -9 M, about 5x10 -9 M, or approximately 1x10 -10 K of M DMore preferably, the human PD-1 agonist antibodies of the disclosure have a cytoplasmic affinity of about 1 x 10 to human PD-1 (i.e., SEQ ID NO: 39) or human PD-1 ECD (e.g., SEQ ID NO: 40), as determined by methods known in the art, including the use of an SPR biosensor at 25°C or 37°C, and / or by methods essentially as described herein. -8 M ~ approx. 5x10 -10 K of M D Even more preferably, such antibodies bind to human PD-1 (i.e., SEQ ID NO: 39) or human PD-1 ECD (e.g., SEQ ID NO: 40) at a binding affinity of about 5x10 as determined by methods known in the art, including the use of an SPR biosensor at 25°C or 37°C, and / or by methods essentially as described herein. -8 M ~ approx. 5x10 -10 Even more preferably, such antibodies will each have an affinity of about 1.0x10 M (as determined by SPR using a BIAcore® 8K essentially as described herein). -4 ~about 1.0x10 -3 and approximately 1.3x10 5 ~Approx. 2.0×10 5 K of human PD-1 ECD (e.g., SEQ ID NO: 40) on and K. off would have value.

[0135] As used herein with respect to the affinity of a human LAG-3 agonist antibody for human LAG-3 (SEQ ID NO: 41) or human LAG-3 ECD, preferably the human LAG-3 ECD set forth in SEQ ID NO: 42, "binds to human LAG-3," unless otherwise indicated, means that the antibody binds to human LAG-3 at a binding affinity of about 1 x 10, as determined by methods known in the art, including, but not limited to, the use of a surface plasmon resonance (SPR) biosensor at 25°C or 37°C, and / or by methods essentially as described herein. -7 M, about 1x10 -8 M, about 1x10 -9 M, about 5x10 -9M, or approximately 1x10 -10 K of M D More preferably, the human LAG-3 agonist antibodies of the present disclosure have a binding affinity to human LAG-3 (i.e., SEQ ID NO: 41) or human LAG-3 (e.g., SEQ ID NO: 42) of about 1 x 10 as determined by methods known in the art, including the use of an SPR biosensor at 25°C or 37°C, and / or by methods essentially as described herein. -8 M ~ approx. 5x10 -10 K of M D Even more preferably, such antibodies bind to human LAG-3 (i.e., SEQ ID NO: 41) or human LAG-3 (e.g., SEQ ID NO: 42) at a binding affinity of about 5x10 as determined by methods known in the art, including the use of an SPR biosensor at 25°C or 37°C, and / or by methods essentially as described herein. -8 M ~ approx. 5x10 -10 Even more preferably, such antibodies will each have an affinity of about 1.0x10 M (as determined by SPR using a BIAcore® 8K essentially as described herein). -4 ~about 1.0x10 -3 and approximately 1.3x10 5 ~Approx. 2.0×10 5 K of human LAG-3ECD (e.g., SEQ ID NO: 40) on and K. off would have value.

[0136] Unless otherwise indicated herein, "LAG-3" refers to human LAG-3 and "PD-1" refers to human PD-1.

[0137] As used herein, the term "binds" refers to the molecular interaction between two molecules, e.g., an antibody of the invention and LAG-3, PD-1, or both LAG-3 and PD-1. The term "bispecific binding" refers to binding to human LAG-3 and human PD-1.

[0138] The terms "selectively bind" or "specifically bind" mean that an antibody of the invention interacts with human LAG-3, or PD-1, or human LAG-3 and human PD-1, more frequently, more rapidly, with a longer duration, with greater affinity, or some combination of the above, than with other substances. In one embodiment, "specifically bind" means that an antibody of the invention binds to human LAG-3, or human PD-1, or both human LAG-3 and human PD-1 with a K of about 0.1 mM or less. D In another embodiment, "specifically binds" means that the antibody of the invention binds to human LAG-3, or human PD-1, or both human LAG-3 and human PD-1 with a K of about 0.01 mM or less. D In another embodiment, "specifically binds" means that an antibody of the invention binds to human LAG-3, or human PD-1, or both human LAG-3 and human PD-1 with a K of about 0.001 mM or less. D In another embodiment, "specifically binds" means that the antibody of the invention binds to human LAG-3, or human PD-1, or both human LAG-3 and human PD-1 with a K of about 0.0001 mM or less. D In another embodiment, the antibody of the invention binds to human PD-1 at a K D Different from K D In another embodiment, the antibody binds to human LAG-3 about 10 times more tightly than it binds to human PD-1.

[0139] In one embodiment, an antibody of the invention is an scFv molecule that binds to human LAG-3, or human PD-1, or both human LAG-3 and human PD-1. In another embodiment, the scFv molecule specifically binds to human LAG-3, or human PD-1, or both human LAG-3 and human PD-1.

[0140] As used herein, the term "substantially pure" refers to a material, e.g., an antibody of the present invention, that is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% free from contaminants.

[0141] Synonyms for "LAG-3" are LAG-3 and CD223.

[0142] As used herein, the term "about" means plus or minus 10 percent of the stated value or range of values. For example, "about" 12 includes values ​​ranging from 10.8 (inclusive) to 13.2 (inclusive); about 10% by weight includes compositions containing 9 (inclusive) to 11% by weight, etc.

[0143] As used herein, the term "adaptive immunity" includes the arm of the immune response, which, in contrast to the innate arm of the immune response, is antigen-specific and refers to a secondary antigen-specific immune response that is enhanced upon restimulation with the same antigen.

[0144] The term "treating" (or "treat" or "treatment") refers to slowing, hindering, suppressing, alleviating, halting, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.

[0145] An "effective amount" refers to the amount of an antibody of the present invention, or a pharmaceutical composition containing such an antibody, that elicits the biological or medical response or desired therapeutic effect in a tissue, system, animal, mammal, or human being sought by a researcher, physician, or other clinician. An effective amount of an antibody may vary depending on factors such as the individual's condition, age, sex, and weight, as well as the ability of the antibody to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the antibody are outweighed by the therapeutically beneficial effects. Such benefits include any one or more of: increased immune tolerance of a transplanted organ; stabilized autoimmune disease or disorder; or amelioration of signs and symptoms of an autoimmune disorder. An effective amount can be readily determined by one of ordinary skill in the art by using known techniques and observing results obtained under similar circumstances. In determining the effective amount for a patient, numerous factors are considered by the attending diagnostician, including, but not limited to, the size, age, and general health of the patient; the particular disease or disorder involved; the extent, involvement, or severity of the disease or disorder; the response of the individual patient, the particular compound administered; the mode of administration; the bioavailability characteristics of the administered preparation; the selected dosing regimen; the use of concomitant medications; and other relevant circumstances.

[0146] Dosage regimens for administering antibodies of the invention may be adjusted to provide the optimum desired response (eg, a therapeutic effect).

[0147] As used herein, the term "effective response" of a patient, or "responsiveness" of a patient to a treatment, refers to a clinical or therapeutic benefit conferred on a patient upon administration of an antibody of the present disclosure. Such benefit may include any one or more of the following: increased immune tolerance of a transplanted organ; stabilized autoimmune disease or disorder; or amelioration of signs and symptoms of an autoimmune disorder.

[0148] A potential advantage of the methods disclosed herein is the potential to provide significant and / or long-term relief in patients suffering from autoimmune disorders with an acceptable safety profile, including acceptable tolerability, toxicity, and / or adverse events, thereby enabling patients to benefit from a comprehensive therapeutic approach. The efficacy of the disclosed treatments can be measured by a variety of endpoints commonly used in evaluating treatments for various autoimmune disorders, including, but not limited to, the American College of Rheumatology (ACR) 20, ACR50, ACR70, Psoriasis Area and Severity Index (PASI) 50, PASI75, PASI90, PASI100, and Systemic Lupus Erythmatosus Disease Activity Index (SLEDAI). Various other approaches to determining the efficacy of any particular therapy disclosed herein can optionally be used, including measuring response through, for example, immune cell activation markers, measures of inflammation, cell cycle-dependent biomarker measurement visualization, and / or pain assessment.

[0149] As used herein, the term "modified human IgG1" refers to a human IgG1 that has been engineered to reduce binding of the human IgG1 to at least one human Fc gamma receptor. Typically, this is performed by mutating residues that result in reduced binding of the antibody to Fc gamma receptors, for example, P329A, L234A, and L235A mutations.

[0150] Methods for assaying LAG-3 activity in vitro are known to those skilled in the art (e.g., Angin M, et al., J. Immunol. 2020;204(4):810-818).

[0151] Methods for assaying PD-1 activity in vitro are known to those skilled in the art (e.g., Carpenito et al., J Immunother Cancer 2018;6(1):31).

[0152] In vivo mouse models of immune activity are well known to those skilled in the art, as shown and disclosed herein (e.g., Vincelette J, et al., Arthritis Res Ther. 2007;9(6):R123. doi:10.1186 / ar2331).

[0153] The DNA molecules of the present disclosure are DNA molecules that comprise a non-naturally occurring polynucleotide sequence that encodes a polypeptide having the amino acid sequence of at least one of the polypeptides in the antibodies of the present disclosure.

[0154] The polynucleotide of the present disclosure can be expressed in host cells after the sequence is operably linked to expression control sequence.Expression vectors are typically replicable in host organisms, either as episomes or as an integrated part of host chromosomal DNA.Generally, expression vectors contain selectable markers, such as tetracycline, neomycin, and dihydrofolate reductase, to allow detection of cells transformed with desired DNA sequence.

[0155] An expression vector containing a polynucleotide sequence of interest (e.g., a polynucleotide encoding an antibody polypeptide of the present invention and an expression control sequence) can be introduced into a host cell by known methods that vary depending on the type of host cell.

[0156] The antibodies of the present disclosure can be readily produced in mammalian host cells, non-limiting examples of which include CHO, NS0, HEK293, or COS cells. The host cells can be cultured using techniques known in the art.

[0157] Various methods of protein purification may be used to purify the antibodies of the present disclosure, and such methods are known in the art and are described, for example, in Deutscher, Methods in Enzymology 182:83-89 (1990) and Scopes, Protein Purification: Principles and Practice, 3rd Edition, Springer, NY (1994).

[0158] The sequences referenced herein are numbered according to the SEQ ID NOs listed in Tables 1a and 1b. The sequences in Tables 1a and 1b are amino acid sequences unless otherwise indicated.

[0159] [Table 1-1]

[0160] [Table 1-2]

[0161] SEQ ID NOs:15 and 29 are identical except that SEQ ID NO:15 contains a lysine at amino acid residue 205 and SEQ ID NO:29 contains an alanine residue at position 205.

[0162] SEQ ID NOs:19 and 30 are identical except that SEQ ID NO:19 contains a lysine at amino acid residue 205 and SEQ ID NO:30 contains an alanine residue at position 205.

[0163] SEQ ID NOs:21 and 31 are identical except that SEQ ID NO:21 contains a lysine at amino acid residue position 328 and SEQ ID NO:31 contains an alanine residue at position 328.

[0164] SEQ ID NOs:23 and 32 are identical except that SEQ ID NO:23 contains a lysine at amino acid residue 324 and SEQ ID NO:32 contains an alanine residue at position 324.

[0165] SEQ ID NO: 35 (LAG-3_CH_X) is a consensus sequence and contains an X residue at amino acid residue position 205, which corresponds to the lysine residue at position 205 of SEQ ID NO: 15 and the alanine residue at position 205 of SEQ ID NO: 29, respectively.

[0166] SEQ ID NO:36 (PD-1_CH_X) is a consensus sequence and contains an X residue at amino acid residue position 205, which corresponds to the lysine residue at position 205 of SEQ ID NO:29 and the alanine residue at position 205 of SEQ ID NO:30, respectively.

[0167] SEQ ID NO: 37 (LAG-3_HC_X) is a consensus sequence and contains an X residue at amino acid residue position 328, which corresponds to the lysine residue at position 328 of SEQ ID NO: 21 and the alanine residue at position 328 of SEQ ID NO: 31, respectively.

[0168] SEQ ID NO: 38 (PD-1_HC_X) is a consensus sequence and contains an X residue at amino acid residue 324, which corresponds to the lysine residue at position 324 of SEQ ID NO: 23 and the alanine residue at position 324 of SEQ ID NO: 32, respectively. [Example]

[0169] Antibody expression and purification The human LAG-3 agonist antibodies and human LAG-3 / human PD-1 dual agonist bispecific antibodies of the present disclosure can be expressed and purified essentially as follows: Suitable host cells, such as HEK293 or CHO, can be transfected, either transiently or stably, with an expression system for antibody secretion using an optimal, predetermined heavy chain:light chain vector ratio, or with a single vector system encoding both the heavy and light chains. The antibodies or bispecific antibodies of the present disclosure can be transfected, either transiently or stably, with an expression system for antibody secretion using one or more DNA molecules encoding the heavy and light chains (in the case of human LAG-3 agonist antibodies of the present disclosure), or a first heavy chain, a first light chain, a second heavy chain, and a second light chain (in the case of human LAG-3 / human PD-1 dual agonist bispecific antibodies of the present disclosure).

[0170] Antibodies can be purified using one of many commonly used techniques. For example, the medium can be conveniently applied to a MabSelect column (GE Healthcare) or a KappaSelect column (GE Healthcare) equilibrated with a compatible buffer, such as phosphate-buffered saline (pH 7.4). The column can be washed to remove nonspecifically bound components. Bound antibodies can be eluted, for example, by a pH gradient (e.g., from 20 mM Tris buffer (pH 7.0) to 10 mM sodium citrate buffer (pH 3.0), or from phosphate-buffered saline (pH 7.4) to 100 mM glycine buffer (pH 3.0)). Antibody fractions can be detected by UV absorbance or SDS-PAGE, and then pooled. Depending on the intended use, further purification is optional. The purified antibodies can be concentrated and / or sterile filtered using common techniques. Soluble aggregates, multimers, and mismatches can be effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, multimodal, affinity, or hydroxyapatite chromatography. Purified antibodies can be immediately frozen at -70°C or lyophilized.

[0171] Binding to human LAG-3 and human PD-1 The binding kinetics and affinity of LAG-3 antibodies to the soluble extracellular domain (ECD) of LAG-3 and bispecific antibodies to soluble LAG-3-ECD and PD-1-ECD (human: Sino Biologicals, catalog no. 10377-H08H; cynomolgus monkey: Sino Biologicals, catalog no. 90311-C08H) were measured by surface plasmon resonance using a Biacore® T200 (Cytiva, Marlborough, MA). Samples were diluted in HBS-EP+ (10 mM Hepes, 150 mM NaCl, 3 mM EDTA, 0.05% Tween-20, pH 7.6) (Teknova catalog no. H8022) containing 5 g / L BSA (Jackson ImmunoResearch catalog no. 001-000-161) running buffer. PrismA Series S Sensor chips (catalog number 29650263) are purchased from Cytiva.

[0172] Binding was assessed by antibody capture using multicycle kinetics, with each cycle performed at either 37°C or 25°C, with a flow rate of 10 μL / min for antibody capture onto the Prism A chip and 100 μL / min for analyte association (120 s contact time) and dissociation (900 s). Each cycle consisted of the following steps: injection of 1 μg / mL of bispecific antibody into HBS-EP+ targeting Rmax values ​​of 125 RU (LAG-3 arm), 294 RU (PD-1 arm), and 63 RU (LAG-3 antibody) onto the flow cell; injection of analyte into HBS-EP+ (concentration range of 62.5 nM to 0.25 nM by two-fold serial dilution for both human PD-1-ECD-His (SEQ ID NO: 40) and human LAG-3-His (SEQ ID NO: 42)); followed by a 900-second dissociation phase and regeneration with 10 μL of 10 mM glycine hydrochloride, pH 1.5, for a 60-second contact time using a flow rate of 10 μL / min. All analyte concentrations were determined using monomeric molecular weight (MW) values. Association rates (k) of LAG-3-ECD and PD-1-ECD were calculated using the analyte-specific kinetics (K). on ) and dissociation rate (k off) were evaluated using double referencing with 0 nM blank subtraction plus reference subtraction of flow cell 1 and fitted to a "1:1 (Langmuir) binding" model in BIAevaluation software version 4.1. D ) into the relation K D =K off / K on The stoichiometry was calculated from the binding rate according to max / RU 捕捉 ] / [MW 分析物 / MW 抗体 ](in the formula, MW 抗体A is 150 kDa). Values ​​are reported as mean ± standard deviation.

[0173] Experiments performed essentially as described above produced the results in Tables 2 and 3. The results in Table 2a demonstrate that antibody E bound to human and cynomolgus monkey LAG-3-ECD. The results in Table 2b demonstrate that antibody F bound to human LAG-3-ECD. The results in Table 2c demonstrate that antibody G bound to human but not cynomolgus monkey LAG-3-ECD at the concentrations tested. The results in Table 3a demonstrate that antibody A bound to human PD-1-ECD, human LAG-3-ECD, cynomolgus monkey PD-1-ECD, and cynomolgus monkey LAG-3-ECD. The results in Table 3b demonstrate that antibody B bound to human PD-1-ECD, human LAG-3-ECD, cynomolgus monkey PD-1-ECD, and cynomolgus monkey LAG-3-ECD. The results in Table 3c demonstrate that antibody C bound to human PD-1-ECD and human LAG-3-ECD. The results in Table 3d demonstrate that antibody D bound to human PD-1-ECD and human LAG-3-ECD.

[0174] [Table 2-1]

[0175] [Table 2-2] a No binding observed up to 1000 nM.

[0176] [Table 2-3] a No binding observed up to 1000 nM.

[0177] [Table 3-1] b SS: Steady-state equilibrium model. Due to fast on- and off-rates, kinetic parameters cannot be fitted.

[0178] [Table 3-2]

[0179] [Table 3-3]

[0180] [Table 3-4]

[0181] Peripheral mononuclear cell binding assay The ability of the human LAG-3 / PD-1 dual agonist antibodies disclosed herein to bind to cell-surface human or cynomolgus monkey LAG-3 / PD-1 can be measured using a flow cytometry assay.

[0182] Briefly, human peripheral blood mononuclear cells (PBMCs) were isolated from healthy individuals at Trima LRS (San Diego Blood Bank; San Diego, CA) using Ficoll (GE Healthcare #17144002) and activated with 0.5 μg / mL plate-bound anti-CD3 (Biolegend #300302) for 3 days at 37°C in 5% CO in complete RPMI (RPMI, Corning #MT10041CV; 10% FCS, Corning #MT35011CV; 1X Glutamax, Gibco #35050061; 1X P / S, Corning #30002CI; 1X BME, Gibco #21985023; 1X MEM, Gibco #11140050; 1X sodium pyruvate, Corning #25-000-CI).

[0183] Cynomolgus monkey PBMCs were isolated from whole blood (BioIVT#NHP01WBK2-0000861) using 90% Ficoll (Corning#21-031-CM) in PBS and activated in RPMI with 2 μg / mL plate-bound anti-CD3 (BD#551916) for 3 days as described above.

[0184] Cells were incubated for 30 minutes at 4°C with 10-12 point, 3-fold serial dilutions of each labeled antibody (Invitrogen #S20026, Invitrogen #C20029) (starting at 30µg / mL in PBS + 2% FCS containing: CD4 eBioscience #12-0047-42 or Biolegend #344634; CD8 eBioscience #25-0088-42; CD3 eBioscience 56-0038-82 for human cells, BD #557917 for cynomolgus monkey cells; CD33 eBioscience 45-0338-42 for human cells; Live / Dead eBioscience #65-0865-18). After thorough washing, cells were processed on a BD Fortessa or Fortessa X20 cytometer and data were analyzed using Flowjo software (BD The percentage of cells bound by each antibody was quantified and graphed using Prism (Graphpad, San Diego, CA).

[0185] Experiments performed essentially as described above produced the data in Table 4. Antibody A and Antibody B bound to cell membrane-expressed human LAG-3 / PD-1 and cynomolgus monkey LAG-3 / PD-1, as shown in Table 4a (n=3). Antibody C and Antibody D bound to cell membrane-expressed human LAG-3 / PD-1 and cynomolgus monkey LAG-3 / PD-1, as shown in Table 4b (n=3). Antibody E and Antibody F bound to cell membrane-expressed human LAG-3, as shown in Table 4c (n=3).

[0186] [Table 4-1]

[0187] [Table 4-2]

[0188] [Table 4-3]

[0189] PD-1 and LAG-3 dimerization assay Induction of PD-1 and LAG-3 dimerization was measured in a cell-based enzyme fragment complementation assay, in which bringing the two receptors into close proximity induces a measurable luminescent signal in a dose-dependent manner.

[0190] The PathHunter® U2OS PD-1 / LAG-3 Dimerization Assay (Eurofins DiscoveRx) containing the cell line and assay components was utilized. The cell line was the U2OS cell line (Eurofins DiscoveRx #83-0009C3) expressing the extracellular domain of human PD-1 (aa 1-199) fused to the EA (enzyme acceptor) subunit of β-galactosidase and the extracellular domain of human LAG-3 (aa 1-477) fused to the PK1 enzyme donor subunit of β-galactosidase. Cells were maintained in AssayComplete™ Cell Culture Kit-103 medium (#92-3103G) supplemented with 250 μg / ml hygromycin B and 500 μg / ml G418.

[0191] After detachment with Cell Detachment Reagent (#92-0009), cell lines were seeded at 5,000 cells / well in 80 μl Cell Plating 5 Reagent (#93-0563R5A) into 96-well white opaque plates (Costar #35-3296). Cells were incubated overnight at 37°C, 90% RH, and 5% CO2. The next day, compounds (5x the final concentration) were serially diluted 1:5 in protein dilution buffer (#92-0023M) in polypropylene plates. Compounds were added to the cells in 20 μl volumes, in triplicate wells per concentration. (The final compound concentration range was 0.00128–100 nM.) Cells were incubated for 30 minutes at 37°C, 90% RH, and 5% CO2. The plate was cooled to room temperature for 10 minutes, then 100 μl / well of Flash substrate / cell assay buffer mixture (from PathHunter® Flash Detection Kit, #93-0247) was added. The plate was incubated at room temperature in the dark for 1 hour without agitation. The plate was sealed with TopSeals (Perkin Elmer), and then luminescence was read on a Biotek Synergy Neo2 multimode reader (0.1 sec per well, PMT gain 135).

[0192] EC50 values ​​for induction of PD-1 and LAG-3 dimerization were calculated in GraphPad Prism 9 using Log-transformed X values. Nonlinear regression (curve-fitting) analysis (sigmoidal dose-response, variable slope) may be performed on log-transformed data to obtain EC50 values.

[0193] In experiments performed essentially as described in this assay, Antibody A induced PD-1 and LAG-3 dimerization in a dose-dependent manner with a mean EC50 of 2.97 nM + / - 1.14 (SE) (Table 5a), while Antibody B had a mean EC50 of 2.47 nM + / - 1.26 (SE) (Table 5b). The combination of the human LAG-3 agonist arm plus the human PD-1 agonist arm did not induce PD-1 and LAG-3 dimerization in this assay.

[0194] The data in Table 5a also show that Antibody A and Antibody B bind to (and bring into close proximity of) human PD-1 and human LAG-3 in a cell-based system.

[0195] The data in Table 5b also show that Antibody C and Antibody D bind to (and bring into close proximity of) human PD-1 and human LAG-3 in a cell-based system.

[0196] The data shown in Tables 5a and 5b are relative luminescence units from one of two experiments.

[0197] [Table 5-1]

[0198] [Table 5-2]

[0199] Inhibition of T-cell proliferation Human LAG-3 / human PD-1 dual agonist The ability of a bispecific antibody to inhibit T cell proliferation can be measured as follows.

[0200] Briefly, human peripheral blood mononuclear cells (PBMCs) were isolated from healthy individuals at Trima LRS (San Diego Blood Bank, San Diego, CA) using Ficoll (GE Healthcare #17144002). Isolated PBMCs were labeled with proliferation dye (eBioscience #65-0842-90) for 30 minutes at room temperature and then thoroughly washed.

[0201] Labeled PBMCs were treated with 10-12 point, 3-fold serial dilutions of each antibody, starting at a final concentration of 30 μg / mL, in complete RPMI (RPMI, Corning #MT10041CV; 10% FCS, Corning #MT35011CV; 1X Glutamax, Gibco #35050061; 1X P / S, Corning #30002CI; 1X BME, Gibco #21985023; 1X MEM, Gibco #11140050; 1X Sodium Pyruvate, Corning #25-000-CI) for 30 minutes at room temperature. Cells were then stimulated with SEB (Toxin Technologies BT2021M) in complete RPMI at a final concentration of 4 ng / mL for 3 days at 37°C with 5% CO2. Cells were then washed extensively.

[0202] Cells were stained for 30 minutes at 4°C with the following: non-competitive PD-1, Biolegend 329904; CD4, eBioscience #12-0047-42 or Biolegend #344634; CD33, eBioscience #45-0338-42; CD8, eBioscience #25-0088-42; internal non-competitive LAG-3 conjugated to Alexa 647, Invitrogen A20186; CD3, eBioscience 56-0038-82; and Live / Dead, eBioscience #65-0865-18. After thorough washing, cells were processed on a BD Fortessa or Fortessa X20 cytometer, and data were analyzed using Flowjo software (BD Biosciences). The percentage of proliferating cells was quantified and graphed using Prism (Graphpad, San Diego, CA).

[0203] In experiments performed essentially as described above, antibodies A-D each inhibited human primary T cell proliferation, as shown in Tables 6a and 6b. Antibodies E and F also each inhibited primary T cell proliferation, as shown in Table 6c.

[0204] [Table 6-1]

[0205] [Table 6-2]

[0206] [Table 6-3]

[0207] Murine graft-versus-host disease (GVHD) in vivo model To demonstrate the immunomodulatory activity of the PD-1 / LAG-3 bispecific antibody of the present invention, a humanized model of xenogeneic GvHD can be utilized. This model is generated by transplanting human PBMCs into immunodeficient mice. Human immune cells recognize the mouse as foreign and initiate an immune response, resulting in a significant increase in human pro-inflammatory cytokines, immune cell activation, proliferation, and extravasation into tissues, ultimately leading to weight loss and multiple organ dysfunction. Importantly, the inflammatory response is driven by human cells, thus allowing human-specific treatments to be investigated in the model.

[0208] Briefly, female NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ, JAX Labs, stock #05557) were housed three per cage at 72°C under a 12-hour light:dark cycle with free access to food and water (n=76). Human PBMCs were isolated from LRS tubes obtained from blood donors (San Diego Blood Bank) using SepMate 50 Ficoll preparation tubes according to the manufacturer's instructions (STEMCELL Technologies, Vancouver, BC). Freshly isolated PBMCs were diluted to 1.2 x 10 in PBS. 8 Suspend the cells / mL and transplant the mice intravenously with 100 µL PBMC suspension on day 0 (1.2 x 10 7 72 mice receive PBMCs and 4 mice remain as non-engrafted controls.

[0209] One day after engraftment, mice are divided into nine weight-matched groups (n = 8 / group) and administered the bispecific antibody subcutaneously at 0.1, 1.0, or 3.0 mg / kg. Dosing continues weekly for the remainder of the experiment. Medical examinations and weight measurements are performed regularly. Mice that have lost 20% of their starting weight or are clearly distressed are euthanized. Common clinical signs in this model are unkempt hair, a hunched body, weakness, and difficulty breathing or moving.

[0210] When the majority of isotype control mice require euthanasia due to disease progression, all mice are sacrificed. For all sacrificed mice, blood is collected into EDTA tubes by cardiac puncture under isoflurane anesthesia. In addition, an interim blood sample on day 10 is obtained from the retro-orbital sinus. Blood from both collections is clarified by centrifugation for human plasma cytokine analysis. Body weight change is calculated as a percentage of their baseline weight: (day (x) weight / day 0 weight). * Weights are calculated as 100. Mice requiring euthanasia before the end of the study are ranked up to the last weight measurement. Plasma cytokines are measured using Mesoscale Discovery Human Th1 / Th2 10-Vplex (Rockville, Maryland) according to the manufacturer's instructions. Data are graphed and statistics are calculated using Prism Software (GraphPad, San Diego, CA). Differences in weight between groups are determined by 2-way RM-ANOVA with Tukey's post-hoc test. Differences in plasma cytokine levels are determined by 1-way ANOVA with Tukey's post-hoc test. Differences between test groups are considered significant if p<0.05.

[0211] Engraftment of human PBMCs from induced GvHD, as evidenced by significant wasting in NSG mice, required study termination on day 36 post-engraftment. Treatment with a human LAG-3 / human PD-1 dual agonist bispecific antibody significantly attenuated disease progression, as measured by a dose-dependent reduction in weight loss in mice. Furthermore, the human LAG-3 / human PD-1 dual agonist bispecific antibody inhibited the significant increase in plasma human pro-inflammatory cytokines associated with disease progression. Thus, the human LAG-3 / human PD-1 dual agonist bispecific antibody can attenuate human immune cell pathogenicity and reduce disease progression in a humanized GvHD model.

[0212] Amino acid and nucleotide sequences SEQ ID NO: 1 (LAG-3 HCDR1 amino acid sequence) KASGYTFTGYYMH

[0213] SEQ ID NO: 2 (LAG-3 HCDR2 amino acid sequence) WINPNSGDTNYAQKFQG

[0214] SEQ ID NO: 3 (LAG-3 HCDR3 amino acid sequence) AREGDSSGWHGGWFDP

[0215] SEQ ID NO: 4 (LAG-3 LCDR1 amino acid sequence) RSSQSLLDSAEGSTYLD

[0216] SEQ ID NO: 5 (LAG-3 LCDR2 amino acid sequence) YTLSYRAS

[0217] SEQ ID NO: 6 (LAG-3 LCDR3 amino acid sequence) MQRVEFPYT

[0218] SEQ ID NO: 7 (PD-1 HCDR1 amino acid sequence) KVSGYSLSKYDMS

[0219] SEQ ID NO: 8 (PD-1 HCDR2 amino acid sequence) IIYTSGYTDYAQEFQG

[0220] SEQ ID NO: 9 (PD-1 HCDR3 amino acid sequence) ATGNPYYTNGFNS

[0221] SEQ ID NO: 10 (PD-1 LCDR1 amino acid sequence) QASQSPNNLLA

[0222] SEQ ID NO: 11 (PD-1 LCDR2 amino acid sequence) YGASDLPS

[0223] SEQ ID NO: 12 (PD-1 LCDR3 amino acid sequence) QNNYYVGPVSYA

[0224] SEQ ID NO: 13 (LAG-3 variable heavy amino acid sequence) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRYAPGQGLEWMGWINPNSGDTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREGDSSGWHGGWFDPWGRGTLVTVSS

[0225] SEQ ID NO: 14 (LAG-3 variable light amino acid sequence) DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSAEGSTYLDWFLRKPGDSPQLLIYTLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQRVEFPYTFGQGTKVEIK

[0226] SEQ ID NO: 15 (LAG-3 constant heavy amino acid sequence) ASTKGPSVFPLAPCSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPDSGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPRRPRVYTLPPSREEMTKNQVSLVCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSVLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0227] SEQ ID NO: 16 (LAG-3 constant light amino acid sequence) RTVAAPSVFIFPPSKEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0228] SEQ ID NO: 17 (PD-1 variable heavy amino acid sequence) QVQLVQSGAEVKKPGASVKVSCKVSGYSLSKYDMSWVRKAPGKGLEWMGIIYTSGYTDYAQEFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCATGNPYYTNGFNSWGQGTLVTVSS

[0229] SEQ ID NO: 18 (PD-1 variable light amino acid sequence) RIQMTQSPSSLSASVGDRVTITCQASQSPNNLLAWYQDKPGKAPKLLIYGASDLPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNNYYVGPVSYAFGGGTKVEIK

[0230] SEQ ID NO: 19 (PD-1 constant heavy amino acid sequence) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVATGPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTDNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLMSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0231] SEQ ID NO: 20 (PD-1 constant light amino acid sequence) GQPKAAPSVTLFPPSSEELQANKATLVCYISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAAWSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC

[0232] SEQ ID NO: 21 (LAG-3 heavy chain amino acid sequence) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRYAPGQGLEWMGWINPNSGDTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREGDSSGWHGGWFDPWGRGTLVTVSSASTKGPSVFPLAPCSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPDSGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRRPRVYTLPPSREEMTKNQVSLVCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSVLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0233] SEQ ID NO: 22 (LAG-3 light chain amino acids) DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSAEGSTYLDWFLRKPGDSPQLLIYTLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQRVEFPYTFGQGTKVEIKRTVAAPSVFIFPPSKEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0234] SEQ ID NO: 23 (PD-1 heavy chain amino acid sequence) QVQLVQSGAEVKKPGASVKVSCKVSGYSLSKYDMSWVRKAPGKGLEWMGIIYTSGYTDYAQEFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCATGNPYYTNGFNSWGQGTLVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVATGAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTDNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLMSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0235] SEQ ID NO: 24 (PD-1 light chain amino acid sequence) RIQMTQSPSSLSASVGDRVTITCQASQSPNNLLAWYQDKPGKAPKLLIYGASDLPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNNYYVGPVSYAFGGGTKVEIKGQPKAAPSVTLFPPSSEELQANKATLVCYISDFYPGAVTVAWKADSSPVKAGVETTPSKQSNNKYAAWSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC

[0236] SEQ ID NO: 25 (LAG-3 heavy chain DNA sequence)

[0237] SEQ ID NO: 26 (LAG-3 light chain DNA sequence) GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCTTGGATAGTGCTGAAGGAAGCACCTATTTGGACTGGTTCCTGAGAAAGCCAGGGGACTCTCCACAGCTCCTGATCTATACGCTTTCCTACCGGGCCTCTGGAGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGCATGCAGAGGGTAGAATTTCCGTACACATTCGGACAGGGAACAAAGGTGGAAATCAAGCGGACCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTAAGGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC

[0238] SEQ ID NO: 27 (PD-1 heavy chain DNA sequence)

[0239] SEQ ID NO: 28 (PD-1 light chain DNA sequence) AGAATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGCCAGGCCAGTCAGAGCCCTAATAACCTCCTGGCCTGGTATCAGGACAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGTGCATCCGATCTGCCATCTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAGAACAATTATTATGTGGGACCAGTGAGCTATGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAGGGCCAGCCTAAAGCTGCCCCTAGCGTTACCCTTTTCCCACCGAGCTCCGAGGAGCTGCAGGCCAATAAAGCAACCTTGGTCTGCTACATATCAGATTTTTACCCTGGCGCCGTGACCGTAGCATGGAAAGCTGATTCATCCCCTGTGAAGGCCGGTGTTGAAACTACAACCCCTTCCAAACAATCTAACAATAAATACGCGGCATGGTCCTACCTGTCCTTGACACCCGAGCAGTGGAAATCTCACAGATCTTACAGCTGCCAGGTCACCCACGAGGGGAGCACTGTGGAGAAGACCGTCGCGCCCACTGAGTGC

[0240] SEQ ID NO: 29 (LAG-3 constant heavy amino acid sequence of antibody B) ASTKGPSVFPLAPCSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPDSGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGPRRPRVYTLPPSREEMTKNQVSLVCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSVLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0241] SEQ ID NO: 30 (PD-1 constant heavy amino acid sequence of antibody B) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVATGAPVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTDNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLMSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0242] SEQ ID NO: 31 (antibody B LAG-3 heavy chain amino acid sequence) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRYAPGQGLEWMGWINPNSGDTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREGDSGWHGGWFDPWGRGTLVTVSSASTKGPSVFPLAPCSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPDSGDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGPRRPRVYTLPPSREEMTKNQVSLVCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSVLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0243] SEQ ID NO: 32 (PD-1 heavy chain amino acid sequence of antibody B) QVQLVQSGAEVKKPGASVKVSCKVSGYSLSKYDMSWVRKAPGKGLEWMGIIYTSGYTDYAQEFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCATGNPYYTNGFNSWGQGTLVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVATGAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTDNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLMSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0244] SEQ ID NO: 33 (LAG-3 heavy chain DNA sequence of antibody B)

[0245] SEQ ID NO: 34 (PD-1 heavy chain DNA sequence of antibody B)

[0246] SEQ ID NO: 35 (LAG-3_CH_X amino acid sequence) ASTKGPSVFPLAPCSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPDSGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCXVSNKALPAPIEKTISKAKGQPRRPRVYTLPPSREEMTKNQVSLVCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSVLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (X is K or A)

[0247] SEQ ID NO: 36 (PD-1_CH_X amino acid sequence) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVATGPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCXVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTDNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLMSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (X is K or A)

[0248] SEQ ID NO: 37 (LAG-3_HC_X amino acid sequence) QVQLVQSGAEVKKPGASVKVSCKASGYTFGYMHWVRYAPGQGLEWMGWINPNSGDTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYCAREGDSSGWHGGWFDPWGRGTLVTVSSASTKGPSVFPLAPCSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPDSGDKTHTC PPCPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCXVSNKALPAPIEKTISKAGQPRRPRVYTLPPSREEMTKNQVSLVCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSVLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Xははれなるるる

[0249] SEQ ID NO: 38 (PD-1_HC_X amino acid sequence) QVQLVQSGAEVKKPGASVKVSCKVSGYSLSKYDMSWVRKAPGKGLEWMGIIYTSGYTDYAQEFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCATGNPYYTNGFNSWGQGTLVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVATGAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCXVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTDNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLMSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(X is K or A)

[0250] SEQ ID NO: 39 (amino acid sequence of human PD-1) MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

[0251] SEQ ID NO: 40 (human PD-1 ECD-His amino acid sequence) LDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQHHHHHHHH

[0252] SEQ ID NO: 41 (human LAG-3 amino acid sequence) MWEAQFLGLLFLQPLWVAPVKPLQPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSSPGAQRSGRAPGALPAGHLLLFLILGVLSLLLLVTGAFGFHLWRRQWRPRRFSALEQGIHPPQAQSKIEELEQEPEPEPEPEPEPEPEPEPEQL

[0253] SEQ ID NO: 42 (human LAG-3 ECD-His amino acid sequence) LQPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSSPGAQRSGRAPGALPAGHGGGGSHHHHHH

[0254] SEQ ID NO: 43 (Cynomolgus monkey LAG-3 ECD-His amino acid sequence) MWEAQFLGLLFLQPLWVAPVKPPQPGAEISVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAPAPGHPPVPGHRPAAPYSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRATVHLRDRALSCRLRLRVGQASMTASPPGSLRTSDWVILNCSFSRPDRPASVHWFRSRGQGRVPVQGSPHHHLAESFLFLPHVGPMDSGLWGCILTYRDGFNVSIMYNLTVLGLEPATPLTVYAGAGSRVELPCRLPPAVGTQSFLTAKWAPPGGGPDLLVAGDNGDFTLRLEDVSQAQAGTYICHIRLQGQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPASGQEHFVWSPLNTPSQRSFSGPWLEAQEAQLLSQPWQCQLHQGERLLGAAVYFTELSSPGAQRSGRAPGALRAGHAAAHHHHHHSGS

[0255] SEQ ID NO: 44 (LAG-3 HCDR1 amino acid sequence) TVSGGSISSYYWS

[0256] SEQ ID NO: 45 (LAG-3 HCDR2 amino acid sequence) RIFTSGTTNYNPELKS

[0257] SEQ ID NO: 46 (PD1 HCDR2 amino acid sequence) IIYTSGYTDYAQKFQG

[0258] SEQ ID NO: 47 (LAG-3 HCDR3 amino acid sequence) ARYDAFDI

[0259] SEQ ID NO: 48 (LAG-3 LCDR1 amino acid sequence) RASQSVSSSYLA

[0260] SEQ ID NO: 49 (LAG-3 LCDR2 amino acid sequence) YGASSRAT

[0261] SEQ ID NO: 50 (LAG-3 LCDR3 amino acid sequence) QQYGSSPIT

[0262] SEQ ID NO: 51 (LAG-3 HCDR1 amino acid sequence) TVSGGSIHSYYWS

[0263] SEQ ID NO: 52 (LAG-3 HCDR2 amino acid sequence) RIFTSGSTNYNPELKS

[0264] SEQ ID NO: 53 (LAG-3 variable heavy amino acid sequence) QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRKPAGKGLEWVGRIFTSGTTNYNPELKSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARYDAFDIWGQGTLVTVSS

[0265] SEQ ID NO: 54 (LAG-3 variable heavy amino acid sequence) QVQLQESGPGLVKPSETLSLTCTVSGGSIHSYYWSWIRKPAGKGLEWVGRIFTSGSTNYNPELKSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARYDAFDIWGQGTLVTVSS

[0266] SEQ ID NO: 55 (PD-1 variable heavy amino acid sequence) QVQLVQSGAEVKKPGASVKVSCKVSGYSLSKYDMSWVRYAPGKGLEWMGIIYTSGYTDYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCATGNPYYTNGFNSWGRGTLVTVSS

[0267] SEQ ID NO: 56 (LAG-3 variable light amino acid sequence) RIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWFQDKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPITFGQGTKVEIK

[0268] SEQ ID NO: 57 (PD-1 variable light amino acid sequence) DIQMTQSPSSLSASVGDRVTITCQASQSPNNLLAWYQRKPGDAPKLLIYGASDLPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNNYYVGPVSYAFGGGTKVEIK

[0269] SEQ ID NO: 58 (LAG-3 constant heavy amino acid sequence) ASTKGPSVFPLAPSSKSTSGGTAALGCLVADYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDERVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGPREPQVYTLPPSRGDMTKNQVQLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0270] SEQ ID NO: 59 (PD-1 constant heavy amino acid sequence) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVATGAPVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGPREPQVSTLPPSREEMTKNQVSLMCLVYGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSVLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0271] SEQ ID NO: 60 (LAG-3 constant light amino acid sequence) RTVAAPSVFIFPPSDKQLKSGTARVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0272] SEQ ID NO: 61 (LAG-3 heavy chain amino acid sequence) QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRKPAGKGLEWVGRIFTSGTTNYNPELKSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARYDAFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVADYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDERVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGPREPQVYTLPPSRGDMTKNQVQLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0273] SEQ ID NO: 62 (LAG-3 heavy chain amino acid sequence) QVQLQESGPGLVKPSETLSLTCTVSGGSIHSYYWSWIRKPAGKGLEWVGRIFTSGSTNYNPELKSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARYDAFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVADYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDERVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGPREPQVYTLPPSRGDMTKNQVQLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0274] SEQ ID NO: 63 (PD-1 heavy chain amino acid sequence) QVQLVQSGAEVKKPGASVKVSCKVSGYSLSKYDMSWVRYAPGKGLEWMGIIYTSGYTDYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCATGNPYYTNGFNSWGRGTLVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVATGAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGPREPQVSTLPPSREEMTKNQVSLMCLVYGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSVLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0275] SEQ ID NO: 64 (LAG-3 light chain amino acids) RIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWFQDKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPITFGQGTKVEIKRTVAAPSVFIFPPSDKQLKSGTARVVCLLNNFYPREAKVQWKVDNALQGSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0276] SEQ ID NO: 65 (PD-1 light chain amino acid sequence) DIQMTQSPSSLSASVGDRVTITCQASQSPNNLLAWYQRKPGDAPKLLIYGASDLPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNNYYVGPVSYAFGGGTKVEIKGQPKAAPSVTLFPPSSEELQANKATLVCYISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAAWSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC

[0277] SEQ ID NO: 66 (LAG-3 heavy chain DNA sequence)

[0278] SEQ ID NO: 67 (LAG-3 heavy chain DNA sequence)

[0279] SEQ ID NO: 68 (PD-1 heavy chain DNA sequence)

[0280] SEQ ID NO: 69 (LAG-3 light chain DNA sequence) Agaattgtgttgacgcagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagcagctacttagcctggttccaggacaaacctggccaggctcccaggctcctcatctatggtgcatccagcagggccactggcatcccagacaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagactggagcctgaagattttgcagtgtattactgtcagcagtacggaagcagtccgataacgttcggccagggaacaaaggtggaaataaagcggaccgtggctgcaccatctgtcttcatcttcccgccatctgataagcagttgaaatctggaactgccagagttgtgtgcctgctgaataacttctatcccagagaggccaaagtaccynoagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgc

[0281] SEQ ID NO: 70 (PD-1 light chain DNA sequence) gacatccagatgacccagtctccatcctccctgtctgcatctgtgggagacagagtcaccatcacttgccaggccagtcagagccctaataacctcctggcctggtatcagAGAaaaccagggGACgcccctaagctcctgatctatggtgcatccgatctgccatctggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcagaacaattattatgtgggaccagtgagctatgctttcggcggagggaccaaggtggagatcaagggccagcctaaagctgcccctagcgttacccttttcccaccgagctccgaggagctgcaggccaataaagcaaccttggtctgctacatatcagatttttaccctggcgccgtgaccgtagcatggaaagctgattcatcccctgtgaaggccggtgttgaaactacaaccccttccaaacaatctaacaataaatacgcggcatggtcctacctgtccttgacacccgagcagtggaaatctcacagatcttacagctgccaggtcacccacgaggggagcactgtggagaagaccgtcgcgcccactgagtgc

Claims

1. 1. An antibody comprising a human LAG-3 binding domain comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprises heavy chain complementarity determining regions (HCDRs): HCDR1 comprising SEQ ID NO:1, HCDR2 comprising SEQ ID NO:2, and HCDR3 comprising SEQ ID NO:3; An antibody wherein the VL comprises light chain complementarity determining regions (LCDRs): LCDR1 comprising SEQ ID NO:4, LCDR2 comprising SEQ ID NO:5, and LCDR3 comprising SEQ ID NO:

6.

2. The antibody of claim 1, wherein the VH comprises SEQ ID NO: 13 and the VL comprises SEQ ID NO:

14.

3. 3. The antibody of claim 1 or claim 2, comprising a light chain (LC) comprising SEQ ID NO: 22 and a heavy chain (HC) comprising SEQ ID NO:

37.

4. The antibody of claim 3, wherein X at position 328 of SEQ ID NO: 37 is a lysine residue.

5. The antibody of claim 3, wherein X at position 328 of SEQ ID NO: 37 is an alanine residue.

6. The antibody of claim 3, comprising (a) an LC comprising SEQ ID NO: 22, and (b) an HC comprising SEQ ID NO:

21.

7. The antibody of claim 3, comprising (a) an LC comprising SEQ ID NO: 22, and (b) an HC comprising SEQ ID NO:

31.

8. The antibody of any one of claims 1 to 7, which is of the human IgG1 or IgG4 isotype.

9. The antibody of claim 8, which is of the human IgG1 isotype.

10. The antibody of claim 1 or claim 2, wherein the antigen-binding domain is a single-chain variable fragment (scFv).

11. A nucleic acid comprising a sequence encoding one or both of SEQ ID NO:22 and SEQ ID NO:

37.

12. 12. The nucleic acid of claim 11, wherein X at position 328 of SEQ ID NO: 37 is a lysine residue.

13. 12. The nucleic acid of claim 11, wherein X at position 328 of SEQ ID NO: 37 is an alanine residue.

14. A vector comprising the nucleic acid of claim 11.

15. A composition comprising a first vector comprising a nucleic acid sequence encoding SEQ ID NO:22 and a second vector comprising a nucleic acid sequence encoding SEQ ID NO:

37.

16. A cell comprising the vector of claim 14.

17. A cell comprising the composition of claim 15.

18. 18. The cell of claim 16 or 17, which is a mammalian cell.

19. 19. A method of producing an antibody, comprising culturing a cell of any one of claims 16 to 18 under conditions in which the antibody is expressed, and then recovering the expressed antibody from the culture medium.

20. An antibody produced by culturing the cell of any one of claims 16 to 18 under conditions in which the antibody is expressed, and then recovering the expressed antibody from the culture medium.

21. A composition comprising an antibody according to any one of claims 1 to 10 and 20.

22. A human LAG-3 / human PD-1 dual agonist bispecific antibody that binds to both human LAG-3 and human PD-1.

23. 23. The human LAG-3 / human PD-1 dual agonist bispecific antibody of claim 22, wherein the bispecific antibody comprises: (a) a first antigen-binding domain that binds to human LAG-3, comprising a first heavy chain variable region (VH1) and a first light chain variable region (VL1), the VH1 comprises an HCDR1 comprising SEQ ID NO:1, an HCDR2 comprising SEQ ID NO:2, and an HCDR3 comprising SEQ ID NO:3; a first antigen-binding domain, wherein the VL1 comprises an LCDR1 comprising SEQ ID NO:4, an LCDR2 comprising SEQ ID NO:5, and an LCDR3 comprising SEQ ID NO:6; and (b) a second antigen-binding domain that binds to human PD-1, comprising a second heavy chain variable region (VH2) and a second light chain variable region (VL2), the VH2 comprises an HCDR1 comprising SEQ ID NO:7, an HCDR2 comprising SEQ ID NO:8, and an HCDR3 comprising SEQ ID NO:9; a second antigen-binding domain, wherein the VL2 comprises an LCDR1 comprising SEQ ID NO: 10, an LCDR2 comprising SEQ ID NO: 11, and an LCDR3 comprising SEQ ID NO: 12; A human LAG-3 / human PD-1 dual agonist bispecific antibody comprising:

24. 24. The human LAG-3 / human PD-1 dual agonist bispecific antibody of claim 23, wherein the VH1 comprises SEQ ID NO: 13 and the VL1 comprises SEQ ID NO:

14.

25. The human LAG-3 / human PD-1 dual agonist bispecific antibody of claim 23 or 24, wherein the VH2 comprises SEQ ID NO: 17 and the VL2 comprises SEQ ID NO:

18.

26. 25. The human LAG-3 / human PD-1 dual agonist bispecific antibody of any one of claims 22 to 24, wherein the antibody comprises two heavy chains and two light chains; the first heavy chain (HC1) comprises the first VH1 and a first heavy chain constant region; the first light chain (LC1) comprises the VL1 and a first light chain constant region; the second heavy chain (HC2) comprises the second VH2 and a second heavy chain constant region; A human LAG-3 / human PD-1 dual agonist bispecific antibody, wherein the second light chain (LC2) comprises the second VL2 and a second light chain constant region.

27. 27. The human LAG-3 / human PD-1 dual agonist bispecific antibody of claim 26, said HC1 comprising SEQ ID NO: 37; the LC1 comprises SEQ ID NO: 22; the HC2 comprises SEQ ID NO: 38, and A human LAG-3 / human PD-1 dual agonist bispecific antibody, wherein the LC2 comprises SEQ ID NO:

24.

28. The human LAG-3 / human PD-1 dual agonist bispecific antibody of claim 27, wherein X at position 328 of SEQ ID NO: 37 is lysine and X at position 324 of SEQ ID NO: 38 is lysine.

29. The human LAG-3 / human PD-1 dual agonist bispecific antibody of claim 27, wherein X at position 328 of SEQ ID NO: 37 is alanine, and X at position 324 of SEQ ID NO: 38 is alanine.

30. 28. The human LAG-3 / human PD-1 dual agonist bispecific antibody of claim 27, comprising a first HC1 comprising SEQ ID NO:21, a first LC1 comprising SEQ ID NO:22, a second HC1 comprising SEQ ID NO:23, and a second LC1 comprising SEQ ID NO:

24.

31. 28. The human LAG-3 / human PD-1 dual agonist bispecific antibody of claim 27, comprising a first HC1 comprising SEQ ID NO: 31, a first LC1 comprising SEQ ID NO: 22, a second HC1 comprising SEQ ID NO: 32, and a second LC1 comprising SEQ ID NO:

24.

32. 24. The human LAG-3 / human PD-1 dual agonist bispecific antibody of claim 23, wherein the first antigen-binding domain is an scFv and the second antigen-binding domain is an scFv.

33. A nucleic acid comprising a sequence encoding one or more of SEQ ID NOs:22, 24, 37, and 38, wherein if amino acid residue 328 of SEQ ID NO:37 is a lysine residue, then the X at position 324 of SEQ ID NO:38 is a lysine residue, and if X at position 328 of SEQ ID NO:37 is an alanine residue, then the X at position 324 of SEQ ID NO:38 is an alanine residue.

34. 1. A cell comprising a nucleic acid comprising a sequence encoding one or more of the polypeptides of SEQ ID NOs: 22, 24, 37 and 38, wherein if X at position 328 of SEQ ID NO: 37 is a lysine residue, then X at position 324 of SEQ ID NO: 38 is a lysine residue, and if X at position 328 of SEQ ID NO: 37 is an alanine residue, then X at position 324 of SEQ ID NO: 38 is an alanine residue, and wherein said cell is capable of expressing said one or more polypeptides.

35. 35. The cell of claim 34, which is a mammalian cell.

36. 36. A method for producing a human LAG-3 / human PD-1 dual agonist antibody, comprising culturing the cell of claim 34 or claim 35 under conditions in which the antibody is expressed, and then recovering the expressed antibody from the culture medium.

37. A human LAG-3 / human PD-1 dual agonist antibody produced by culturing the cell of claim 34 or claim 35 under conditions in which the antibody is expressed, and then recovering the expressed antibody from the culture medium.

38. A pharmaceutical composition comprising the human LAG-3 / human PD-1 dual agonist antibody of any one of claims 22 to 32, and a pharmaceutically acceptable excipient, diluent, or carrier.

39. 33. A method of treating an autoimmune disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the human LAG-3 / human PD-1 dual agonist bispecific antibody of any one of claims 22 to 32.

40. 40. The method of claim 39, wherein the autoimmune disease is rheumatoid arthritis, ulcerative colitis, type 1 diabetes, or systemic lupus erythematosus.

41. The human LAG-3 / human PD-1 dual agonist bispecific antibody of any one of claims 22 to 32 for use in therapy.

42. The human LAG-3 / human PD-1 dual agonist bispecific antibody of any one of claims 22 to 32 for use in the treatment of an autoimmune disease.

43. 43. The use of claim 42, wherein the autoimmune disease is rheumatoid arthritis, ulcerative colitis, type 1 diabetes, or systemic lupus erythematosus.

44. 33. A pharmaceutical composition comprising the human LAG-3 / human PD-1 dual agonist bispecific antibody of any one of claims 22 to 32 for use in the treatment of rheumatoid arthritis, ulcerative colitis, type 1 diabetes, or systemic lupus erythematosus.

45. Use of the human LAG-3 / human PD-1 dual agonist bispecific antibody of any one of claims 23 to 32 in the manufacture of a medicament for treating rheumatoid arthritis, ulcerative colitis, type 1 diabetes, or systemic lupus erythematosus.

Images