CD40 x CD40 bispecific antigen-binding molecule and its use

Abstract

This disclosure provides antigen-binding molecules that bind to CD40, such as bispecific antigen-binding molecules that bind to two different epitopes of CD40. In some embodiments, the antigen-binding molecules inhibit CD40L-induced activation and significantly impair CD40 in the absence of CD40L. Methods of using antigen-binding molecules to treat CD40-mediated diseases are also provided. In one embodiment, this disclosure provides antigen-binding molecules that bind to CD40, such as monospecific antibodies, bispecific antibodies, or multispecific antibodies. In some embodiments, the antigen-binding molecule is a bispecific antigen-binding molecule, such as a bispecific antibody.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 521,408, filed on 16 June 2023, the entire contents of said Provisional Patent Application are incorporated herein by reference.

[0002] Sequence listing reference This application includes a sequence listing submitted electronically in XML format, which is incorporated herein by reference in its entirety. The above XML copy, created on 13 June 2024, is named 118003-50020.XML and has a size of 99,202 bytes.

[0003] This disclosure relates to an antigen-binding molecule that binds to CD40 and inhibits CD40L-induced activation, and to a method of using the same for treating, for example, autoimmune diseases. [Background technology]

[0004] CD40 is a cell surface receptor that is part of the tumor necrosis factor (TNF) receptor superfamily. CD40 is expressed on antigen-presenting cells such as B cells, macrophages, and dendritic cells, as well as on some non-immune cells and tumors (Dakal et al, Immunobiology 2020, 225:151899). The interaction between CD40 and its ligand CD40L provides costimulatory signaling, which is essential for the survival of many cell types and is required for immune response functions such as germinal center formation, antibody responses to T-dependent antigens, and the "licensing" of dendritic cells to mature and become capable of inducing T cell activation and differentiation (see, e.g., Kawabe et al., Immunity 1994, 1:167-178, Elgueta et al., Immunol. Rev. 2009, 229:152-172).

[0005] CD40-CD40L signaling is thought to be involved in autoimmune states primarily caused by autoantibodies, such as systemic rheumatic diseases in which autoantibodies play a crucial role in disease progression (e.g., multiple sclerosis, autoimmune nephritis, rheumatoid arthritis, Sjögren's syndrome, and systemic lupus erythematosus), as well as non-rheumatic diseases containing autoantibody components (e.g., myasthenia gravis, Graves' disease, and neuromyelitis optica) (see, e.g., Karnell et al., Adv Drug Delivery Rev. 2019, 141:92-103). Furthermore, since CD40-CD40L signaling is essential for the activation of antigen-presenting cells, blocking antigen presentation by dendritic cells or B cells may affect the CD8+ T cell response in some diseases, such as multiple sclerosis (see, e.g., Denic et al., Expert Opin Ther Targets 2013, 17:1053-1066). Alterations in CD40-CD40L signaling are thought to be involved in cardiovascular disease and other diseases and conditions such as transplantation (see, for example, Dakal et al, Immunobiology 2020, 225:151899, Pamukcu et al., Ann. Med. 2011, 43:331;340, Pinelli et al., Immunotherapy 2015, 7:399-410). Early clinical trials using monoclonal antibodies targeting CD40L provided biological validation for inhibiting the CD40-CD40L signaling pathway, but also revealed unexpected safety concerns due to thromboembolic complications (see, e.g., Karnell et al., Adv Drug Delivery Rev. 2019, 141:92-103). Several monoclonal antibodies targeting CD40 are currently in clinical development. No serious safety events have been reported with these antibodies, however, CD40 antibodies failed to meet their primary endpoints in several Phase 2a trials (Kumar et al., Biopharm. Drug Dispos. 2018, 39:245-255, Visvanathan et al., Ann Rheum Dis. 2019, 78:754-760). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Dakal et al, Immunobiology 2020,225:151899 [Non-Patent Document 2] Kawabe et al.,Immunity 1994,1:167-178 [Non-Patent Document 3] Elgueta et al.,Immunol.Rev.2009,229:152-172 [Non-Patent Document 4] Karnell et al.,Adv Drug Delivery Rev.2019,141:92-103 [Overview of the project] [Means for solving the problem]

[0007] In one embodiment, the disclosure provides an antigen-binding molecule that binds to CD40, such as a monospecific antibody, a bispecific antibody, or a multispecific antibody. In some embodiments, the antigen-binding molecule is a bispecific antigen-binding molecule, such as a bispecific antibody. In some embodiments, the bispecific antigen-binding molecule is (a) A first antigen-binding domain (D1) that binds to the first epitope of human CD40, (b) comprising a second antigen-binding domain (D2) that binds to a second epitope of human CD40.

[0008] In some embodiments, the bispecific antigen-binding molecule comprises a D1 domain and a D2 domain that do not compete with each other for binding to human CD40.

[0009] In some embodiments, the bispecific antigen-binding molecule exhibits one or more of the following properties: (i) When measured by surface plasmon resonance at 25°C, K is less than 25 nM. DIt binds to human CD40. (ii) When measured by surface plasmon resonance at 37°C, K is less than 70 nM. D It binds to human CD40. (iii) When measured by surface plasmon resonance at 25°C, the dissociation half-life (t) exceeds 75 minutes. 1 / 2 ) binds to human CD40, (iv) EC of approximately 10 nM or less 50 It binds to human CD40-expressing cells based on its value. (v) Inhibit the binding of human CD40 monomer to CD40L, (vi) inhibit CD40 ligand (CD40L)-induced activation, and / or (vii) In the absence of CD40L, do not significantly operate CD40.

[0010] In some embodiments, the bispecific antigen-binding molecule inhibits CD40L-induced activation. In some embodiments, the bispecific antigen-binding molecule inhibits CD40L-induced activation and significantly deactivates CD40 in the absence of CD40L.

[0011] In some embodiments, the bispecific antigen-binding molecule comprises a D1 domain and a D2 domain, each of which comprises a heavy chain immunoglobulin variable region containing a set of three heavy chain complementarity-determining region sequences, HCDR1, HCDR2, and HCDR3, selected from the group consisting of the following: (a) HCDR1 containing the amino acid sequence of SEQ ID NO: 4, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 8 (b) HCDR1 containing the amino acid sequence of SEQ ID NO: 24, HCDR2 containing the amino acid sequence of SEQ ID NO: 26, and HCDR3 containing the amino acid sequence of SEQ ID NO: 28, (c) HCDR1 containing the amino acid sequence of SEQ ID NO: 34, HCDR2 containing the amino acid sequence of SEQ ID NO: 36, and HCDR3 containing the amino acid sequence of SEQ ID NO: 38.

[0012] In some embodiments, the D1 domain and the D2 domain each include a light chain immunoglobulin variable region comprising a set of three light chain complementarity determining region sequences LCDR1, LCDR2, and LCDR3, where LCDR1 comprises the amino acid sequence of SEQ ID NO: 12, LCDR2 comprises the amino acid sequence AAS, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the D1 domain includes a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 10.

[0013] In some embodiments, the D1 domain includes the following: (a) HCDR1 containing the amino acid sequence of SEQ ID NO: 4, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, HCDR3 containing the amino acid sequence of SEQ ID NO: 8, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence of SEQ ID NO: 16, or (b) HCDR1 containing the amino acid sequence of SEQ ID NO: 24, HCDR2 containing the amino acid sequence of SEQ ID NO: 26, HCDR3 containing the amino acid sequence of SEQ ID NO: 28, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence of SEQ ID NO: 16.

[0014] In some embodiments, the D1 domain includes HCDR1 containing the amino acid sequence of SEQ ID NO: 4, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, HCDR3 containing the amino acid sequence of SEQ ID NO: 8, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, the D1 domain includes a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO: 2.

[0015] In some embodiments, the D1 domain includes HCDR1 containing the amino acid sequence of SEQ ID NO: 24, HCDR2 containing the amino acid sequence of SEQ ID NO: 26, HCDR3 containing the amino acid sequence of SEQ ID NO: 28, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, the D1 domain includes HCVR containing the amino acid sequence of SEQ ID NO: 22.

[0016] In some embodiments, the D2 domain includes HCDR1 containing the amino acid sequence of SEQ ID NO: 34, HCDR2 containing the amino acid sequence of SEQ ID NO: 36, HCDR3 containing the amino acid sequence of SEQ ID NO: 38, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, the D2 domain includes HCVR containing the amino acid sequence of SEQ ID NO: 32. In some embodiments, the D2 domain includes LCVR containing the amino acid sequence of SEQ ID NO: 10.

[0017] In some embodiments, the bispecific antigen-binding molecule includes the following: HCDR1 containing the amino acid sequence of SEQ ID NO: 4, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, HCDR3 containing the amino acid sequence of SEQ ID NO: 8, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and D1 containing LCDR3 containing the amino acid sequence of SEQ ID NO: 16, and HCDR1 containing the amino acid sequence of SEQ ID NO: 34, HCDR2 containing the amino acid sequence of SEQ ID NO: 36, HCDR3 containing the amino acid sequence of SEQ ID NO: 38, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and D2 containing LCDR3 containing the amino acid sequence of SEQ ID NO: 16.

[0018] In some embodiments, D1 includes an HCVR containing the amino acid sequence of SEQ ID NO: 2 and an LCVR containing the amino acid sequence of SEQ ID NO: 10, and D2 includes an HCVR containing the amino acid sequence of SEQ ID NO: 32 and an LCVR containing the amino acid sequence of SEQ ID NO: 10.

[0019] In some embodiments, the bispecific antigen-binding molecule includes the following: HCDR1 containing the amino acid sequence of SEQ ID NO: 24, HCDR2 containing the amino acid sequence of SEQ ID NO: 26, HCDR3 containing the amino acid sequence of SEQ ID NO: 28, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and D1 containing LCDR3 containing the amino acid sequence of SEQ ID NO: 16, and It includes HCDR1 containing the amino acid sequence of SEQ ID NO: 34, HCDR2 containing the amino acid sequence of SEQ ID NO: 36, HCDR3 containing the amino acid sequence of SEQ ID NO: 38, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and D2 containing LCDR3 containing the amino acid sequence of SEQ ID NO: 16.

[0020] In some embodiments, D1 includes an HCVR containing the amino acid sequence of SEQ ID NO: 22 and an LCVR containing the amino acid sequence of SEQ ID NO: 10, and D2 includes an HCVR containing the amino acid sequence of SEQ ID NO: 32 and an LCVR containing the amino acid sequence of SEQ ID NO: 10.

[0021] In some embodiments, the bispecific antigen-binding molecule includes a D1 domain comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 34, HCDR2 containing the amino acid sequence of SEQ ID NO: 36, HCDR3 containing the amino acid sequence of SEQ ID NO: 38, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, the D1 domain includes HCVR containing the amino acid sequence of SEQ ID NO: 32. In some embodiments, the D1 domain includes LCVR containing the amino acid sequence of SEQ ID NO: 10.

[0022] In some embodiments, the bispecific antigen-binding molecule includes a D2 domain comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 4, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, HCDR3 containing the amino acid sequence of SEQ ID NO: 8, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, the D2 domain includes HCVR containing the amino acid sequence of SEQ ID NO: 2. In some embodiments, the D2 domain includes LCVR containing the amino acid sequence of SEQ ID NO: 10.

[0023] In some embodiments, the bispecific antigen-binding molecule includes the following: HCDR1 containing the amino acid sequence of SEQ ID NO: 34, HCDR2 containing the amino acid sequence of SEQ ID NO: 36, HCDR3 containing the amino acid sequence of SEQ ID NO: 38, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and D1 containing LCDR3 containing the amino acid sequence of SEQ ID NO: 16, and HCDR1 containing the amino acid sequence of SEQ ID NO: 4, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, HCDR3 containing the amino acid sequence of SEQ ID NO: 8, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and D2 containing LCDR3 containing the amino acid sequence of SEQ ID NO: 16.

[0024] In some embodiments, D1 includes an HCVR containing the amino acid sequence of SEQ ID NO: 32 and an LCVR containing the amino acid sequence of SEQ ID NO: 10, and D2 includes an HCVR containing the amino acid sequence of SEQ ID NO: 2 and an LCVR containing the amino acid sequence of SEQ ID NO: 10.

[0025] In some embodiments, the bispecific antigen-binding molecule is a bispecific antibody. In some embodiments, the bispecific antibody comprises a human IgG heavy chain constant region. In some embodiments, the human IgG heavy chain constant region is isotype IgG4 or IgG1. In some embodiments, the human IgG heavy chain constant region comprises one or more modifications that reduce binding to the Fc receptor (e.g., one or more modifications in the hinge region and / or CH region).

[0026] In some embodiments, the bispecific antigen-binding molecule comprises D1, which includes the following: A heavy chain containing the amino acid sequence of SEQ ID NO: 42 and a light chain containing the amino acid sequence of SEQ ID NO: 20, or A heavy chain containing the amino acid sequence of SEQ ID NO: 46 and a light chain containing the amino acid sequence of SEQ ID NO: 20, or A heavy chain containing the amino acid sequence of SEQ ID NO: 48 and a light chain containing the amino acid sequence of SEQ ID NO: 20, or A heavy chain containing the amino acid sequence of SEQ ID NO: 52 and a light chain containing the amino acid sequence of SEQ ID NO: 20, or A heavy chain containing the amino acid sequence of SEQ ID NO: 58 and a light chain containing the amino acid sequence of SEQ ID NO: 20.

[0027] In some embodiments, the bispecific antigen-binding molecule includes D2, which comprises the following: A heavy chain containing the amino acid sequence of SEQ ID NO: 44 and a light chain containing the amino acid sequence of SEQ ID NO: 20, or A heavy chain containing the amino acid sequence of SEQ ID NO: 50 and a light chain containing the amino acid sequence of SEQ ID NO: 20, or A heavy chain containing the amino acid sequence of SEQ ID NO: 60 and a light chain containing the amino acid sequence of SEQ ID NO: 20.

[0028] In some embodiments, the bispecific antigen-binding molecule is a bispecific antibody comprising (i) D1 comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 42 and a light chain containing the amino acid sequence of SEQ ID NO: 20, and (ii) D2 comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 44 and a light chain containing the amino acid sequence of SEQ ID NO: 20.

[0029] In some embodiments, the bispecific antigen-binding molecule is a bispecific antibody comprising (i) D1 comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 46 and a light chain containing the amino acid sequence of SEQ ID NO: 20, and (ii) D2 comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 44 and a light chain containing the amino acid sequence of SEQ ID NO: 20.

[0030] In some embodiments, the bispecific antigen-binding molecule is a bispecific antibody comprising (i) D1 comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 48 and a light chain containing the amino acid sequence of SEQ ID NO: 20, and (ii) D2 comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 50 and a light chain containing the amino acid sequence of SEQ ID NO: 20.

[0031] In some embodiments, the bispecific antigen-binding molecule is a bispecific antibody comprising (i) D1, which comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 52 and a light chain containing the amino acid sequence of SEQ ID NO: 20, and (ii) D2, which comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 50 and a light chain containing the amino acid sequence of SEQ ID NO: 20.

[0032] In some embodiments, the bispecific antigen-binding molecule is a bispecific antibody comprising (i) D1, which comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 58 and a light chain containing the amino acid sequence of SEQ ID NO: 20, and (ii) D2, which comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 60 and a light chain containing the amino acid sequence of SEQ ID NO: 20.

[0033] In some embodiments, the bispecific antigen-binding molecule is a bispecific antibody having the amino acid sequence REGN16334. In some embodiments, the bispecific antigen-binding molecule is a bispecific antibody having the amino acid sequence REGN16335. In some embodiments, the bispecific antigen-binding molecule is a bispecific antibody having the amino acid sequence REGN16431. In some embodiments, the bispecific antigen-binding molecule is a bispecific antibody having the amino acid sequence REGN16432. In some embodiments, the bispecific antigen-binding molecule is a bispecific antibody having the amino acid sequence REGN20484.

[0034] In another embodiment, a pharmaceutical composition is provided comprising an antigen-binding molecule that binds to CD40. In some embodiments, the pharmaceutical composition comprises a bispecific antigen-binding molecule disclosed herein and a pharmaceutically acceptable carrier.

[0035] In another embodiment, the disclosure provides a nucleic acid molecule comprising a nucleotide sequence encoding an antigen-binding molecule that binds to CD40 (e.g., a single-specific or bispecific antigen-binding molecule disclosed herein). In some embodiments, the nucleic acid molecule comprises one or more nucleotide sequences encoding a bispecific antigen-binding molecule disclosed herein. In some embodiments, the nucleic acid molecule comprises one or more nucleotide sequences listed in Table 36.

[0036] In another embodiment, the disclosure provides an expression vector and / or host cell comprising one or more nucleic acid molecules containing nucleotide sequences encoding an antigen-binding molecule that binds to CD40 (e.g., monospecific or bispecific antigen-binding molecules disclosed herein). In some embodiments, the expression vector and / or host cell comprises nucleic acid molecules containing one or more nucleotide sequences listed in Table 36.

[0037] In another embodiment, the Disclosure provides a method for producing antigen-binding molecules that bind to CD40 (e.g., monospecific or bispecific antigen-binding molecules disclosed herein). In some embodiments, the method includes culturing host cells disclosed herein under conditions that enable the production of antigen-binding molecules and recovering the antigen-binding molecules thus produced.

[0038] In another embodiment, the present disclosure provides a method for inhibiting CD40L-induced signaling. In some embodiments, the method involves contacting cells expressing CD40 with an antigen-binding molecule or pharmaceutical composition disclosed herein (e.g., a bispecific antigen-binding molecule containing a sequence described in any of Tables 1-4 or 36, or a pharmaceutical composition containing a bispecific antigen-binding molecule containing a sequence described in any of Tables 1-4 or 36).

[0039] In another embodiment, the Disclosure provides a method for treating, improving, or preventing a CD40-mediated disease or condition in a subject. In some embodiments, the method involves administering a therapeutically effective amount of an antigen-binding molecule or pharmaceutical composition disclosed herein (e.g., a bispecific antigen-binding molecule containing a sequence described in any of Tables 1-4 or 36, or a pharmaceutical composition containing a bispecific antigen-binding molecule containing a sequence described in any of Tables 1-4 or 36) to a subject. In some embodiments, the CD40-mediated disease or condition is an autoimmune disease or condition, an inflammatory disease or condition, a cardiovascular disease or condition, or an organ transplant. In some embodiments, CD40-mediated diseases or conditions include autoimmune thyroid disease, autoimmune hemolytic anemia, Crohn's disease, diabetes mellitus, experimental autoimmune encephalomyelitis (EAE), focal segmental glomerulosclerosis (FSGS), glomerulonephritis, graft-versus-host disease (GVHD), hidradenitis suppurativa (HS), immune thrombocytopenia, inflammatory bowel disease, inflammatory neuropathy (e.g., acute inflammatory demyelinating polyneuropathy (AIDP) or chronic inflammatory demyelinating polyneuropathy). These include disorders (CIDP), Kawasaki disease, lupus nephritis, mixed connective tissue disease (MCTD), multiple sclerosis, myasthenia gravis, neuromyelitis optica spectrum disorder, organ transplantation, pemphigus, primary biliary cirrhosis, psoriasis, psoriatic arthritis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Sjögren's syndrome, skin grafting, systemic lupus erythematosus, systemic sclerosis, transplant rejection, vasculitis, ANCA-associated vasculitis, ulcerative colitis, or Wegener's granulomatosis. In some embodiments, the method further comprises administering a second therapeutic agent to the target.

[0040] In another embodiment, the Disclosure provides an antigen-binding molecule (e.g., a bispecific antigen-binding molecule) or a pharmaceutical composition comprising such antigen-binding molecule for use in inhibiting CD40L-induced signaling. In some embodiments, the antigen-binding molecule is a bispecific antigen-binding molecule (e.g., a bispecific antibody) comprising a sequence described in any of Tables 1-4 or 36.

[0041] In another embodiment, the Disclosure provides an antigen-binding molecule (e.g., a bispecific antigen-binding molecule) or a pharmaceutical composition comprising such antigen-binding molecule for use in the treatment, improvement, or prevention of CD40-mediated diseases or conditions in a subject. In some embodiments, the antigen-binding molecule is a bispecific antigen-binding molecule (e.g., a bispecific antibody) comprising a sequence described in any of Tables 1-4 or 36. In some embodiments, the CD40-mediated disease or condition is an autoimmune disease or condition, an inflammatory disease or condition, a cardiovascular disease or condition, or an organ transplant. In some embodiments, CD40-mediated diseases or conditions include autoimmune thyroid disease, autoimmune hemolytic anemia, Crohn's disease, diabetes mellitus, experimental autoimmune encephalomyelitis (EAE), focal segmental glomerulosclerosis (FSGS), glomerulonephritis, graft-versus-host disease (GVHD), hidradenitis suppurativa (HS), immune thrombocytopenia, inflammatory bowel disease, inflammatory neuropathy (e.g., acute inflammatory demyelinating polyneuropathy (AIDP) or chronic inflammatory demyelinating polyneuropathy). These include disorders (CIDP), Kawasaki disease, lupus nephritis, mixed connective tissue disease (MCTD), multiple sclerosis, myasthenia gravis, neuromyelitis optica spectrum disorder, organ transplantation, pemphigus, primary biliary cirrhosis, psoriasis, psoriatic arthritis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Sjögren's syndrome, skin grafting, systemic lupus erythematosus, systemic scleroderma, transplant rejection, vasculitis, ANCA-associated vasculitis, ulcerative colitis, or Wegener's granulomatosis.

[0042] In another embodiment, the Disclosure provides antigen-binding molecules (e.g., bispecific antigen-binding molecules) disclosed herein for use in the manufacture of pharmaceuticals for treating, improving, or preventing CD40-mediated diseases or conditions in a subject. In some embodiments, the antigen-binding molecule is a bispecific antigen-binding molecule (e.g., a bispecific antibody) comprising a sequence described in any of Tables 1-4 or 36. In some embodiments, the CD40-mediated disease or condition is an autoimmune disease or condition, an inflammatory disease or condition, a cardiovascular disease or condition, or an organ transplant. In some embodiments, CD40-mediated diseases or conditions include autoimmune thyroid disease, autoimmune hemolytic anemia, Crohn's disease, diabetes mellitus, experimental autoimmune encephalomyelitis (EAE), focal segmental glomerulosclerosis (FSGS), glomerulonephritis, graft-versus-host disease (GVHD), hidradenitis suppurativa (HS), immune thrombocytopenia, inflammatory bowel disease, inflammatory neuropathy (e.g., acute inflammatory demyelinating polyneuropathy (AIDP) or chronic inflammatory demyelinating polyneuropathy). These include disorders (CIDP), Kawasaki disease, lupus nephritis, mixed connective tissue disease (MCTD), multiple sclerosis, myasthenia gravis, neuromyelitis optica spectrum disorder, organ transplantation, pemphigus, primary biliary cirrhosis, psoriasis, psoriatic arthritis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Sjögren's syndrome, skin grafting, systemic lupus erythematosus, systemic scleroderma, transplant rejection, vasculitis, ANCA-associated vasculitis, ulcerative colitis, or Wegener's granulomatosis.

[0043] Other embodiments will become apparent by examining the detailed description below. [Brief explanation of the drawing]

[0044] [Figure 1A] This study demonstrates the effect of a CD40×CD40 bispecific antibody on IL-6 production in human B cells derived from three different donors, in the presence of a certain amount of CD40L. [Figure 1B] This study demonstrates the effect of a CD40×CD40 bispecific antibody on IL-6 production in human B cells derived from three different donors, in the presence of a certain amount of CD40L. [Figure 1C]This study demonstrates the effect of a CD40×CD40 bispecific antibody on IL-6 production in human B cells derived from three different donors, in the presence of a certain amount of CD40L. [Figure 1D] This study demonstrates the effect of a CD40×CD40 bispecific antibody on IL-6 production in human B cells derived from two different donors, in the presence of a certain amount of CD40L. [Figure 1E] This study demonstrates the effect of a CD40×CD40 bispecific antibody on IL-6 production in human B cells derived from two different donors, in the presence of a certain amount of CD40L. [Figure 2A] This study demonstrates the effect of a CD40×CD40 bispecific antibody on IL10 production in human B cells derived from three different donors, in the presence of a certain amount of CD40L. [Figure 2B] This study demonstrates the effect of a CD40×CD40 bispecific antibody on IL10 production in human B cells derived from three different donors, in the presence of a certain amount of CD40L. [Figure 2C] This study demonstrates the effect of a CD40×CD40 bispecific antibody on IL10 production in human B cells derived from three different donors, in the presence of a certain amount of CD40L. [Figure 2D] This study demonstrates the effect of a CD40×CD40 bispecific antibody on IL10 production in human B cells derived from two different donors, in the presence of a certain amount of CD40L. [Figure 2E] This study demonstrates the effect of a CD40×CD40 bispecific antibody on IL10 production in human B cells derived from two different donors, in the presence of a certain amount of CD40L. [Figure 3A] This study demonstrates the effect of a CD40×CD40 bispecific antibody on TNFα production in human B cells derived from three different donors, in the presence of a certain amount of CD40L. [Figure 3B] This study demonstrates the effect of a CD40×CD40 bispecific antibody on TNFα production in human B cells derived from three different donors, in the presence of a certain amount of CD40L. [Figure 3C]This study demonstrates the effect of a CD40×CD40 bispecific antibody on TNFα production in human B cells derived from three different donors, in the presence of a certain amount of CD40L. [Figure 3D] This study demonstrates the effect of a CD40×CD40 bispecific antibody on TNFα production in human B cells derived from two different donors, in the presence of a certain amount of CD40L. [Figure 3E] This study demonstrates the effect of a CD40×CD40 bispecific antibody on TNFα production in human B cells derived from two different donors, in the presence of a certain amount of CD40L. [Figure 3F] This paper presents an analysis of the agonist activity of CD40×CD40 bispecific antibodies, measured by IL-6 production in human B cells derived from two different donors. [Figure 3G] This paper presents an analysis of the agonist activity of CD40×CD40 bispecific antibodies, measured by IL-6 production in human B cells derived from two different donors. [Figure 4A] This study demonstrates the effect of a CD40×CD40 bispecific antibody on IL-12 / IL-23p40 production from human monocyte-derived dendritic cells from two different donors in the presence of a certain amount of CD40L. [Figure 4B] This study demonstrates the effect of a CD40×CD40 bispecific antibody on IL-12 / IL-23p40 production from human monocyte-derived dendritic cells from two different donors in the presence of a certain amount of CD40L. [Figure 5A] This paper presents an analysis of the agonist activity of CD40×CD40 bispecific antibodies, measured by IL-6 production in human B cells derived from two different donors. [Figure 5B] This paper presents an analysis of the agonist activity of CD40×CD40 bispecific antibodies, measured by IL-6 production in human B cells derived from two different donors. [Figure 6A] This paper presents an analysis of the agonist activity of CD40×CD40 bispecific antibodies, measured by IL10 production in human B cells derived from two different donors. [Figure 6B]This paper presents an analysis of the agonist activity of CD40×CD40 bispecific antibodies, measured by IL10 production in human B cells derived from two different donors. [Figure 7] The experimental timeline using the NP-KLH immunization model disclosed in Example 9 is shown. [Figure 8A] This shows the effect of CD40×CD40 bispecific antibodies on the frequency of NP-positive germinal center B cells. *p<0.05. [Figure 8B] This shows the effect of CD40×CD40 bispecific antibodies on the frequency of NP IgG1 titers in mouse serum. *p<0.05, **p<0.005. [Figure 9A] This shows the effect of CD40×CD40 bispecific antibodies on the frequency of NP-positive germinal center B cells. [Figure 9B] This study demonstrates the effect of CD40×CD40 bispecific antibodies on the frequency of NP IgG1 titers in mouse serum. [Figure 10] The experimental timeline using the mouse EAE model disclosed in Example 10 is shown. [Figure 11A] The mean EAE symptom scores of mice treated with REGN16431 or REGN16432 are shown. [Figure 11B] This shows the mean EAE symptom score of mice treated with REGN16334 or REGN16335. [Figure 11C] This shows the percentage (%) of initial body weight of mice treated with REGN16431 or REGN16432. [Figure 11D] This shows the percentage (%) of initial body weight of mice treated with REGN16334 or REGN16335. [Figure 12A] This shows the mean EAE symptom score of mice treated with CD40×CD40 bispecific antibodies. [Figure 12B] This shows the percentage (%) of initial body weight of mice treated with CD40×CD40 bispecific antibody. [Figure 13] This shows the cryoEM reconstruction of CD40 composited with Fab arms 30027P2, 21519P2, and 21520P2. [Modes for carrying out the invention]

[0045] definition Before describing the present invention, please understand that the present invention is not limited to the specific methods and experimental conditions described, as these may vary. Furthermore, please understand that the scope of the present invention is limited only by the appended claims, and therefore the terms used herein are intended solely to describe specific embodiments and are not intended to limit them.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this invention pertains.

[0047] As used herein, the term “about” means that, when used in reference to a particular enumerated number, that value may vary by no more than 1% from the enumerated value. For example, as used herein, the expression “about 100” includes 99 and 101, as well as all values ​​in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0048] The term "CD40," as used herein, refers to differentiation antigen group 40 (CD40 / TNFRSF5), a costimulatory cell surface receptor that is part of the tumor necrosis factor (TNF) receptor superfamily. In some embodiments, CD40 is human CD40. In some embodiments, the CD40 protein comprises the amino acid sequence of human CD40 described in UniProt accession number Q09LL4.

[0049] The term "antigen-binding molecule" includes antibodies and antigen-binding fragments of antibodies (including multispecific antibodies, such as bispecific antibodies).

[0050] As used herein, the term "antibody" refers to an antigen-binding molecule or molecular complex that includes a set of complementarity determining regions (CDRs) that specifically bind to or interact with a particular antigen (e.g., CD40). As used herein, the term "antibody" includes immunoglobulin molecules containing four polypeptide chains interconnected by disulfide bonds, namely two heavy (H) chains and two light (L) chains, as well as multimers thereof (e.g., IgM). In a typical antibody, each heavy chain includes a heavy chain variable region (referred to herein as HCVR or V H for short) and a heavy chain constant region. The heavy chain constant region includes three domains designated C H 1, C H 2, and C H 3. Each light chain includes a light chain variable region (referred to herein as LCVR or V L for short) and a light chain constant region. The light chain constant region includes one domain (C L 1). The V H and V L regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) and more conserved regions called framework regions (FRs) that are interspersed therebetween. Each V H and V L is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments, the FRs of an antibody (or antigen-binding portion thereof) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence can be defined based on the alignment analysis of two or more CDRs.

[0051] Methods and techniques for identifying CDRs within HCVR amino acid sequences and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within specified HCVR amino acid sequences and / or LCVR amino acid sequences as disclosed herein. Exemplary conventions that can be used to identify CDR boundaries include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition (extended Chothia or Martin), the IMGT definition, and the Honneger definition (AHo). Generally speaking, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, for example, Kabat et al., “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991), Chothia et al., J Mol Biol (1987), 4:901-17, Al-Lazikani et al., J.Mol.Biol. 273:927-948 (1997), and Martin et al., Proc.Natl.Acad.Sci.USA 86:9268-9272 (1989). Also see Dondelinger et al., Front.Immunol. (2018), 9:2278, doi:10.3389 / fimmu.2018.02278. Public databases are also available to identify CDR sequences within antibodies.

[0052] As used herein, the term “antibody” also includes the antigen-binding fragment of a complete antibody molecule. As used herein, terms such as “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, and “antigen-binding domain” include any naturally occurring, enzymatically obtained, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be derived from complete antibody molecules using any suitable standard technique, such as protein digestion or recombinant genetic engineering techniques, including manipulation and expression of DNA encoding the antibody variable domain and optionally a constant domain. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (e.g., including phage antibody libraries), or can be synthesized. DNA may be sequenced and manipulated using chemical or molecular biological techniques to, for example, position one or more variable domains and / or constant domains into a suitable structure, or to introduce codons, create cysteine ​​residues, or modify, add, or delete amino acids.

[0053] Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single-chain Fv(scFv) molecules, (vi) dAb fragments, and (vii) the hypervariable regions of antibodies (e.g., isolated complementarity-determining regions (CDRs) such as the CDR3 peptide), or minimal recognition units consisting of amino acid residues mimicking the restricted FR3-CDR3-FR4 peptide. Other manipulated molecules, such as domain-specific antibodies, single-domain antibodies, domain deletion antibodies, chimeric antibodies, CDR-implanted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also included within the expression “antigen-binding fragment” as used herein.

[0054] The antigen-binding fragment of an antibody typically contains at least one variable domain. The variable domain may be of any size or amino acid composition and generally contains at least one CDR adjacent to or in-frame with one or more framework sequences. L V associated with the domain H In antigen-binding fragments having a domain, V H Domain and V L Domains can be located relative to each other in any suitable arrangement. For example, the variable region may be a dimer, V H -V H , V H -V L , or V L -V L It may contain dimers. Alternatively, the antigen-binding fragment of the antibody may contain monomer V H or V L You may include a domain name.

[0055] In certain embodiments, the antigen-binding fragment of the antibody may include at least one variable domain covalently bound to at least one constant domain. Non-limiting and exemplary structures of the variable and constant domains found within the antigen-binding fragment of the antibody include (i)V H -C H 1. (ii)V H -C H 2, (iii)V H -C H 3, (iv)V H -C H 1-C H 2. (v)V H -C H 1-C H 2-C H 3. (vi)V H -C H 2-C H 3. (vii)V H -C L (viii)V L -C H 1. (ix)V L -C H 2, (x)V L -C H 3. (xi)VL -C H 1-C H 2. (xii)V L -C H 1-C H 2-C H 3. (xiii)V L -C H 2-C H 3, and (xiv)V L -C L Examples include: In any structure of the variable domain and the constant domain (including any of the exemplary structures listed above), the variable domain and the constant domain may be directly linked to each other, or they may be linked by a complete or partial hinge region or linker region. The hinge region may consist of at least two amino acids (e.g., 5, 10, 15, 20, 40, 60 or more), resulting in the formation of a flexible or semi-flexible linkage between adjacent variable domains and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragments of the antibody may be linked to each other and / or one or more monomers V H Or V L The structure may include a homodimer or heterodimer (or other polymer) of any of the variable domain and stationary domain structures listed above, which are non-covalently associated with the domain (for example, by disulfide bonds).

[0056] As used herein, the term “antibody” also includes multispecific (e.g., bispecific) antibodies. A multispecific antibody or antigen-binding fragment of an antibody typically comprises at least two distinct variable domains, each of which can specifically bind to a separate antigen or to a different epitope on the same antigen. In some embodiments, a multispecific antibody (e.g., bispecific antibody) has an arm that binds to a first epitope of the antigen and an arm that binds to a second epitope on the same antigen.

[0057] Any multispecific antibody format can be adapted for use in the context of the antibodies or antigen-binding fragments of antibodies of this disclosure using common techniques available in the art. For example, this disclosure includes a bispecific antibody in which one arm of immunoglobulin is specific to a first epitope of CD40 and the other arm of immunoglobulin is specific to a second epitope of CD40. Exemplary bispecific formats that can be used in the context of this disclosure include, for example, scFv-based or diabody bispecific formats, IgG-scFv fusions, bivariable domain (DVD)-Ig, quadromas, knob-into-holes, common light chains (e.g., common light chains with knob-in-holes), CrossMab, CrossFab, (SEED) bodies, leucine zippers, Duobody, IgG1 / IgG2, dual-acting Fab (DAF)-IgG, and Mab 2 Bispecificity formats include, but are not limited to, Klein et al. 2012, mAbs 4:6, 1-11 and the references cited therein for an overview of the aforementioned formats. Bispecificity antibodies can also be constructed using peptide / nucleic acid bonds, for example, using non-natural amino acids with orthogonal chemical reactivity to generate site-specific antibody-oligonucleotide conjugates, which then self-assemble into a multimeric complex with a defined composition, valency, and geometric shape (see, for example, Kazane et al., J.Am.Chem.Soc. [Epub:Dec.4,2012]).

[0058] The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Nevertheless, the human antibodies of this disclosure may include, for example, amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo) in the CDR, particularly CDR3. However, the term "human antibody," as used herein, is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as mouse, has been transplanted into a human framework sequence.

[0059] The term “recombinant antibody,” as used herein, is intended to include all antibodies prepared, expressed, produced, or isolated by recombinant means. This term includes, but is not limited to, antibodies expressed using recombinant expression vectors introduced into host cells (e.g., Chinese hamster ovary (CHO) cells) or cell expression systems, antibodies isolated from recombinant combinatorial human antibody libraries, and antibodies isolated from non-human animals (e.g., mice, e.g., mice transgenic for human immunoglobulin genes) (see, for example, Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295). In some embodiments, the recombinant antibody is a recombinant human antibody. In some embodiments, the recombinant human antibody has a variable region and a constant region derived from a human germline immunoglobulin sequence. However, in certain embodiments, such a recombinant human antibody undergoes in vitro mutagenesis (or in vivo somatic mutagenesis if an animal transgenic for a human Ig sequence is used), and therefore the V of the recombinant antibody H Region and V L The amino acid sequence of the region is human germline V H Array and V LThese are sequences that originate from and are related to other sequences, but may not naturally exist in the human antibody germline repertoire in vivo.

[0060] "Isolated antibodies" refer to antibodies identified, isolated, and / or recovered from at least one component of the natural environment. For example, antibodies isolated or removed from at least one component of an organism, or from tissues or cells in which antibodies are naturally present or naturally produced, are "isolated antibodies." Isolated antibodies also include in situ antibodies in recombinant cells. Isolated antibodies are antibodies that have undergone at least one purification or isolation step. According to certain embodiments, isolated antibodies may substantially contain no other cellular material and / or chemical substances.

[0061] Terms such as "specifically binding" mean that an antibody or its antigen-binding fragment forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is at least about 1 × 10⁻⁶ -6 M or less, for example, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 It can be characterized by the equilibrium dissociation constant of M (K D (A smaller value indicates stronger binding.) Methods for determining whether an antibody specifically binds to an antigen are known in the art, and include, for example, equilibrium dialysis, surface plasmon resonance (e.g., BIACORE®), biolayer interferometry assays (e.g., Octet® HTX biosensor), and solution affinity ELISA. In some embodiments, specific binding is measured in surface plasmon resonance assays, for example, at 25°C or 37°C. An antibody or antigen-binding fragment that specifically binds to an antigen from one species may or may not cross-react to other antigens, such as orthologous antigens from another species.

[0062] "K DAs used herein, the term "equilibrium dissociation constant" refers to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0063] As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that enables real-time analysis of biomolecular interactions by detecting changes in protein concentration within a biosensor matrix, for example, using a BIACORE® system (Cytiva, Marlborough, MA).

[0064] As used herein, the term “epitope” refers to an antigenic determinant that interacts with a specific antigen-binding site known as a paratope in the variable region of an antibody molecule. A single antigen may have more than one epitope. Therefore, different antibodies may bind to different regions on an antigen and may have different biological effects. The term “epitope” also refers to a site on an antigen to which B cells and / or T cells respond. It also refers to the region of the antigen to which an antibody binds. Epitopes can be either linear or discontinuous (e.g., conformational). A linear epitope is an epitope produced by adjacent amino acid residues in a polypeptide chain. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. In certain embodiments, an epitope may include a determinant that is a chemically active surface group of a molecule, such as an amino acid, a sugar side chain, a phosphoryl group, or a sulfonyl group, and in certain embodiments, it may have certain three-dimensional structural properties and / or certain charge properties. Epitopes may also be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and have residues that directly contribute to the affinity of the interaction. Epitopes typically contain at least three, more commonly, at least five, or at least eight to ten amino acids within their unique spatial structure.

[0065] Methods for determining the epitopes of antigen-binding proteins (e.g., antibodies or antigen-binding fragments) include alanine scan mutation analysis, peptide blot analysis (Reineke, Methods Mol Biol 2004, 248:443-463), peptide cleavage analysis, crystallographic studies, and NMR analysis. Furthermore, methods such as epitope exclusion, epitope extraction, and chemical modification of antigens can be used (Tomer, Prot Sci 2000, 9:487-496). Another method that can be used to identify amino acids within polypeptides that antigen-binding proteins (e.g., antibodies or antigen-binding fragments) interact with is hydrogen / deuterium exchange (HDX), detected by mass spectrometry. See, for example, Ehring, Analytical Biochemistry 1999, 267:252-259 and Engen and Smith, Analytical Chem 2001, 73:256A-265A.

[0066] The term “compete” as used in relation to competition for binding means that one antigen-binding protein (e.g., an antibody or antigen-binding fragment) binds to an antigen, and inhibits or blocks the binding of another antigen-binding protein (e.g., an antibody or antigen-binding fragment) to the same antigen. Unless otherwise specified, this term also includes competition between two antigen-binding proteins (e.g., antibodies) in both directions, i.e., the first antigen binds to the antigen and blocks the binding by the second antibody, and vice versa. Thus, in some embodiments, competition occurs in one such direction. In some embodiments, the first antigen-binding protein (e.g., an antibody) and the second antigen-binding protein (e.g., an antibody) may bind to the same epitope. Alternatively, the first and second antigen-binding proteins (e.g., antibodies) may bind to different epitopes, which may or may not overlap, and the binding of one antigen-binding protein may inhibit or block the binding of the second antigen-binding protein, for example, via steric hindrance. Competition among antigen-binding proteins can be measured by methods known in the art, for example, by a real-time label-free biolayer interferometry assay.

[0067] The terms “substantially identical” or “substantially identical” as used with respect to nucleic acids or fragments indicate that, when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, at least about 90% of the nucleotide bases, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% are nucleotide sequence identical, as measured by any well-known algorithm of sequence identity such as FASTA, BLAST, or GAP, which are discussed below. A nucleic acid molecule having substantial identity with respect to a reference nucleic acid molecule may, in some cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0068] When applied to polypeptides, the terms “substantial identity” and “substantially identical” mean that, when two peptide sequences are optimally aligned, they share at least about 90% sequence identity, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, non-identical residue positions are distinguished by conservative amino acid substitutions. A “conservative amino acid substitution” is when an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of the protein.

[0069] Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conserved amino acid substitutions. For example, GCG software includes programs such as GAP and BESTFIT, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species, or between a wild-type protein and its mutant protein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment and sequence identity percentage of the best overlap region between the query sequence and the search sequence (Pearson, 2000, above). Another preferred algorithm for comparing the sequences of this disclosure with a database containing numerous sequences from different biological origins is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters (see, for example, Altschul et al., 1990, J.Mol.Biol.215:403-410 and 1997 Nucleic Acids Res.25:3389-3402).

[0070] A “variant” of a polypeptide (such as an immunoglobulin, VH, VL, heavy chain, light chain, or CDR) containing an amino acid sequence specifically described herein means a polypeptide containing an amino acid sequence that is at least approximately 70% to 99.9% (e.g., at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9%) identical to a reference polypeptide sequence (e.g., listed in the following sequence listings) when compared using the BLAST algorithm, where the algorithm parameters are selected to give the greatest possible match between each sequence over the entire length of each reference sequence. In some embodiments, a polypeptide variant includes a polypeptide having the amino acid sequence of a reference polypeptide sequence (e.g., listed in the following sequence listings) with the exception of one or more (e.g., 1 to 10, or less than 20, or less than 10) missense mutations (e.g., conservative substitutions), nonsense mutations, deletions, or insertions.

[0071] CD40 antigen binding molecule In one embodiment, the disclosure relates to antigen-binding molecules (including monospecific antibodies, bispecific antibodies, and multispecific antibodies) that bind to CD40. In some embodiments, the antigen-binding molecule is a monospecific CD40 antibody. In some embodiments, the antigen-binding molecule is a multispecific (e.g., bispecific) antibody. The multispecific antibody may be specific to a different epitope of one target polypeptide, or it may contain antigen-binding domains specific to two or more target polypeptides. See, for example, Tutt et al., 1991, J.Immunol. 147:60-69 and Kufer et al., 2004, Trends Biotechnol. 22:238-244. In some embodiments, the CD40 antibody of the disclosure may be ligated to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or a fragment thereof can be functionally linked (e.g., by chemical bonding, gene fusion, non-covalent association, or other means) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody having a second binding specificity. In some embodiments, the multispecific antibody includes an antigen-binding domain specific to CD40 and an antigen-binding domain specific to another antigen (i.e., not CD40). In some embodiments, the multispecific antibody includes an antigen-binding domain specific to a first epitope of CD40 and an antigen-binding domain specific to a second epitope of CD40.

[0072] Single-specificity CD40 antibody In some embodiments, the disclosure provides a monospecific CD40 antibody or its antigen-binding fragment that specifically binds to CD40 (e.g., human CD40). In some embodiments, the CD40 antibody or its antigen-binding fragment comprises the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set contained within an HCVR / LCVR amino acid sequence pair selected from the group consisting of 2 / 10, 22 / 10, and 32 / 10. In some embodiments, the CDRs within the HCVR and / or LCVR are identified according to the Kabat definition. In some embodiments, the CDRs within the HCVR and / or LCVR are identified according to the Chothia definition. In some embodiments, the CDRs within the HCVR and / or LCVR are identified according to the AbM definition. In some embodiments, the CDRs within the HCVR and / or LCVR are identified according to the IMGT definition.

[0073] In some embodiments, the CD40 antibody or its antigen-binding fragment includes the following: (a) HCDR1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 24, and 34, (b) HCDR2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 26, and 36, (c) HCDR3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 28, and 38, (d) LCDR1 containing the amino acid sequence of SEQ ID NO: 12, (e) LCDR2 containing the amino acid sequence AAS, and (f) LCDR3 containing the amino acid sequence of SEQ ID NO: 16.

[0074] In some embodiments, the CD40 antibody or antigen-binding fragment includes the following: (a) HCDR1 containing the amino acid sequence of SEQ ID NO: 4, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, HCDR3 containing the amino acid sequence of SEQ ID NO: 8, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence of SEQ ID NO: 16, or (b) HCDR1 containing the amino acid sequence of SEQ ID NO: 24, HCDR2 containing the amino acid sequence of SEQ ID NO: 26, HCDR3 containing the amino acid sequence of SEQ ID NO: 28, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence of SEQ ID NO: 16, or (c) HCDR1 containing the amino acid sequence of SEQ ID NO: 34, HCDR2 containing the amino acid sequence of SEQ ID NO: 36, HCDR3 containing the amino acid sequence of SEQ ID NO: 38, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence of SEQ ID NO: 16.

[0075] In some embodiments, the CD40 antibody comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 4, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, HCDR3 containing the amino acid sequence of SEQ ID NO: 8, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, the CD40 antibody comprises HCDR1 consisting of the amino acid sequence of SEQ ID NO: 4, HCDR2 consisting of the amino acid sequence of SEQ ID NO: 6, HCDR3 consisting of the amino acid sequence of SEQ ID NO: 8, LCDR1 consisting of the amino acid sequence of SEQ ID NO: 12, LCDR2 consisting of the amino acid sequence AAS, and LCDR3 consisting of the amino acid sequence of SEQ ID NO: 16.

[0076] In some embodiments, the CD40 antibody comprises an HCVR having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2, and / or an LCVR having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10. In some embodiments, the CD40 antibody comprises an HCVR containing or consisting of the amino acid sequence of SEQ ID NO: 2. In some embodiments, the CD40 antibody comprises an LCVR containing or consisting of the amino acid sequence of SEQ ID NO: 10.

[0077] In some embodiments, the CD40 antibody comprises a heavy chain containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 18, and / or a light chain containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 20. In some embodiments, the CD40 antibody comprises a heavy chain containing or comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the CD40 antibody comprises a light chain containing or comprising the amino acid sequence of SEQ ID NO: 20.

[0078] In some embodiments, the CD40 antibody comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 24, HCDR2 containing the amino acid sequence of SEQ ID NO: 26, HCDR3 containing the amino acid sequence of SEQ ID NO: 28, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, the CD40 antibody comprises HCDR1 consisting of the amino acid sequence of SEQ ID NO: 24, HCDR2 consisting of the amino acid sequence of SEQ ID NO: 26, HCDR3 consisting of the amino acid sequence of SEQ ID NO: 28, LCDR1 consisting of the amino acid sequence of SEQ ID NO: 12, LCDR2 consisting of the amino acid sequence AAS, and LCDR3 consisting of the amino acid sequence of SEQ ID NO: 16.

[0079] In some embodiments, the CD40 antibody comprises an HCVR having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 22, and / or an LCVR having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10. In some embodiments, the CD40 antibody comprises an HCVR containing or consisting of the amino acid sequence of SEQ ID NO: 22. In some embodiments, the CD40 antibody comprises an LCVR containing or consisting of the amino acid sequence of SEQ ID NO: 10.

[0080] In some embodiments, the CD40 antibody comprises a heavy chain containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 28, and / or a light chain containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 20. In some embodiments, the CD40 antibody comprises a heavy chain containing or comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments, the CD40 antibody comprises a light chain containing or comprising the amino acid sequence of SEQ ID NO: 20.

[0081] In some embodiments, the CD40 antibody comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 34, HCDR2 containing the amino acid sequence of SEQ ID NO: 36, HCDR3 containing the amino acid sequence of SEQ ID NO: 38, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, the CD40 antibody comprises HCDR1 consisting of the amino acid sequence of SEQ ID NO: 34, HCDR2 consisting of the amino acid sequence of SEQ ID NO: 36, HCDR3 consisting of the amino acid sequence of SEQ ID NO: 38, LCDR1 consisting of the amino acid sequence of SEQ ID NO: 12, LCDR2 consisting of the amino acid sequence AAS, and LCDR3 consisting of the amino acid sequence of SEQ ID NO: 16.

[0082] In some embodiments, the CD40 antibody comprises an HCVR having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 32, and / or an LCVR having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10. In some embodiments, the CD40 antibody comprises an HCVR containing or consisting of the amino acid sequence of SEQ ID NO: 32. In some embodiments, the CD40 antibody comprises an LCVR containing or consisting of the amino acid sequence of SEQ ID NO: 10.

[0083] In some embodiments, the CD40 antibody comprises a heavy chain containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 40, and / or a light chain containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 20. In some embodiments, the CD40 antibody comprises a heavy chain containing or comprising the amino acid sequence of SEQ ID NO: 40. In some embodiments, the CD40 antibody comprises a light chain containing or comprising the amino acid sequence of SEQ ID NO: 20.

[0084] In some embodiments, the CD40 antibody has at least 85% sequence identity to 30027P2, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In some embodiments, the CD40 antibody has the amino acid sequence of 30027P2.

[0085] In some embodiments, the CD40 antibody has at least 85% sequence identity to 21519P2, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In some embodiments, the CD40 antibody has the amino acid sequence of 21519P2.

[0086] In some embodiments, the CD40 antibody has at least 85% sequence identity to 21520P2, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In some embodiments, the CD40 antibody contains or consists of the amino acid sequence of 21520P2.

[0087] CD40×CD40 bispecific antigen binding molecule This disclosure also provides multispecific antigen-binding molecules that specifically bind to CD40. In some embodiments, the antigen-binding molecule is a bispecific antigen-binding molecule, e.g., a bispecific antibody. In some embodiments, the bispecific antigen-binding molecule includes a first antigen-binding domain (D1) that binds to a first epitope of CD40 (e.g., human CD40) and a second antigen-binding domain (D2) that binds to a second epitope of CD40 (e.g., human CD40). In some embodiments, D1 and D2 do not compete with each other for binding to CD40 (e.g., human CD40). In some embodiments, D1 and D2 compete with each other for binding to CD40 (e.g., human CD40).

[0088] In some embodiments, the bispecific antigen-binding molecule comprises two distinct heavy-chain immunoglobulin variable regions, where at least one heavy-chain immunoglobulin variable region comprises an HCDR1-HCDR2-HCDR3 amino acid sequence set contained within an HCVR amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 22, and 32. In some embodiments, each heavy-chain immunoglobulin variable region comprises an HCDR1-HCDR2-HCDR3 amino acid sequence set contained within an HCVR amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 22, and 32. In some embodiments, the CDRs within the HCVR are identified according to the Kabat definition. In some embodiments, the CDRs are identified according to the Chothia definition. In some embodiments, the CDRs are identified according to the AbM definition. In some embodiments, the CDRs are identified according to the IMGT definition.

[0089] In some embodiments, one or more of the heavy chain immunoglobulin variable regions include a set of HCDR sequences selected from the group consisting of the following: (a) HCDR1 containing the amino acid sequence of SEQ ID NO: 4, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 8 (b) HCDR1 containing the amino acid sequence of SEQ ID NO: 24, HCDR2 containing the amino acid sequence of SEQ ID NO: 26, and HCDR3 containing the amino acid sequence of SEQ ID NO: 28, (c) HCDR1 containing the amino acid sequence of SEQ ID NO: 34, HCDR2 containing the amino acid sequence of SEQ ID NO: 36, and HCDR3 containing the amino acid sequence of SEQ ID NO: 38.

[0090] In some embodiments, the bispecific antigen-binding molecule comprises (i) an antigen-binding domain comprising a heavy-chain immunoglobulin variable region including HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, HCDR2 comprising the amino acid sequence of SEQ ID NO: 6, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 8, and (ii) an antigen-binding domain comprising a heavy-chain immunoglobulin variable region including HCDR1 comprising the amino acid sequence of SEQ ID NO: 24, HCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 28.

[0091] In some embodiments, the bispecific antigen-binding molecule comprises (i) an antigen-binding domain comprising a heavy-chain immunoglobulin variable region including HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, HCDR2 comprising the amino acid sequence of SEQ ID NO: 6, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 8, and (ii) an antigen-binding domain comprising a heavy-chain immunoglobulin variable region including HCDR1 comprising the amino acid sequence of SEQ ID NO: 34, HCDR2 comprising the amino acid sequence of SEQ ID NO: 36, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 38.

[0092] In some embodiments, the bispecific antigen-binding molecule comprises (i) an antigen-binding domain comprising a heavy chain immunoglobulin variable region including HCDR1 comprising the amino acid sequence of SEQ ID NO: 24, HCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 28, and (ii) an antigen-binding domain comprising a heavy chain immunoglobulin variable region including HCDR1 comprising the amino acid sequence of SEQ ID NO: 34, HCDR2 comprising the amino acid sequence of SEQ ID NO: 36, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 38.

[0093] In some embodiments, the bispecific antigen-binding molecule includes a common light chain variable region. In some embodiments, the light chain variable region includes the HCDR1-HCDR2-HCDR3 amino acid sequence set contained within the LCVR amino acid sequence of SEQ ID NO: 10. In some embodiments, the light chain variable region includes LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence of SEQ ID NO: 16.

[0094] In some embodiments, the bispecific antigen-binding molecule comprises D1, which binds to a first epitope of human CD40, where the D1 domain comprises a heavy immunoglobulin chain comprising: (a) HCDR1 containing the amino acid sequence of SEQ ID NO: 4, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 8, or (b) HCDR1 containing the amino acid sequence of SEQ ID NO: 24, HCDR2 containing the amino acid sequence of SEQ ID NO: 26, and HCDR3 containing the amino acid sequence of SEQ ID NO: 28.

[0095] In some embodiments, the D1 domain includes HCDR1 containing the amino acid sequence of SEQ ID NO: 4, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 8. In some embodiments, the D1 domain includes HCDR1 consisting of the amino acid sequence of SEQ ID NO: 4, HCDR2 consisting of the amino acid sequence of SEQ ID NO: 6, and HCDR3 consisting of the amino acid sequence of SEQ ID NO: 8. In some embodiments, the D1 domain includes an HCVR containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO: 2. In some embodiments, the D1 domain includes the amino acid sequence of SEQ ID NO: 2, or an HCVR consisting of it.

[0096] In some embodiments, the D1 domain comprises a heavy chain containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO: 46. In some embodiments, the D1 domain comprises the amino acid sequence of SEQ ID NO: 46 or a heavy chain comprising the same.

[0097] In some embodiments, the D1 domain comprises a heavy chain containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO: 52. In some embodiments, the D1 domain comprises the amino acid sequence of SEQ ID NO: 52 or a heavy chain comprising the same.

[0098] In some embodiments, the D1 domain further comprises a light chain immunoglobulin chain comprising LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence of SEQ ID NO: 16.

[0099] In some embodiments, the D1 domain includes an LCVR containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO: 10. In some embodiments, the D1 domain includes an LCVR containing the amino acid sequence of SEQ ID NO: 10.

[0100] In some embodiments, the D1 domain includes a light chain containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO: 20. In some embodiments, the D1 domain includes a light chain containing the amino acid sequence of SEQ ID NO: 20.

[0101] In some embodiments, the D1 domain includes HCDR1 containing the amino acid sequence of SEQ ID NO: 24, HCDR2 containing the amino acid sequence of SEQ ID NO: 26, and HCDR3 containing the amino acid sequence of SEQ ID NO: 28. In some embodiments, the D1 domain includes HCDR1 consisting of the amino acid sequence of SEQ ID NO: 24, HCDR2 consisting of the amino acid sequence of SEQ ID NO: 26, and HCDR3 consisting of the amino acid sequence of SEQ ID NO: 28. In some embodiments, the D1 domain includes an HCVR containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO: 22. In some embodiments, the D1 domain includes the amino acid sequence of SEQ ID NO: 22, or an HCVR consisting of it.

[0102] In some embodiments, the D1 domain comprises a heavy chain containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO: 42. In some embodiments, the D1 domain comprises the amino acid sequence of SEQ ID NO: 42 or a heavy chain comprising the same.

[0103] In some embodiments, the D1 domain comprises a heavy chain containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO: 46. In some embodiments, the D1 domain comprises the amino acid sequence of SEQ ID NO: 46 or a heavy chain comprising the same.

[0104] In some embodiments, the D1 domain further comprises a light chain immunoglobulin chain comprising LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence of SEQ ID NO: 16.

[0105] In some embodiments, the D1 domain includes an LCVR containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO: 10. In some embodiments, the D1 domain includes an LCVR containing the amino acid sequence of SEQ ID NO: 10.

[0106] In some embodiments, the D1 domain includes a light chain containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO: 20. In some embodiments, the D1 domain includes a light chain containing the amino acid sequence of SEQ ID NO: 20.

[0107] In some embodiments, the bispecific antigen-binding molecule comprises D2 which binds to a second epitope of human CD40, where the D2 domain comprises a heavy immunoglobulin chain comprising HCDR1 comprising the amino acid sequence of SEQ ID NO: 34, HCDR2 comprising the amino acid sequence of SEQ ID NO: 36, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 38. In some embodiments, the D2 domain comprises HCDR1 consisting of the amino acid sequence of SEQ ID NO: 34, HCDR2 consisting of the amino acid sequence of SEQ ID NO: 36, and HCDR3 consisting of the amino acid sequence of SEQ ID NO: 38. In some embodiments, the D2 domain comprises an HCVR comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO: 32. In some embodiments, the D2 domain comprises the amino acid sequence of SEQ ID NO: 32 or an HCVR comprising the same.

[0108] In some embodiments, the D2 domain comprises a heavy chain containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO: 44. In some embodiments, the D2 domain comprises the amino acid sequence of SEQ ID NO: 44 or a heavy chain comprising the same.

[0109] In some embodiments, the D2 domain comprises a heavy chain containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO: 50. In some embodiments, the D2 domain comprises the amino acid sequence of SEQ ID NO: 50 or a heavy chain comprising the same.

[0110] In some embodiments, the D2 domain further comprises a light chain immunoglobulin chain comprising LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence of SEQ ID NO: 16.

[0111] In some embodiments, the D2 domain includes an LCVR containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO: 10. In some embodiments, the D2 domain includes an LCVR containing the amino acid sequence of SEQ ID NO: 10.

[0112] In some embodiments, the D2 domain includes a light chain containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO: 20. In some embodiments, the D2 domain includes a light chain containing the amino acid sequence of SEQ ID NO: 20.

[0113] In some embodiments, the bispecific antigen-binding molecule comprises a D1 domain that binds to a first epitope of human CD40 and a D2 domain that binds to a second epitope of human CD40, wherein the D1 domain comprises a heavy immunoglobulin chain comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 34, HCDR2 containing the amino acid sequence of SEQ ID NO: 36, and HCDR3 containing the amino acid sequence of SEQ ID NO: 38. In some embodiments, the D1 domain comprises HCDR1 consisting of the amino acid sequence of SEQ ID NO: 34, HCDR2 consisting of the amino acid sequence of SEQ ID NO: 36, and HCDR3 consisting of the amino acid sequence of SEQ ID NO: 38. In some embodiments, the D1 domain comprises an HCVR containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO: 32. In some embodiments, the D1 domain comprises the amino acid sequence of SEQ ID NO: 32 or an HCVR consisting thereof.

[0114] In some embodiments, the D1 domain comprises a heavy chain containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO: 58. In some embodiments, the D1 domain comprises the amino acid sequence of SEQ ID NO: 58 or a heavy chain comprising the same.

[0115] In some embodiments, the D2 domain comprises a heavy immunoglobulin chain comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 4, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 8. In some embodiments, the D2 domain comprises HCDR1 consisting of the amino acid sequence of SEQ ID NO: 4, HCDR2 consisting of the amino acid sequence of SEQ ID NO: 6, and HCDR3 consisting of the amino acid sequence of SEQ ID NO: 8. In some embodiments, the D2 domain comprises an HCVR containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO: 2. In some embodiments, the D2 domain comprises the amino acid sequence of SEQ ID NO: 2 or an HCVR consisting of it.

[0116] In some embodiments, the D2 domain comprises a heavy chain containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO: 60. In some embodiments, the D2 domain comprises the amino acid sequence of SEQ ID NO: 60 or a heavy chain comprising the same.

[0117] In some embodiments, the D1 domain and / or D2 domain further comprises a light chain immunoglobulin chain comprising LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, the D1 domain and / or D2 domain comprises an LCVR containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO: 10. In some embodiments, the D1 domain and / or D2 domain comprises an LCVR containing the amino acid sequence of SEQ ID NO: 10. In some embodiments, the D1 domain and / or D2 domain include a light chain containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO: 20. In some embodiments, the D1 domain and / or D2 domain include a light chain containing the amino acid sequence of SEQ ID NO: 20.

[0118] In some embodiments, the bispecific antigen-binding molecule includes the following: A D1 domain that binds to the first epitope of human CD40, wherein the D1 domain comprises an immunoglobulin chain comprising the amino acid sequence of SEQ ID NO: 4 or HCDR1, the amino acid sequence of SEQ ID NO: 6 or HCDR2, the amino acid sequence of SEQ ID NO: 8 or HCDR3, the amino acid sequence of SEQ ID NO: 12 or HCDR1, the amino acid sequence AAS or HCDR2, and the amino acid sequence of SEQ ID NO: 16 or HCDR3, and A D2 domain that binds to a second epitope of human CD40, wherein the D2 domain comprises an immunoglobulin chain comprising the amino acid sequence of SEQ ID NO: 34 or HCDR1, the amino acid sequence of SEQ ID NO: 36 or HCDR2, the amino acid sequence of SEQ ID NO: 38 or HCDR3, the amino acid sequence of SEQ ID NO: 12 or LCDR1, the amino acid sequence AAS or LCDR2, and the amino acid sequence of SEQ ID NO: 16 or LCDR3.

[0119] In some embodiments, the bispecific antigen-binding molecule includes the following: A D1 domain comprising an HCVR containing an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2 (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%), and an LCVR containing an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 10 (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%), and A D2 domain comprising an HCVR containing an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 32 (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%), and an LCVR containing an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 10 (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%).

[0120] In some embodiments, the bispecific antigen-binding molecule comprises a D1 domain comprising an HCVR containing or consisting of the amino acid sequence of SEQ ID NO: 2, and an LCVR containing or consisting of the amino acid sequence of SEQ ID NO: 10, and a D2 domain comprising an HCVR containing or consisting of the amino acid sequence of SEQ ID NO: 32, and an LCVR containing or consisting of the amino acid sequence of SEQ ID NO: 10.

[0121] In some embodiments, the bispecific antigen-binding molecule comprises a D1 domain comprising a heavy chain containing or consisting of the amino acid sequence of SEQ ID NO: 46 and a light chain containing or consisting of the amino acid sequence of SEQ ID NO: 20, and a D2 domain comprising a heavy chain containing or consisting of the amino acid sequence of SEQ ID NO: 44 and a light chain containing or consisting of the amino acid sequence of SEQ ID NO: 20.

[0122] In some embodiments, the bispecific antigen-binding molecule comprises a D1 domain comprising a heavy chain containing or consisting of the amino acid sequence of SEQ ID NO: 52 and a light chain containing or consisting of the amino acid sequence of SEQ ID NO: 20, and a D2 domain comprising a heavy chain containing or consisting of the amino acid sequence of SEQ ID NO: 50 and a light chain containing or consisting of the amino acid sequence of SEQ ID NO: 20.

[0123] In some embodiments, the bispecific antigen-binding molecule includes the following: A D1 domain that binds to the first epitope of human CD40, wherein the D1 domain comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 24, HCDR2 containing or consisting of the amino acid sequence of SEQ ID NO: 26, HCDR3 containing or consisting of the amino acid sequence of SEQ ID NO: 28, LCDR1 containing or consisting of the amino acid sequence of SEQ ID NO: 12, LCDR2 containing or consisting of the amino acid sequence AAS, and LCDR3 containing or consisting of the amino acid sequence of SEQ ID NO: 16, and A D2 domain that binds to a second epitope of human CD40, wherein the D2 domain comprises HCDR1 containing or consisting of the amino acid sequence of SEQ ID NO: 34, HCDR2 containing or consisting of the amino acid sequence of SEQ ID NO: 36, HCDR3 containing or consisting of the amino acid sequence of SEQ ID NO: 38, LCDR1 containing or consisting of the amino acid sequence of SEQ ID NO: 12, LCDR2 containing or consisting of the amino acid sequence AAS, and LCDR3 containing or consisting of the amino acid sequence of SEQ ID NO: 16.

[0124] In some embodiments, the bispecific antigen-binding molecule includes the following: A D1 domain comprising an HCVR containing an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2 (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%), and an LCVR containing an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 10 (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%), and A D2 domain comprising an HCVR containing an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 32 (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%), and an LCVR containing an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 10 (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%).

[0125] In some embodiments, the bispecific antigen-binding molecule comprises a D1 domain comprising an HCVR containing or consisting of the amino acid sequence of SEQ ID NO: 2, and an LCVR containing or consisting of the amino acid sequence of SEQ ID NO: 10, and a D2 domain comprising an HCVR containing or consisting of the amino acid sequence of SEQ ID NO: 32, and an LCVR containing or consisting of the amino acid sequence of SEQ ID NO: 10.

[0126] In some embodiments, the bispecific antigen-binding molecule comprises a D1 domain comprising a heavy chain containing or consisting of the amino acid sequence of SEQ ID NO: 42 and a light chain containing or consisting of the amino acid sequence of SEQ ID NO: 20, and a D2 domain comprising a heavy chain containing or consisting of the amino acid sequence of SEQ ID NO: 44 and a light chain containing or consisting of the amino acid sequence of SEQ ID NO: 20.

[0127] In some embodiments, the bispecific antigen-binding molecule comprises a D1 domain comprising a heavy chain containing or consisting of the amino acid sequence of SEQ ID NO: 48 and a light chain containing or consisting of the amino acid sequence of SEQ ID NO: 20, and a D2 domain comprising a heavy chain containing or consisting of the amino acid sequence of SEQ ID NO: 50 and a light chain containing or consisting of the amino acid sequence of SEQ ID NO: 20.

[0128] In some embodiments, the bispecific antigen-binding molecule includes the following: A D1 domain that binds to the first epitope of human CD40, wherein the D1 domain comprises an immunoglobulin chain comprising the amino acid sequence of SEQ ID NO: 34 or HCDR1, the amino acid sequence of SEQ ID NO: 36 or HCDR2, the amino acid sequence of SEQ ID NO: 38 or HCDR3, the amino acid sequence of SEQ ID NO: 12 or HCDR1, the amino acid sequence AAS or HCDR2, and the amino acid sequence of SEQ ID NO: 16 or HCDR3, and A D2 domain that binds to a second epitope of human CD40, wherein the D2 domain comprises an immunoglobulin chain comprising the amino acid sequence of SEQ ID NO: 4 or HCDR1, the amino acid sequence of SEQ ID NO: 6 or HCDR2, the amino acid sequence of SEQ ID NO: 8 or HCDR3, the amino acid sequence of SEQ ID NO: 12 or HCDR1, the amino acid sequence AAS or HCDR2, and the amino acid sequence of SEQ ID NO: 16 or HCDR3.

[0129] In some embodiments, the bispecific antigen-binding molecule includes the following: A D1 domain comprising an HCVR containing an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 32 (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%), and an LCVR containing an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 10 (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%), and A D2 domain comprising an HCVR containing an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2 (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%), and an LCVR containing an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 10 (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%).

[0130] In some embodiments, the bispecific antigen-binding molecule comprises a D1 domain comprising an HCVR containing or consisting of the amino acid sequence of SEQ ID NO: 32 and an LCVR containing or consisting of the amino acid sequence of SEQ ID NO: 10, and a D2 domain comprising an HCVR containing or consisting of the amino acid sequence of SEQ ID NO: 2 and an LCVR containing or consisting of the amino acid sequence of SEQ ID NO: 10.

[0131] In some embodiments, the bispecific antigen-binding molecule comprises a D1 domain comprising a heavy chain containing or consisting of the amino acid sequence of SEQ ID NO: 58 and a light chain containing or consisting of the amino acid sequence of SEQ ID NO: 20, and a D2 domain comprising a heavy chain containing or consisting of the amino acid sequence of SEQ ID NO: 60 and a light chain containing or consisting of the amino acid sequence of SEQ ID NO: 20.

[0132] The bispecific antigen-binding molecules disclosed herein may be bispecific antibodies. In some cases, the bispecific antibody includes a human IgG heavy chain constant region. In some cases, the human IgG heavy chain constant region is isotype IgG1. In some cases, the human IgG heavy chain constant region is isotype IgG4.

[0133] The first antigen-binding domain and the second antigen-binding domain may be linked directly or indirectly to each other to form the bispecific antigen-binding molecule of this disclosure. Alternatively, the first antigen-binding domain and the second antigen-binding domain may each be linked to a separate multimerizing domain. The association of one multimerizing domain with another facilitates the association between the two antigen-binding domains, thereby forming the bispecific antigen-binding molecule. As used herein, “multimerizing domain” is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to associate with a second multimerizing domain of the same or similar structure or configuration. For example, the multimerizing domain may be a polypeptide containing an immunoglobulin CH3 domain. Non-limiting examples of multimerizing components are the Fc portion of immunoglobulins (including the CH2-CH3 domain), e.g., the Fc domain of IgG selected from isotypes IgG1, IgG2, IgG3, and IgG4, and any allotype within each isotype group.

[0134] In some embodiments, the bispecific antigen-binding molecule of the present disclosure comprises two multimerization domains, for example, two Fc domains, each individually part of a distinct antibody heavy chain. The first and second multimerization domains may be of the same IgG isotype (e.g., IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4, etc.). Alternatively, the first and second multimerization domains may be of different IgG isotypes (e.g., IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.).

[0135] In some embodiments, the multimerizing domain is an Fc fragment or an amino acid sequence of 1 to about 200 amino acids in length, containing at least one cysteine ​​residue. In other embodiments, the multimerizing domain is a cysteine ​​residue or a short cysteine-containing peptide. Other multimerizing domains include peptides or polypeptides containing or consisting of a leucine zipper, a helix-loop motif, or a coiled-coil motif.

[0136] In some embodiments, the bispecific antigen-binding molecule is a CD40×CD40 bispecific antibody having at least 85% sequence identity to REGN16334, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In some embodiments, the bispecific antigen-binding molecule is a CD40×CD40 bispecific antibody having the amino acid sequence of REGN16334.

[0137] In some embodiments, the bispecific antigen-binding molecule is a CD40×CD40 bispecific antibody having at least 85% sequence identity to REGN16335, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In some embodiments, the bispecific antigen-binding molecule is a bispecific antibody having the amino acid sequence of REGN16335.

[0138] In some embodiments, the bispecific antigen-binding molecule is a CD40×CD40 bispecific antibody having at least 85% sequence identity to REGN16431, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In some embodiments, the bispecific antigen-binding molecule is a CD40×CD40 bispecific antibody having the amino acid sequence of REGN16431.

[0139] In some embodiments, the bispecific antigen-binding molecule is a CD40×CD40 bispecific antibody having at least 85% sequence identity to REGN16432, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In some embodiments, the bispecific antigen-binding molecule is a CD40×CD40 bispecific antibody having the amino acid sequence of REGN16432.

[0140] In some embodiments, the bispecific antigen-binding molecule is a CD40×CD40 bispecific antibody having at least 85% sequence identity to REGN20484, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In some embodiments, the bispecific antigen-binding molecule is a CD40×CD40 bispecific antibody having the amino acid sequence of REGN20484.

[0141] Array variant The antigen-binding molecules of this disclosure may include one or more amino acid substitutions, insertions, and / or deletions in the framework region and / or CDR region of the heavy chain variable domain and / or light chain variable domain, compared to the corresponding germline sequence from which each antigen-binding domain originates. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from publicly available antibody sequence databases. The antigen-binding molecules of this disclosure may include antigen-binding fragments derived from any of the exemplary amino acid sequences disclosed herein, where one or more amino acids in one or more framework regions and / or CDR regions are mutated to corresponding residues(may be) of the germline sequence from which the antibody originates, or to corresponding residues(may be) of another human germline sequence, or to conserved amino acid substitutions of corresponding germline residues(may be) (such sequence changes are collectively referred to herein as “germline mutations”). Those skilled in the art can readily construct a number of antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof, starting from the heavy chain variable region sequences and light chain variable region sequences disclosed herein. In a particular embodiment, V H Domain and / or V LAll framework residues and / or CDR residues within the domain are mutated to revert to residues found in the original germline sequence from which the antigen-binding domain originally originated. In other embodiments, only certain residues, for example, only mutant residues found in the first eight amino acids of FR1 or the last eight amino acids of FR4, or only mutant residues found in CDR1, CDR2, or CDR3, are mutated to revert to the original germline sequence. In other embodiments, one or more framework residues and / or CDR residues are mutated to corresponding residues in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antigen-binding domain originally originated). Furthermore, the antigen-binding domain may contain any combination of two or more germline mutations within the framework region and / or CDR region, for example, certain individual residues are mutated to corresponding residues in a particular germline sequence, while other specific residues different from the original germline sequence are maintained or mutated to corresponding residues in a different germline sequence. Once obtained, antigen-binding domains containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties, or reduced immunogenicity. Bispecific antigen-binding molecules containing one or more antigen-binding domains obtained by this general method are included within this disclosure.

[0142] This disclosure also includes antigen-binding molecules comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein, wherein one or both antigen-binding domains have one or more conserved substitutions. For example, the present invention includes antigen-binding molecules comprising antigen-binding domains having HCVR, LCVR, and / or CDR amino acid sequences having, for example, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or more conserved amino acid substitutions with respect to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. A “conservative amino acid substitution” is defined as the substitution of an amino acid residue with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of the protein. Examples of amino acids having side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conserved amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative permutation is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445. A "moderately conservative" permutation is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0143] This disclosure also includes antigen-binding molecules comprising an antigen-binding domain having an HCVR, LCVR, and / or CDR amino acid sequence substantially identical to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. In some embodiments, the antigen-binding molecule comprises an HCVR, LCVR, and / or CDR amino acid sequence having at least 85% sequence identity with respect to the sequences disclosed in Table 36, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In some embodiments, the antigen-binding molecule comprises an HCVR, LCVR, and / or CDR amino acid sequence having at least 85% sequence identity to the sequences disclosed in Table 36, e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, where the amino acid residue(s) difference from the sequences disclosed in Table 36 is a conserved substitution or a moderately conserved substitution.

[0144] Antigen-binding proteins containing Fc modification In some embodiments, the CD40 antigen-binding molecules disclosed herein (e.g., CD40 × CD40 bispecific antigen-binding molecules disclosed in any one of Tables 1-4) include an Fc domain containing one or more modifications or mutations that enhance or reduce antibody binding to the FcRn receptor. For example, the disclosure includes antigen-binding molecules containing one or more mutations in the CH2 and / or CH3 regions of the Fc domain, where the mutation(s) increase the affinity of the Fc domain to FcRn in an acidic environment (e.g., in endosomes where the pH is in the range of about 5.5 to about 6.0). Such mutations may prolong the serum half-life of the antibody when administered to animals.

[0145] Non-restrictive examples of such Fc modifications include modifications at position 250 (e.g., E or Q), positions 250 and 428 (e.g., L or F), position 252 (e.g., L / Y / F / W or T), position 254 (e.g., S or T), and position 256 (e.g., S / R / Q / E / D or T), or modifications at position 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y), or modifications at position 250 and / or 428, or modifications at position 307 or 308 (e.g., 308F, V308F) and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications, 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications, 433K (e.g., H433K) and 434 (e.g., 434Y) modifications, 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications, 250Q and 428L modifications (e.g., T250Q and M428L), and 307 and / or 308 modifications (e.g., 308F or 308P). See, for example, Ko et al., BioDrugs 2021, 35:147-157.

[0146] In a particular embodiment, the CD40×CD40 bispecific antigen-binding molecule includes an Fc domain comprising one or more mutant pairs or groups selected from the group consisting of 250Q and 248L (e.g., T250Q and M248L), 252Y, 254T and 256E (e.g., M252Y, S254T and T256E), 428L and 434S (e.g., M428L and N434S), and 433K and 434F (e.g., H433K and N434F).

[0147] In some embodiments, the CD40×CD40 bispecific antigen-binding molecules of this disclosure include a modified Fc domain with reduced effector function. As used herein, “modified Fc domain with reduced effector function” means any Fc portion of an immunoglobulin that has been modified, mutated, cleaved, etc., compared to a wild-type naturally occurring Fc domain, and as a result, molecules containing the modified Fc exhibit a reduced severity or degree of at least one effect selected from the group consisting of cell death (e.g., ADCC and / or CDC), complement activation, phagocytosis, and opsonization, compared to a comparator molecule containing a wild-type naturally occurring version of the Fc portion. In certain embodiments, “modified Fc domain with reduced effector function” is an Fc domain in which binding to an Fc receptor (e.g., FcγR) is reduced or attenuated.

[0148] In certain embodiments, a modified Fc domain with reduced binding to an Fc receptor (e.g., an Fcγ receptor, e.g., FcγRI, FcγRIIA, FcγRIIB, or FcγRIIIA) is a variant IgG1 Fc or variant IgG4 Fc that includes one or more substitutions or modifications in the hinge region and / or CH region (e.g., CH2). For example, a modified Fc domain may include a variant IgG1 Fc in which at least one amino acid in the IgG1 Fc hinge region and / or CH region is substituted with a corresponding amino acid from the IgG2 Fc hinge region and / or CH region. Alternatively, a modified Fc domain may include a variant IgG4 Fc in which at least one amino acid in the IgG4 Fc hinge region and / or CH region is substituted with a corresponding amino acid from the IgG2 Fc hinge region and / or CH region. In some embodiments, a modified Fc domain includes modifications in which each of positions 233-236 according to EU numbering is occupied by G or not occupied. Non-limiting and exemplary modified Fc regions that can be used in the context of this disclosure are described in U.S. Patent No. 11,518,807 (the disclosure of which is incorporated herein by reference in its entirety), and any functionally equivalent variants of the modified Fc regions described therein can also be used. Other modified Fc domains and Fc modifications that can be used in the context of this disclosure include any of the modifications described in U.S. 8,697,396, U.S. 10,988,537, U.S. 2014 / 0171623, U.S. 2014 / 0134162, U.S. 2014 / 0243504, and WO 2014 / 043361, the respective disclosures of which are incorporated herein by reference.

[0149] In certain embodiments, the bispecific antigen-binding molecules disclosed herein include an immunoglobulin heavy chain that is a heterodimer (i.e., differing by at least one amino acid) and has different affinities to affinity reagents such as protein A. In some embodiments, one of the heavy chains includes one or more modifications to the Fc domain that reduce or eliminate the binding of the Fc domain to protein A. In some embodiments, one of the heavy chains includes an H435R / Y436F (according to the EU numbering system) substitution in the CH3 region. Non-limiting and exemplary modified Fc regions that can be used in the context of this disclosure are described in U.S. Patent No. 8,586,713, the disclosure of which is incorporated herein by reference in its entirety.

[0150] It is intended that all possible combinations of the aforementioned Fc domain mutations and other mutations within the antibody variable domain disclosed herein fall within the scope of this disclosure.

[0151] Polynucleotides, vectors, and host cells In another aspect, the disclosure provides nucleic acid molecules comprising one or more polynucleotide sequences encoding an antigen-binding molecule disclosed herein, as well as vectors encoding such polynucleotide sequences (e.g., expression vectors), and host cells into which such vectors have been introduced.

[0152] The polynucleotides disclosed herein may encode all or part of the antigen-binding molecules, antibodies, or antigen-binding fragments disclosed throughout this disclosure. In some cases, a single polynucleotide may encode both the HCVR and LCVR of an antibody or antigen-binding fragment (for example, defined by reference to the CDR contained within the HCVR and LCVR as defined by their respective amino acid sequences, or by reference to the amino acid sequences of the CDRs of the HCVR and LCVR, or by reference to the amino acid sequences of the HCVR and LCVR, respectively), or the HCVR and LCVR may be encoded by separate polynucleotides (i.e., a pair of polynucleotides). In the latter case, where the HCVR and LCVR are encoded by separate polynucleotides, the polynucleotides may be combined into a single vector or contained in separate vectors (i.e., a pair of vectors). In either case, the host cell used to express the polynucleotide(s) or vector(s) may contain the complete complement of the components for generating the antibody or its antigen-binding fragment. For example, a host cell may contain separate vectors encoding the HCVR and LCVR of the antibody or its antigen-binding fragment discussed above or herein. Similarly, polynucleotides (one or more) and vectors (one or more) may be used to express the full-length heavy chain and full-length light chain of the antibody discussed above or herein. For example, a host cell may contain a single vector having polynucleotides encoding both the heavy chain and light chain of the antibody, or the host cell may contain separate vectors having polynucleotides encoding the heavy chain and light chain of the antibody discussed above or herein.

[0153] In some embodiments, the nucleic acid molecule comprises one or more polynucleotide sequences encoding an antigen-binding molecule as disclosed in any of Tables 1-4. In some embodiments, the nucleic acid molecule comprises one or more polynucleotide sequences as described in Table 36.

[0154] In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence encoding CD40 HCVR, including HCDR1, HCDR2, and HCDR3 of SEQ ID NOs. 4, 6, and 8, SEQ ID NOs. 24, 26, and 28, or SEQ ID NOs. 34, 36, and 38, respectively. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence encoding CD40 HCVR, including or consisting of the sequence of SEQ ID NOs. 2, SEQ ID NOs. 22, or SEQ ID NOs. 32.

[0155] In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence encoding LCVR, comprising LCDR1 containing or consisting of the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence encoding LCVR, comprising the sequence of SEQ ID NO: 10.

[0156] In some embodiments, compositions comprising one or more nucleic acid molecules disclosed herein are provided. For example, in some embodiments, the composition comprises a first nucleic acid molecule comprising a polynucleotide sequence encoding HCVR and / or LCVR of a first antigen-binding molecule that binds to a first epitope of CD40, and a second nucleic acid molecule comprising a polynucleotide sequence encoding HCVR and / or LCVR of a second antigen-binding molecule that binds to a second epitope of CD40. In some embodiments, the composition comprises a first nucleic acid molecule comprising a polynucleotide sequence encoding HCVR of a first antigen-binding molecule that binds to a first epitope of CD40, a second nucleic acid molecule comprising a polynucleotide sequence encoding LCVR of a first antigen-binding molecule that binds to a first epitope of CD40, a third nucleic acid molecule comprising a polynucleotide sequence encoding HCVR of a second antigen-binding molecule that binds to a second epitope of CD40, and a fourth nucleic acid molecule comprising a polynucleotide sequence encoding LCVR of a second antigen-binding molecule that binds to a second epitope of CD40. In some embodiments, the HCVR sequences of the first and second antigen-binding molecules are selected from CD40 HCVR containing HCDR1, HCDR2, and HCDR3 of SEQ ID NOs. 4, 6, and 8, respectively; CD40 HCVR containing HCDR1, HCDR2, and HCDR3 of SEQ ID NOs. 24, 26, and 28, respectively; and CD40 HCVR containing HCDR1, HCDR2, and HCDR3 of SEQ ID NOs. 34, 36, and 38, respectively. In some embodiments, the HCVR sequences of the first and second antigen-binding molecules are selected from SEQ ID NOs. 2, 22, and 32. In some embodiments, the LCVR sequences of the first and second antigen-binding molecules each contain the amino acid sequence of SEQ ID NO. 12, or LCDR1 consisting thereof, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence of SEQ ID NO. 16. In some embodiments, the LCVR sequences of the first and second antigen-binding molecules each contain the sequence of SEQ ID NO. 10.

[0157] In another embodiment, the Disclosure also provides recombinant expression vectors carrying one or more nucleic acid molecules disclosed herein, as well as host cells into which such vectors have been introduced. In some embodiments, the host cell is a prokaryotic cell (e.g., Escherichia coli). In some embodiments, the host cell is a eukaryotic cell, e.g., a non-human mammalian cell (e.g., Chinese hamster ovary (CHO) cell). The Disclosure also provides a method for producing antigen-binding molecules of the Disclosure by culturing host cells under conditions that enable the production of antigen-binding molecules and recovering the antigen-binding molecules thus produced.

[0158] Characterization of CD40 x CD40 bispecific antigen-binding molecules This disclosure includes antibodies that bind to human CD40 with high affinity and their antigen-binding fragments, such as bispecific antigen-binding molecules that bind to two different epitopes of CD40. In some embodiments, the antibody and its antigen-binding fragment (e.g., bispecific antigen-binding molecule) bind to CD40 and inhibit CD40L-induced activation, but do not possess agonist activity and / or cytotoxic effector function.

[0159] In some embodiments, the present disclosure measures K levels less than approximately 75 nM (e.g., at 25°C or 37°C) when measured by surface plasmon resonance using an assay format such as that described in Example 2 of this specification. DThe antigen-binding molecules of this disclosure include bispecific antigen-binding molecules that bind to human CD40 (for example, bispecific antibodies having the amino acid sequence of REGN16334, REGN16335, REGN16431, REGN16432, or REGN20484, as disclosed herein). In certain embodiments, the antigen-binding molecules of this disclosure, when measured by surface plasmon resonance using, for example, the assay format defined in Example 2 herein or substantially similar assays, have a magnitude of less than approximately 75 nM, less than approximately 70 nM, less than approximately 60 nM, less than approximately 50 nM, less than approximately 40 nM, less than approximately 30 nM, less than approximately 25 nM, less than approximately 20 nM, less than approximately 15 nM, less than approximately 10 nM, less than approximately 5 nM, less than approximately 1 nM, and less than approximately 5 K levels below 00 pM, approximately below 400 pM, approximately below 300 pM, approximately below 200 pM, approximately below 100 pM, approximately below 90 pM, approximately below 80 pM, approximately below 70 pM, approximately below 60 pM, approximately below 50 pM, approximately below 40 pM, approximately below 30 pM, approximately below 20 pM, approximately below 10 pM, approximately below 5 pM, approximately below 4 pM, approximately below 2 pM, approximately below 1 pM, approximately below 0.5 pM, approximately below 0.2 pM, approximately below 0.1 pM, or approximately below 0.05 pM. D It then binds to human CD40.

[0160] In some embodiments, the disclosure includes bispecific antigen-binding molecules with improved half-lives compared to monospecific antibodies (e.g., parent CD40 antibodies) (e.g., bispecific antibodies having the amino acid sequences of the bispecific antibodies disclosed herein, e.g., REGN16334, REGN16335, REGN16431, REGN16432, or REGN20484). In some embodiments, the disclosure includes bispecific antigen-binding molecules with improved half-lives (t) of more than approximately 70 minutes when measured by surface plasmon resonance at 25°C using, for example, the assay format defined in Example 2 herein, or substantially similar assays. 1 / 2) comprises a bispecific antigen-binding molecule (e.g., a bispecific antibody disclosed herein) that binds to human CD40. In certain embodiments, the antibody or antigen-binding fragment of this disclosure, when measured by surface plasmon resonance at 25°C using the assay format defined in Example 2 herein or substantially similar assays, has a duration of more than about 75 minutes, more than about 80 minutes, more than about 85 minutes, or more than about 90 minutes. 1 / 2 It then binds to human CD40.

[0161] In some embodiments, the Disclosure includes a bispecific antigen-binding molecule that inhibits the binding of human CD40 (e.g., the monomeric form of hCD40) to human CD40L (e.g., a bispecific antibody having the amino acid sequence of REGN16334, REGN16335, REGN16431, REGN16432, or REGN20484, as disclosed herein). In some embodiments, the inhibition of CD40 binding to CD40L is measured using an ELISA-based blocking assay as described in Example 4 herein. In some embodiments, the bispecific antigen-binding molecule inhibits the binding of CD40 (e.g., the hCD40 monomer) to human CD40L by at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, or more, using, for example, the assay format defined in Example 4 herein, or substantially similar assays.

[0162] In some embodiments, the Disclosure includes a bispecific antigen-binding molecule that specifically interacts with (e.g., binds to) cells expressing CD40 (e.g., a bispecific antibody having the amino acid sequence of REGN16334, REGN16335, REGN16431, REGN16432, or REGN20484, as disclosed herein). The degree to which the antigen-binding molecule binds to cells expressing CD40 can be evaluated by flow cytometry, as shown in Example 5 below. For example, in some embodiments, the Disclosure provides a CD40 × CD40 bispecific antibody that specifically binds to cells expressing CD40 on the cell surface (e.g., primary human B cells, or human B cell lines such as Ramos 2G6.4C10). In some embodiments, the Disclosure provides an EC of about 10 nM or less, e.g., about 0.5 nM to about 10 nM, as measured by flow cytometry or substantially similar assays as described in Example 5. 50 Values ​​such as approximately 1 nM, 1.5 nM, 2 nM, 2.5 nM, 3 nM, 3.5 nM, 4 nM, 4.5 nM, 5 nM, 5.5 nM, 6 nM, 6.5 nM, 7 nM, 7.5 nM, 8 nM, 8.5 nM, 9 nM, 9.5 nM, or 10 nM EC 50 This invention provides a CD40 × CD40 bispecific antibody that binds to CD40-expressing cells or cell lines at specific values.

[0163] In some embodiments, the disclosure includes a bispecific antigen-binding molecule that inhibits CD40L-induced activation (e.g., a bispecific antibody having the amino acid sequence of REGN16334, REGN16335, REGN16431, REGN16432, or REGN20484, as disclosed herein). In some embodiments, CD40L-induced activation is measured using a reporter assay, e.g., a luciferase-based reporter assay that quantitatively evaluates receptor activation in CD40-expressing cells by measuring downstream gene expression. In some embodiments, the reporter assay is the assay described in Example 6 herein. In some embodiments, CD40L-induced activation is measured by a cytokine secretion assay (e.g., secretion of IL-6, IL-10, IL-23, or TNFα) in the presence of CD40L. In some embodiments, the cytokine secretion assay is performed in primary cells expressing CD40 (e.g., human B cells). In some embodiments, the cytokine secretion assay is performed in a stable cell line, for example, a cell line expressing a reporter gene such as CD40 and luciferase. In some embodiments, the cytokine secretion assay is the assay described in Example 7 herein. In some embodiments, the CD40 × CD40 bispecific antibody inhibits CD40L-induced activation (e.g., reporter gene expression or cytokine secretion) by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more, compared to a control or reference value.

[0164] In some embodiments, the disclosure includes a bispecific antigen-binding molecule that significantly inhibits CD40 in the absence of CD40L (e.g., a bispecific antibody having the amino acid sequence of REGN16334, REGN16335, REGN16431, REGN16432, or REGN20484, as disclosed herein). As used herein, “significantly inhibits CD40 in the absence of CD40L” means that the level of CD40 activation, as measured by downstream gene expression or cytokine secretion, is less than 15%, e.g., less than 13%, less than 10%, less than 8%, or less than 6%, in the presence of the bispecific antigen-binding molecule and in the absence of CD40L. In some embodiments, CD40 agonism is measured by a reporter assay, e.g., a luciferase-based reporter assay that quantitatively evaluates receptor activation in CD40-expressing cells by measuring downstream gene expression. In some embodiments, CD40 agonism is measured by a cytokine secretion assay (e.g., secretion of IL-6, IL-10, IL-23, or TNFα) in the absence of CD40L. In some embodiments, the assay is the assay described in Example 6 or Example 8 herein.

[0165] Epitope mapping and related technologies In some embodiments, the epitope on CD40 to which the antigen-binding molecule of this disclosure binds (e.g., the first epitope of human CD40 to which the first antigen-binding domain (D1) binds, or the second epitope of human CD40 to which the second antigen-binding domain (D2) binds) may consist of a single continuous sequence of three or more amino acids (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) of the CD40 protein. Alternatively, the epitope may consist of multiple discontinuous amino acids (or amino acid sequences) of CD40. The term “epitope,” as used herein, refers to an antigenic determinant that interacts with a specific antigen-binding site known as a paratope in the variable region of an antibody molecule. A single antigen may have two or more epitopes. Therefore, different antibodies may bind to different regions on an antigen and may have different biological effects. Epitopes can be conformational or linear. Conformational epitopes are produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are epitopes produced by adjacent amino acid residues in a polypeptide chain. In certain circumstances, epitopes may include sugar, phosphoryl, or sulfonyl groups on the antigen.

[0166] Various techniques known to those skilled in the art can be used to determine whether the antigen-binding domain of an antibody "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques that can be used to determine the epitope or binding domain of a particular antibody or antigen-binding domain include, for example, conventional cross-blocking assays as described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), point mutagenesis (e.g., alanine scan mutagenesis, arginine scan mutagenesis, etc.), peptide blot analysis (Reineke, 2004, Methods Mol Biol 248:443-463), protease protection, and peptide cleavage analysis. Furthermore, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify the amino acids within a polypeptide with which an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. Generally speaking, the hydrogen / deuterium exchange method involves deuterizing the target protein and then conjugating an antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water to induce hydrogen-deuterium exchange at all residues except those protected by the antibody (which remain deuterium-labeled). After dissociating the antibody, protease cleavage and mass spectrometry are performed on the target protein to identify the deuterium-labeled residues corresponding to the specific amino acids with which the antibody interacts. See, for example, Ehring (1999) Analytical Biochemistry 267(2):252-259 and Engen and Smith (2001) Anal. Chem. 73:256A-265A. X-ray crystallography can also be used to identify the amino acids within the polypeptide with which the antibody interacts.

[0167] In some embodiments, the disclosure includes CD40 antibodies and CD40×CD40 bispecific antibodies that bind to the same epitope(s) as any of the specific exemplary antibodies described herein (e.g., antibodies comprising any of the amino acid sequences listed in Table 36 below). In some embodiments, the disclosure includes CD40 antibodies and CD40×CD40 bispecific antibodies that compete for binding to the exemplary antibodies described herein (e.g., antibodies comprising any of the amino acid sequences listed in Table 36 below).

[0168] In some embodiments, the Disclosure provides a CD40 × CD40 bispecific antibody comprising a first antigen-binding domain (D1) that binds to a first epitope of human CD40 and a second antigen-binding domain (D1) that binds to a second epitope of human CD40, wherein the first and second epitopes are different epitopes of CD40. In some embodiments, the first and second epitopes are non-overlapping epitopes.

[0169] In some embodiments, the disclosure provides a CD40 × CD40 bispecific antibody comprising a first antigen-binding domain (D1) that binds to a first epitope of human CD40 and a second antigen-binding domain (D1) that binds to a second epitope of human CD40, wherein D1 and D2 do not compete with each other for binding to human CD40.

[0170] Those skilled in the art can determine, by using common methods known in the art, whether a particular antigen-binding molecule (e.g., an antibody) or its antigen-binding domain binds to the same epitope as the reference antigen-binding molecule of the disclosure, or whether it competes for binding to the reference antigen-binding molecule of the disclosure. For example, to determine whether a test antibody binds to the same CD40 epitope as the reference bispecific antigen-binding molecule of the disclosure, the reference bispecific molecule is first bound to the CD40 protein. Then, the ability of the test antibody to bind to the CD40 molecule is evaluated. If the test antibody can bind to CD40 after saturated binding with the reference bispecific antigen-binding molecule, it can be concluded that the test antibody binds to a different CD40 epitope than the reference bispecific antigen-binding molecule. On the other hand, if the test antibody cannot bind to the CD40 molecule after saturated binding with the reference bispecific antigen-binding molecule, the test antibody may bind to the same CD40 epitope as the epitope to which the reference bispecific antigen-binding molecule of the disclosure binds. Subsequently, additional routine experiments (e.g., peptide mutation and binding analysis) can be performed to confirm whether the observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference bispecific antigen-binding molecule, or whether steric hindrance (or other phenomenon) is the cause of the observed lack of binding. This type of experiment can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. According to certain embodiments of this disclosure, for example, if, as measured by a competitive binding assay, one antigen-binding protein in 1, 2, 5, 10, 20, or 100-fold excess inhibits the binding of the other antigen-binding protein by at least 50%, preferably 75%, 90%, or 99%, then the two antigen-binding proteins bind to the same (or overlapping) epitopes (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502). Alternatively, if all essentially all amino acid mutations in an antigen that reduce or eliminate the binding of one antigen-binding protein also reduce or eliminate the binding of the other antigen-binding protein, then the two antigen-binding proteins are considered to bind to the same epitope.If a subset of amino acid mutations that reduces or eliminates the binding of one antigen-binding protein also reduces or eliminates the binding of the other antigen-binding protein, then the two antigen-binding proteins are considered to have a "duplicate epitope."

[0171] To determine whether an antibody or its antigen-binding domain competes for binding to a reference antigen-binding molecule, the above binding method is performed in two directions. In the first direction, the reference antigen-binding molecule is bound to the CD40 protein under saturated conditions, and then the binding of the test antibody to the CD40 molecule is evaluated. In the second direction, the test antibody is bound to the CD40 molecule under saturated conditions, and then the binding of the reference antigen-binding molecule to the CD40 molecule is evaluated. In both directions, if only the first (saturated) antigen-binding molecule can bind to the CD40 molecule, it is concluded that the test antibody and the reference antigen-binding molecule compete for binding to CD40. As will be understood by those skilled in the art, an antibody competing for binding to a reference antigen-binding molecule does not necessarily have to bind to the same epitope as the reference antibody, and may sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0172] Preparation of antigen-binding domains and construction of multispecific antigen-binding molecules Antigen-binding domains specific to a particular antigen can be prepared by any antibody production technique known in the art. Once obtained, the two different antigen-binding domains can be appropriately positioned relative to each other using conventional methods to produce the bispecific antigen-binding molecules of this disclosure (a discussion of exemplary bispecific antibody formats that can be used to construct the bispecific antigen-binding molecules of this disclosure is provided elsewhere in this specification). In certain embodiments, one or more of the individual components of the bispecific antigen-binding molecule (e.g., heavy and light chains) are derived from a chimeric antibody, a humanized antibody, or a fully human antibody. Methods for producing such antibodies are known in the art. For example, one or more of the heavy and / or light chains of the bispecific antigen-binding molecules of this disclosure can be prepared using VELOCIMMUNE® technology. Using VELOCIMMUNE® technology (or any other human antibody production technique), a high-affinity chimeric antibody against a particular antigen (e.g., CD40) having a human variable region and a mouse constant region is first isolated. Antibodies are characterized and selected based on desirable properties, including affinity, selectivity, and epitopes. The mouse constant region is replaced with a desired human constant region to generate fully human heavy and / or light chains that can be incorporated into bispecific antigen-binding molecules.

[0173] In some embodiments, genetically modified animals may be used to construct human bispecific antigen-binding molecules. For example, a genetically modified mouse incapable of rearranging and expressing an endogenous mouse immunoglobulin light chain variable sequence may be used, in which case the mouse expresses only one or two human light chain variable domains encoded by a human immunoglobulin sequence operably linked to a mouse kappa constant gene at the endogenous mouse kappa locus. Using such a genetically modified mouse, a fully human bispecific antigen-binding molecule can be constructed containing two different heavy chains that associate with the same light chain containing a variable domain derived from one of two different human light chain variable region gene segments (for example, see US2011 / 0195454, whose entire contents are incorporated herein by reference, for a detailed discussion of such a modified mouse and its use for constructing a bispecific antigen-binding molecule). As used herein, “completely human” means an antigen-binding molecule, such as an antibody, or its antigen-binding fragment or immunoglobulin domain, which, over the entire length of each polypeptide of the antigen-binding molecule, antibody, its antigen-binding fragment or immunoglobulin domain, contains an amino acid sequence encoded by DNA derived from a human sequence. In some cases, a completely human sequence is derived from a protein endogenous to humans. In other cases, a completely human protein or protein sequence contains a chimeric sequence in which each component sequence is derived from a human sequence. While not bound by either theory, chimeric proteins or chimeric sequences are generally designed to minimize the generation of immunogenic epitopes at the junctions of component sequences compared to, for example, any wild-type human immunoglobulin region or domain.

[0174] biological equivalent The present invention encompasses antigen-binding molecules having different amino acid sequences from those of the described antibodies but retaining the ability to bind to CD40. Such variant molecules include the addition, deletion, or substitution of one or more amino acids compared to the parent sequence, but exhibit biological activity essentially equivalent to that of the described antigen-binding molecules. Similarly, nucleic acid sequences encoding antigen-binding molecules of this disclosure include sequences encoding antigen-binding molecules that are essentially biologically equivalent to those disclosed herein, but include the addition, deletion, or substitution of one or more nucleotides compared to the disclosed sequences.

[0175] This disclosure includes any exemplary antigen-binding molecules described herein and any antigen-binding molecules that are bioequivalent to them. Two antigen-binding proteins (e.g., bispecific antibodies) are considered bioequivalent or drug equivalents if, for example, they are administered in the same molar dose under similar experimental conditions, either as a single dose or multiple doses, and there is no significant difference in absorption rate and degree of absorption. Some antibodies are considered equivalent or drug equivalents if they have equivalent degrees of absorption but not equivalent absorption rates; however, such differences in absorption rates may be considered bioequivalent because they are intentional, reflected in the labeling, and are not considered medically important to the particular drug under study, for example, because they are not essential for achieving effective drug concentrations in the body in chronic use.

[0176] In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically significant differences in their safety, purity, and potency.

[0177] In one embodiment, the two antigen-binding proteins are bioequivalent if the patient can switch between the first antigen-binding protein (e.g., a reference product) and the second antigen-binding protein (e.g., a biological product) one or more times, and the risk of clinically significant changes in immunogenicity or side effects such as decreased efficacy does not increase to an extent that would be expected compared to continuing treatment without such switching.

[0178] In one embodiment, two antigen-binding proteins are bioequivalent if they both act by a common mechanism of action (one or more) for the conditions of use (one or more) (as far as such mechanisms of action are known).

[0179] Bioequivalence can be demonstrated by in vivo and / or in vitro methods. Non-limiting examples of bioequivalence measurements include, for example, (a) in vivo studies in humans or other mammals measuring the concentration of an antibody or its metabolites in blood, plasma, serum, or other biological fluids as a function of time; (b) in vitro studies that correlate with and reasonably predict human in vivo bioavailability data; (c) in vivo studies in humans or other mammals measuring the appropriate acute pharmacological effect of an antibody (or its target) as a function of time; and (d) well-controlled clinical trials to establish the safety, efficacy, or bioavailability or bioequivalence of an antibody.

[0180] Bioequivalent variants of the exemplary bispecific antigen-binding molecules described herein can be constructed, for example, by making various substitutions of residues or sequences, or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, deleting or replacing cysteine ​​residues that are not essential for biological activity with other amino acids can prevent the formation of unwanted or inaccurate intramolecular disulfide bonds during regeneration. In other embodiments, bioequivalent antibodies may include the exemplary bispecific antigen-binding molecules described herein that include amino acid changes that alter the glycosylation properties of the antibody, such as mutations that eliminate or remove glycosylation.

[0181] Pharmaceutical composition In another embodiment, the present disclosure provides a pharmaceutical composition comprising a CD40 × CD40 bispecific antigen-binding molecule (e.g., a bispecific antibody) as disclosed herein. The pharmaceutical composition is formulated with one or more pharmaceutically acceptable vehicles, carriers, and / or excipients. Various pharmaceutically acceptable carriers and excipients are known in the art; see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. In some embodiments, the carrier is suitable for intravenous, intramuscular, oral, intraperitoneal, intrathecal, transdermal, topical, or subcutaneous administration.

[0182] In some embodiments, the pharmaceutical composition includes injectable formulations, such as dosage forms for intravenous injection, subcutaneous injection, intradermal and intramuscular injection, and intravenous infusion. These injectable formulations can be prepared by known methods. For example, an injectable formulation may be prepared by dissolving, suspending, or emulsifying the antibody or a salt thereof in a sterile aqueous or oily medium conventionally used for injection. Examples of aqueous injectable media include physiological saline, isotonic solutions containing glucose and other adjuvants, which may be used in combination with suitable solubilizers such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Examples of oily media include sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injectable formulations thus prepared can be filled into suitable ampoules.

[0183] The dose of antigen-binding molecules administered to a patient in accordance with this disclosure may vary depending on the patient's age and size, symptoms, condition, and route of administration. The dose is typically calculated based on body weight or body surface area. The frequency and duration of treatment may be adjusted depending on the severity of the condition. Effective doses and schedules for administering the pharmaceutical compositions disclosed herein can be determined empirically, for example, by monitoring the patient's progression through periodic evaluations and adjusting the dose accordingly. Furthermore, interspecies scaling of doses can be performed using methods known in the art (see, for example, Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0184] Various delivery systems (e.g., liposomes, microparticles, microcapsule encapsulation, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis) are known and can be used to administer pharmaceutical compositions (see, for example, Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions may be administered by any convenient route, for example, by injection or bolus injection, by absorption via epithelial or mucocutaneous junctions (e.g., oral mucosa, rectal and intestinal mucosa), or in combination with other bioactive agents. In some embodiments, the pharmaceutical compositions disclosed herein are administered intravenously. In some embodiments, the pharmaceutical compositions disclosed herein are administered subcutaneously.

[0185] In some embodiments, the antigen-binding molecules or pharmaceutical compositions comprising the antigen-binding molecules disclosed herein are contained in a container. Thus, in another embodiment, a container containing the antigen-binding molecules or pharmaceutical compositions disclosed herein is provided. For example, in some embodiments, the antibody or pharmaceutical composition is contained in a container selected from the group consisting of glass vials, syringes, pen-type delivery devices, and auto-injectors.

[0186] In some embodiments, the antigen-binding molecules or pharmaceutical compositions of the Disclosure are delivered, for example, subcutaneously or intravenously, using a standard needle and syringe. In some embodiments, the syringe is a pre-filled syringe. In some embodiments, a pen-type delivery device or auto-injector is used to deliver the pharmaceutical compositions of the Disclosure (e.g., for subcutaneous delivery). Pen-type delivery devices may be reusable or disposable. Reusable pen-type delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen-type delivery device can then be reused. Disposable pen-type delivery devices do not have replaceable cartridges. Rather, disposable pen-type delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. When the pharmaceutical composition in the reservoir is empty, the entire device is discarded.

[0187] Examples of suitable pen-type and auto-injector delivery devices include AUTOPEN® (Owen Mumford, Inc., Woodstock, UK), DISETRONIC® pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25® pen, HUMALOG® pen, HUMALIN 70 / 30® pen (Eli Lilly and Co., Indianapolis, INN), NOVOPEN® I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR® (Novo Nordisk, Copenhagen, Denmark), BD® pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN®, OPTIPEN PRO®, and OPTIPEN®. Examples of disposable pen-type delivery devices applicable to the subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, STARLET (trademark) and OPTICLIK (trademark) (sanofi-aventis, Frankfurt, Germany). Examples of, but are not limited to, SOLOSTAR (trademark) pen (sanofi-aventis), FLEXPEN (trademark) (Novo Nordisk), KWIKPEN (trademark) (Eli Lilly), SURECLICK (trademark) auto-injector (Amgen, Thousand Oaks, CA), PENLET (trademark) (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA (trademark) pen (Abbott Labs, Abbott Park, IL).

[0188] In some embodiments, the pharmaceutical composition is delivered using a controlled-release system. In one embodiment, a pump may be used (see Langer, Sefton, 1987, CRC Crit.Ref.Biomed.Eng.14:201 above). In another embodiment, a polymer material may be used. See, for example, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Press, Boca Raton, Florida. In yet another embodiment, the controlled-release system may be placed near the target of the composition, thereby requiring only a fraction of the systemic dose (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, vol.2, pp.115-138 above). Other controlled-release systems are described in the review by Langer, 1990, Science 249:1527-1533.

[0189] In some embodiments, the pharmaceutical compositions for use described herein are prepared into unit dose dosage forms suitable for the dose of the active ingredient. Such unit dose dosage forms include, for example, tablets, pills, capsules, injections (ampoules), and suppositories. In some embodiments, the amount of antigen-binding molecules contained in the dosage form is about 5 to about 1000 mg, for example, about 5 to about 500 mg, about 5 to about 100 mg, or about 10 to about 250 mg.

[0190] therapeutic use In another embodiment, the Disclosure provides a method using the antigen-binding molecule of the Disclosure (e.g., the CD40×CD40 bispecific antigen-binding molecule disclosed herein). In some embodiments, the Disclosure provides a method for inhibiting CD40L-induced activation of CD40 in or on cells expressing CD40 (e.g., B cells, dendritic cells, monocytes, platelets, or macrophages). In some embodiments, the method involves contacting CD40-expressing cells with the CD40×CD40 bispecific antigen-binding molecule disclosed herein.

[0191] In some embodiments, the Disclosure provides a method for treating, improving, or preventing a CD40-mediated disease or condition by administering a therapeutically effective amount of a CD40 antigen-binding molecule, such as a CD40 × CD40 bispecific antigen-binding molecule, to a subject in need. A CD40-mediated disease or condition, as used herein, is any disease or condition caused by or exacerbated by the activity of CD40 (e.g., activation of downstream signaling by CD40 binding to its ligand, CD40L). In some embodiments, the CD40-mediated disease or condition is caused by mutations in CD40 or CD40L, or in genes in the CD40 signaling pathway.

[0192] In some embodiments, the CD40-mediated disease or condition is an autoimmune disease or condition, an inflammatory disease or condition, a cardiovascular disease or condition, or an organ transplantation. In some embodiments, the CD40-mediated disease or condition is an autoimmune disease or condition. In some embodiments, the CD40-mediated disease or condition is Addison's disease, autoimmune hemolytic anemia, autoimmune thyroid disease (e.g., thyroiditis, Graves' disease, or Hashimoto's thyroiditis), Crohn's disease, diabetes (e.g., type 1 diabetes), experimental autoimmune encephalomyelitis (EAE), focal segmental glomerulosclerosis (FSGS), glomerulonephritis, Guillain-Barré syndrome, graft-versus-host disease (GVHD) (e.g., acute GVHD or chronic GVHD), hemolytic anemia, hidradenitis suppurativa (HS), immune thrombocytopenia, inflammatory bowel disease, inflammatory neuropathy (e.g., acute inflammatory demyelinating polyneuropathy (AIDP) or chronic inflammatory demyelinating polyneuropathy (CIDP)), Kawasaki disease, lupus nephritis, mixed connective tissue disease (MCTD), multiple sclerosis, myasthenia gravis, neuromyelitis optica spectrum disorder, organ transplantation (e.g., kidney transplantation or liver transplantation), pemphigus, primary biliary cirrhosis, psoriasis, psoriatic arthritis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Sjögren's syndrome, skin transplantation, spondyloarthritis, systemic lupus erythematosus, systemic sclerosis, transplant rejection, vasculitis, ANCA-associated vasculitis (e.g., granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and / or eosinophilic GPA (EGPA)), ulcerative colitis, or Wegener's granulomatosis.

[0193] In some embodiments, administration of a CD40 antigen-binding molecule, such as a CD40×CD40 bispecific antigen-binding molecule (e.g., disclosed in any one of Tables 1-4), prevents or delays an increase in disease symptoms or progression of the disease in a subject having a CD40-mediated disease or condition.

[0194] Dosage and Administration Regimen In some embodiments, the amount of the CD40 antigen-binding molecule, such as a CD40×CD40 bispecific antigen-binding molecule (e.g., bispecific antibody), administered to a subject according to the methods disclosed herein is a therapeutically effective amount. As used herein, the phrase "therapeutically effective amount" means an amount that produces the desired effect for which it is administered.

[0195] In some embodiments, the CD40 antigen-binding molecule (e.g., CD40×CD40 bispecific antibody) is administered to a subject at a dose based on body weight. A "dose based on body weight" (e.g., a dose in units of mg / kg) is a dose of the antigen-binding molecule that varies according to the body weight of the subject.

[0196] In other embodiments, the CD40 antigen-binding molecule (e.g., CD40×CD40 bispecific antibody) is administered at a fixed dose. A "fixed dose" (e.g., a dose in units of mg) means that one dose of the antigen-binding molecule is used for all subjects regardless of any specific subject-related factor (e.g., body weight). In one particular embodiment, the fixed dose of the antigen-binding molecule is based on a predetermined body weight or age.

[0197] Typically, an appropriate dose of the antigen-binding molecule can range from about 0.001 to about 200.0 milligrams per kilogram of the recipient's body weight, generally from about 1 to 50 mg per kilogram of body weight. For example, the antigen-binding molecule can be administered at about 0.1 mg / kg, about 0.2 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 3 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 40 mg / kg, or about 50 mg / kg per single dose. Intermediate values and ranges of the recited values are also intended to be part of the present disclosure.

[0198] In some embodiments, the antigen-binding molecule is administered in a fixed dose of approximately 5 mg to approximately 2500 mg. In some embodiments, the antigen-binding molecule is administered in doses of approximately 5 mg, approximately 10 mg, approximately 15 mg, approximately 20 mg, approximately 25 mg, approximately 30 mg, approximately 50 mg, approximately 75 mg, approximately 100 mg, approximately 125 mg, approximately 150 mg, approximately 175 mg, 200 mg, approximately 225 mg, approximately 250 mg, approximately 275 mg, approximately 300 mg, approximately 325 mg, approximately 350 mg, approximately 375 mg, approximately 400 mg, approximately 425 mg, approximately 450 mg, approximately 475 mg, and approximately 500 mg. It is administered in fixed doses of mg, approximately 525 mg, approximately 550 mg, approximately 575 mg, approximately 600 mg, approximately 625 mg, approximately 650 mg, approximately 675 mg, approximately 700 mg, approximately 725 mg, approximately 750 mg, approximately 775 mg, approximately 800 mg, approximately 825 mg, approximately 850 mg, approximately 875 mg, approximately 900 mg, approximately 925 mg, approximately 950 mg, approximately 975 mg, approximately 1000 mg, approximately 1500 mg, approximately 2000 mg, or approximately 2500 mg. Intermediate values ​​and ranges of the listed values ​​are also intended to be part of this disclosure.

[0199] In some embodiments, the CD40 antigen-binding molecule (e.g., a CD40 × CD40 bispecific antibody) is administered to the subject at a frequency of approximately four times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, once every twelve weeks, or less, as long as a therapeutic response is obtained.

[0200] In some embodiments, multiple doses of a CD40 antigen-binding molecule (e.g., a CD40 × CD40 bispecific antibody) are administered to a subject over a defined period of time. In some embodiments, the method of the present disclosure includes sequential administration of multiple doses of antigen-binding molecules to a subject. As used herein, “sequential administration” means that each dose of the antigen-binding molecule is administered to the subject at different times, for example, on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). In some embodiments, the method of the present disclosure includes sequentially administering a single initial dose of the antigen-binding molecule, followed by one or more secondary doses of the antigen-binding molecule, and optionally followed by one or more tertiary doses of the antigen-binding molecule to a patient.

[0201] The terms “initial dose,” “secondary doses,” and “tertiary doses” refer to the chronological order of administration of antigen-binding molecules. Therefore, the “initial dose” is the amount administered at the start of the treatment regimen (also called the “loading dose”), the “secondary dose” is the amount administered after the initial dose, and the “tertiary dose” is the amount administered after the secondary dose. In some embodiments, the initial dose, secondary dose, and tertiary dose may all contain the same amount of antigen-binding molecules, but may differ in terms of administration frequency. In some embodiments, the amounts of antigen-binding molecules in the initial dose, secondary dose, and / or tertiary dose may differ from each other during the course of treatment (e.g., adjusted up or down as needed). In certain embodiments, one or more doses (e.g., 1, 2, 3, 4, or 5) are administered as a “loading dose” at the start of the treatment regimen, followed by subsequent doses (e.g., “maintenance doses”) at a less frequent frequency. In some embodiments, the initial dose and one or more secondary doses each contain the same amount of antigen-binding molecules. In other embodiments, the initial dose comprises a first amount of antigen-binding molecule, and one or more secondary doses each comprise a second amount of antigen-binding molecule. For example, the first amount of antigen-binding molecule may be 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 5 times, or more of the second amount of antigen-binding molecule.

[0202] In some embodiments, each secondary and / or tertiary dose is administered 1 to 14 weeks after the immediately preceding dose (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5 weeks or more). The term "immediately preceding dose," as used herein, means the dose of the antigen-binding molecule administered to the patient in a sequence of multiple doses without an intermediate dose before the next dose in that sequence.

[0203] The methods of the present disclosure may include administering to a patient any number of secondary and / or tertiary doses of an antigen-binding molecule. For example, in one particular embodiment, only a single secondary dose is administered to the patient. In another embodiment, two or more secondary doses (e.g., two, three, four, five, six, seven, eight or more) are administered to the patient. Similarly, in one particular embodiment, only a single tertiary dose is administered to the patient. In another embodiment, two or more tertiary doses (e.g., two, three, four, five, six, seven, eight or more) are administered to the patient.

[0204] In some embodiments involving multiple secondary doses, each secondary dose is administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient one, two, three, or four weeks after the previous dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose is administered at the same frequency as the other tertiary doses. Alternatively, the frequency with which secondary and / or tertiary doses are administered to the patient may vary during the treatment regimen. The administration frequency may also be adjusted by the physician during the course of treatment according to the individual patient's needs after clinical examinations.

[0205] Combination therapy In yet another embodiment, the disclosure includes compositions and therapeutic formulations comprising any exemplary antibody and bispecific antigen-binding molecule described herein in combination with one or more additional therapeutic agents, and methods of treatment comprising administering such combination to a subject in need. In some embodiments, the additional therapeutic agent(s) are immunomodulators or anti-inflammatory agents. In some embodiments, the additional therapeutic agent(s) are immunosuppressive therapies. In some embodiments, the additional therapeutic agent(s) are surgical procedures.

[0206] Exemplary additional therapeutic agents that can be administered in combination with, or in combination with, the antigen-binding molecule of the present invention include, for example, other CD40-CD40L inhibitors (e.g., CD40 antibodies such as iscarimab [CFZ-533], preserumab [ASKP1240], BI 655064, cd5D12, or FFP104, or CD40L antibodies or antigen-binding proteins such as dapyrolizumab pegol, dazodalibep [VIB4920], flexalimab [INX-021], letrizumab [BMS-986004], MR-1, luprizumab [BG9588], tegopulvert [AT-1501], or tralizumab [IDEC-131]); B-cell depletion agents (e.g., CD19 antibody, CD20 antibody, Direct agents such as CD22 antibodies, or indirect agents such as BLyS inhibitors or APRIL inhibitors); plasma cell depletion agents (e.g., B cell maturation antigen (BCMA) targeting agents, BCMA x CD3 bispecific antibodies, CD38 antibodies (e.g., daratumumab), proteasome inhibitors, histone deacetylase inhibitors, B cell activator (BAFF) inhibitors, APRIL inhibitors); steroids (e.g., betamethasone, clobetasol, dexamethasone, fluocinol Corticosteroids such as nonide, halobetazole, hydrocortisone, methylprednisolone, prednisone, prednisolone, or triamcinolone, used topically, systemically, or orally, or inhaled corticosteroids; nonsteroidal topical agents, such as, but not limited to, PDE4 inhibitors or calcineurin inhibitors; nonsteroidal anti-inflammatory drugs (NSAIDs), such as, but not limited to, celecoxib, diclofenac, etodontol Lac, fenprofen, flurbiprofen, ibuprofen, ketoprofen, meclofenamet, meloxicam, nabumetone, naproxen, oxaprozin, piroxicam, rofecoxib, salicylate, sulfasalazine, sulindac, or tolmetin; anti-inflammatory antibodies or biological agents (e.g., TNFα antibodies or biological agents, e.g., adalimumab, certolizumab, etanercept, golimumab, or infliximab);IL-1 antibodies or biological agents, e.g., but not limited to LY2189102, anakinra, canakinumab, gevokizumab, or lilonacept; IL-6 / IL-6R antibodies or biological agents, e.g., but not limited to sarilumab, siltuximab, or tocilizumab; IL-17A / IL-17R antibodies or biological agents, e.g., but not limited to bimekizumab, brodalumab, ixekizumab, or ce Cukinumab; or IL-12 / IL-23 antibodies or biologics, e.g., but not limited to AMG139, BI655066, brazicumab, briankizumab, guselkumab, mirikizumab, risankizumab, tildrakizumab, or ustekinumab); JAK inhibitors, e.g., but not limited to abrocitinib, baricitinib, fedratinib, filgotinib, ruxolitinib, tofacitinib, or upada Examples include citinib; immunosuppressants (e.g., systemic immunosuppressants, e.g., methotrexate, cyclophosphamide, mizoribine, chlorambucil, cyclosporine, mycophenolate mofetil, or azathioprine); disease-modifying antirheumatic drugs (DMARDs), e.g., apremilast, azathioprine, baricitinib, cyclophosphamide, cyclosporine, hydroxychloroquine, leflunomide, methotrexate, mycophenolate mofetil, sulfasalazine, or tofacitinib; therapeutic proteins administered extracorporeally, such as enzymes (e.g., enzyme replacement therapy); radiotherapy; chemotherapy; intravenous immunoglobulin therapy; or surgery or surgical procedures (e.g., splenectomy, lymphadenectomy, thyroidectomy, plasmapheresis, leukocytapheresis, or cell, tissue, or organ transplantation).

[0207] In some embodiments, the CD40 antigen-binding molecules disclosed herein (e.g., CD40 × CD40 bispecific antigen-binding molecules, e.g., those disclosed in any one of Tables 1 to 4) can be administered in combination with or in combination with a B-cell depletion agent and / or a plasma cell depletion agent. In some embodiments, the B-cell depletion agent is a drug that directly targets B cells, e.g., a drug that binds to B-cell surface molecules. In some embodiments, the B-cell depletion agent is a CD19 antibody (e.g., MEDI-551), a CD20 antibody (e.g., rituximab, ocrelizumab, or ofatumumab), or a CD22 antibody (e.g., epratuzumab). In some embodiments, the depletion agent is a drug that indirectly targets B cells and / or plasma cells, e.g., by targeting B-cell or plasma cell survival factors. In some embodiments, the B-cell depletion agent is a BLyS / BAFF inhibitor (e.g., belimumab, BR3-Fc, or AMG-623), an APRIL inhibitor (e.g., atacicept), or a BLyS receptor 3 / BAFF receptor inhibitor (e.g., an anti-BR3 antibody). In some embodiments, the depletion agent is a BCMA targeting agent, e.g., a BCMA × CD3 bispecific antibody, a chimeric antigen receptor for BCMA, or a BCMA antibody conjugated to a cytotoxic drug (e.g., limbocertamab, REGN5459, pacanarotamab, or tecristamab).

[0208] Additional therapeutic active ingredients(s) may be administered immediately before, simultaneously with, or immediately after administration of the antigen-binding molecule of the present invention. For the purposes of this disclosure, such administration regimens are considered to administer the antigen-binding molecule "in combination" with the additional therapeutic active ingredients.

[0209] This disclosure includes pharmaceutical compositions in which the antigen-binding molecule of the present invention is co-formulated with one or more additional therapeutic active ingredients as described elsewhere in this specification. [Examples]

[0210] The following examples are presented to provide a complete disclosure and description to those skilled in the art of how to make and use the methods and compositions of the present disclosure, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to the numerical values used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

[0211] Example 1: Construction of a CD40×CD40 Bispecific Antibody Generation of the parental CD40 antibody Antibodies against CD40 were obtained by immunizing VELOCIMMUNE® mice (i.e., engineered mice containing DNA encoding human immunoglobulin heavy and kappa chain variable regions) with human CD40 antigen (human CD40 extracellular domain with a C-terminal MMH tag, SEQ ID NO: 53).

[0212] After immunization, antibodies were directly isolated from antigen-positive mouse B cells as described, for example, in U.S. Patent No. 7,582,298, which is incorporated herein by reference. Using this method, fully human CD40 antibodies (i.e., antibodies having human variable domains and human constant domains) were obtained. The antibodies generated using this method were characterized and selected for desired properties including affinity, selectivity, etc.

[0213] The CD40 antibodies generated using this method include antibodies named 30027P2, 21519P2, and 21520P2. Certain biological properties of the exemplary CD40 antibodies generated according to the method of this example are described in detail in the examples described below. [Table 1] [Table 2]

[0214] Generation of CD40 x CD40 bispecific antibodies Bispecific antibodies comprising a first CD40-binding arm ("first antigen-binding domain") and a second CD40-binding arm ("second antigen-binding domain") were constructed using standard methods, wherein the two CD40-binding arms contained distinct HCVRs paired with a common light chain. For example, as described in U.S. Patent No. 11,518,807, two different heavy chain constant regions were used for each CD40-binding arm. Exemplary CD40 × CD40 bispecific antibodies were generated according to this embodiment, and they contained the sequences shown in Tables 3-4 below.

[0215] As shown in Table 3, for REGN16334 and REGN16431, the first CD40 binding arm ("D1") contains the HCVR sequence of the parent antibody 21519P2, and the second CD40 binding arm ("D2") contains the HCVR sequence of the parent antibody 21520P2. REGN16334 and REGN16431 have different constant region modifications to reduce Fc receptor binding and effector function.

[0216] For REGN16335 and REGN16432, the first CD40-binding arm ("D1") contains the HCVR sequence of the parent antibody 30027P2, and the second CD40-binding arm ("D2") contains the HCVR sequence of the parent antibody 21520P2. REGN16335 and REGN16432 have different constant region modifications to reduce Fc receptor binding and effector function.

[0217] For REGN20484, the first CD40-binding arm ("D1") contains the HCVR sequence of the parent antibody 21520P2, and the second CD40-binding arm ("D2") contains the HCVR sequence of the parent antibody 30027P2.

[0218] REGN16334, REGN16335, REGN16431, REGN16432, and REGN20484 all share a common light chain sequence. [Table 3] [Table 4]

[0219] Example 2: Biacore binding kinetics of CD40 bivalent parent antibody and CD40×CD40 bispecific antibody Equilibrium dissociation constant (K) of CD40 bivalent and bispecific monoclonal antibodies (mAbs) D The CD40 bivalent parent antibody and the CD40×CD40 bispecific antibody were measured using a real-time surface plasmon resonance (SPR) based Biacore 4000 biosensor. All binding tests were performed at 25°C and 37°C in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% (v / v) surfactant Tween®-20, pH 7.4 (HBS-ET) electrophoresis buffer. The Biacore CM5 sensor surface was first derivatized by amine coupling with monoclonal mouse anti-human Fc antibody (REGN2567) to capture the CD40 bivalent parent antibody and the CD40×CD40 bispecific antibody. Different concentrations of CD40 reagent, human CD40 extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag ("hCD40-MMH", REGN3094, SEQ ID NO: 53), monkey CD40 extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag ("mfCD40-MMH", REGN3097, SEQ ID NO: 54), and mouse CD40 extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag ("mCD40-MMH", REGN3098, SEQ ID NO: 55) were injected at concentrations ranging from 3.7 nM to 100 nM or 3.3 nM to 90 nM in a series of 3-fold dilutions prepared in HBS-ET electrophoresis buffer at a flow rate of 30 μL / min for 4 minutes or 50 μL / min for 5 minutes. The dissociation of different CD40 reagents bound to the CD40 bivalent parent antibody and the CD40×CD40 bispecific antibody was monitored for 10 minutes in HBS-ET electrophoresis buffer. At the end of each cycle, the capture surfaces of the CD40 bivalent parent antibody and the CD40×CD40 bispecific antibody were regenerated by injecting 20 mM H3PO4 for 12 seconds.

[0220] The association rate (ka) and dissociation rate (kd) were determined by fitting the real-time binding sensorgram to a mass transfer-limited 1:1 binding model using Scrubber 2.0c curve fitting software. The association-dissociation equilibrium constant (K D ) and dissociation half-life (t1 / 2) were calculated from the kinetic rates as follows: K D (M) = k d / k a , and t 1 / 2 (min) = [ln(2) / (60 * k d )]<0001xx4><0001xx5><0001xx6>The binding kinetic parameters of different CD40 reagents to the CD40 bivalent parental antibody and CD40×CD40 bispecific antibody at 25 °C and 37 °C are shown in Tables 5 - 10. <0001xx7><0001xx8><0001xx9>As shown in Table 5, at 25 °C, the CD40 bivalent parental antibody and CD40×CD40 bispecific antibody bound to hCD40-MMH with K D values of 101 pM - 25.1 nM. At 25 °C, the CD40 bivalent parental antibody bound to mfCD40-MMH with K D values of 901 pM - 205 nM (Table 6). At 25 °C, the CD40 bivalent parental antibody did not bind to mCD40-MMH (Table 7). <0001xx0><0001xx1><0001xx2>As shown in Table 8, at 37 °C, the CD40 bivalent parental antibody and CD40×CD40 bispecific antibody bound to hCD40-MMH with KD values of 101 pM - 69.9 nM. At 37 °C, the CD40 bivalent parental antibody bound to mfCD40-MMH with a KD value of 3.16 nM (Table 9). At 37 °C, the CD40 bivalent parental antibody did not bind to mCD40-MMH (Table 10). <0001xx3><0001xx4>

Table 5

Table 6

Table 7

[0224] Example 3: Cross-competition between different CD40 monoclonal antibodies Binding competition between different CD40 monoclonal antibodies (mAbs) was measured using a real-time label-free biolayer interferometry (BLI) assay on the Octet HTX biosensor platform (Pall ForteBio Corp.). In addition to parent antibodies 21519P2, 21520P2, and 30027P2, a comparative CD40 antibody (REGN11209) was also tested. This comparative antibody contains the heavy and light chain sequences of iscarimab (see US8,828,396). The entire experiment was performed at 25°C with plate shaking at a speed of 1000 rpm in 10 mM HEPES buffer (pH 7.4, HBS-EP) containing 150 mM NaCl, 3 mM EDTA, 1 mg / mL BSA, 0.02% NaN3, and 0.05% v / v surfactant Tween®-20.

[0225] To evaluate the ability of one antibody to compete with another for binding to CD40, first, recombinant human CD40 extracellular domain (hCD40-MMH, SEQ ID NO: 53), approximately 0.47 nm to 0.54 nm in size and expressed with C-terminal myc-myc-hexahistidine, was captured on an Octet biosensor chip (Fortebio Inc, #18-5122) coated with anti-Penta-His antibody by immersing it in a well containing a 10 μg / mL solution of hCD40-MMH for 90 seconds. Next, the biosensor chip with the captured antigen was saturated with a first CD40 monoclonal antibody (hereinafter referred to as "mAb-1") by immersing it in a well containing a 50 μg / mL solution of mAb-1 for 4 minutes. Subsequently, the biosensor chip was immersed in a well containing a 50 μg / mL solution of a second CD40 monoclonal antibody (hereinafter referred to as "mAb-2") for 3 minutes. The biosensor chip was washed in HBS-EBT buffer between each step of the experiment. The binding response was monitored in real time, and the binding response at the end of each step was recorded. The binding response of mAb-2 to hCD40-MMH pre-complexed with mAb-1 was compared to the binding response of hCD40-MMH alone (isotype control sample). If pre-bound mAb-1 reduced the binding of mAb-2 by more than 50%, mAb-1 was considered a competitor to mAb-2.

[0226] The competitors of each antibody tested are summarized in Table 11 below. Parent antibodies 21519P2 and 21520P2 were found to compete with each other for binding to hCD40-MMH, but not with 30027P2. [Table 11]

[0227] Example 4: ELISA assay to evaluate the blocking activity of a CD40×CD40 bispecific antibody To determine the ability of a C40xCD40 bispecific antibody to block the binding of hCD40 monomers to hCD40L coated on plates, we developed an ELISA-based blockade assay. The recombinant human CD40-mmH protein used in the experiment (hCD40-MMH, REGN3094, SEQ ID NO: 53) contained a portion of the human CD40 extracellular domain (amino acids P20-R193) fused to 2×Myc peptide and 6×histidine at the C-terminus of human CD40, and the extracellular domain had amino acids E108-L261 with 9xHis-2x(SGGG)-IGER at the N-terminus. Human CD40L (accession number NM_000074.2) (9His-hCD40L) was commercially available from Biolegend.

[0228] In the blockade assay, 9His-hCD40L was passively adsorbed onto a 96-well microtiter plate at a concentration of 5 μg / mL in PBS at 4°C overnight. Subsequently, nonspecific binding sites were blocked using a PBS solution containing 0.5% (w / v) BSA. In a separate 96-well microtiter plate, a fixed amount of 40 nM hCD40-mmH was pre-mixed with one of the following antibodies in PBS + 0.5% BSA at a concentration ranging from 977 pM to 1 μM: (1) CD40 × CD40 bispecific antibodies (REGN16431, REGN16432, REGN16634, and REGN16335), (2) parental CD40 mAb (a bivalent antibody containing two fragment antigen-binding [Fab] arms identical to one of the anti-CD40 Fabs of the CD40 × CD40 antibody), (3) CD40x unrelated antibody (a bivalent antibody incorporating one Fab arm identical to one of the anti-CD40 Fabs of the CD40 × CD40 antibody and another Fab arm specific to an unrelated antigen), and (4) human IgG4 isotype control antibodies with Fc mutations (REGN7540 and REGN4513). The fixation concentration of hCD40-mmH is set to the concentration (EC) that produces 50% of the maximum binding to 9His-hCD40L adhered to the plate. 50It was selected to be close to the (value). After 1 hour of incubation, the antibody-antigen complex was transferred to a microtiter plate coated with 9His-hCD40L. After incubating for 1 hour at room temperature, the plate was washed, and the hCD40-mmH protein bound to the plate was detected using a horseradish peroxidase (HRP)-conjugated goat anti-c-Myc antibody. Next, the plate was developed using a TMB substrate solution (BD Biosciences) according to the manufacturer's recommended procedure, and the absorbance (OD 450 ) at 450 nm was measured on a SpectraMax i3x plate reader.

[0229] The binding data was analyzed using a sigmoid (4-parameter logistic) dose-response model with GraphPad Prism (trademark) software. The IC 50 value, defined as the antibody concentration required to block 50% of the 40 nM hCD40-mmH binding to 9His-hCD4oL coated on the plate, was measured to indicate the blocking efficacy.The percent blockade of the tested antibodies at the highest test concentration (1 μM) was calculated based on the formula shown below.

Equation

[0230] Results The ability of CD40×CD40 bispecific antibodies to block the binding of human CD40 monomers to human CD40L coated on plates was evaluated using a sandwich ELISA-based blockade assay. The results are shown in Table 12. As shown in Table 12, each of the CD40×CD40 bispecific antibodies REGN16431, REGN16432, REGN16334, and REGN16335 blocked hCD40 binding to hCD40L in a concentration-dependent manner, showing 93% to 98% blockade at the highest antibody concentration tested (1 μM). The IC50 of these bispecific antibodies 50 The values ​​are similar, around 32 nM. The four parental anti-CD40 antibodies (H4sH21519P2, H4sH21520P2, REGN17288, REGN17544) showed maximum blockade in the range of 91% to 94% and IC50 in the range of 52 nM to 71 nM. 50 The values ​​were shown. Four CD40× unrelated antibodies (REGN17551, REGN17552, REGN17548, RENG17549) also inhibited hCD40 binding to hCD40L with blockade rates ranging from 83% to 87%. Two parental CD40 antibodies (H4sH30027P2 and REGN17289) and two CD40× unrelated antibodies (REGN17553 and REGN17550) showed minimal blocking activity of approximately 10% and were classified as non-blockers. In this experiment, two human IgG4 isotype control antibodies with Fc mutations (REGN7540, REGN4513) did not show blocking activity as expected. [Table 12] NBL: Non-blocking (blocking rate of 50% or less) ND: Undetermined (IC 50 (No sigmoid curve fitting was observed to calculate the value.)

[0231] Example 5: Binding of CD40 × CD40 bispecific antibody to cell surface CD40 as measured by flow cytometry. Flow cytometry was used to evaluate the ability of CD40×CD40 bispecific antibodies to bind to human CD40 (hCD40) or cynomolgus monkey CD40 (mfCD40) expressing cells. Human embryonic kidney 293 (HEK293) cells, which stably express the luciferase reporter gene under the control of activated B cell nuclear factor kappa light chain enhancer (NF-κB) and highly sensitive green fluorescent protein (HEK293 / D9), were manipulated to express hCD40 (accession number P25942-1) or mfCD40 (accession number XP_005569274.1) by introducing either a neomycin-resistant pRG984 plasmid encoding full-length hCD40 (amino acid M1-Q277, HEK293 / D9 / hCD40) or a neomycin-resistant pRG984 plasmid encoding full-length mfCD40 (amino acid M1-Q282, HEK293 / D9 / mfCD40) into these cells. The binding of anti-CD40 antibodies to endogenously expressed hCD40 on the cell surface was evaluated using Ramos 2G6.4C10 cells. CD40-negative HEK293 / D9 cells, in which CD40 expression was not detected by flow cytometry using a commercially available CD40 antibody, were included as a background binding control.

[0232] The experiment was carried out according to the following procedure: HEK293 / D9 / hCD40 cells, HEK293 / D9 / mfCD40 cells, or HEK293 / D9 cells were subjected to Ca 2+ / Mg 2+ The cells were rinsed once in 1x PBS buffer without enzymes and incubated with enzyme-free cell dissociation solution at 37°C for 10 minutes to detach from the flask. The dissociated cells or Ramos 2G6.4G10 suspension cells were washed with 1x PBS and counted using a Cellometer® Auto T4 cell counter (Nexcelom Bioscience, Lawrence, MA). The cells were then counted at a rate of 1 × 10⁶ cells per 1 mL in 1x PBS. 7The cells were resuspended in individual cells and stained separately with 2.5 μM CellTrace® reagent (Invitrogen, Carlsbad, CA) at room temperature (RT) for 20 minutes to generate a unique fluorescent signature for each cell line (CellTrace® CFSE was used for Ramos 2G6.4C10 cells, CellTrace® Violet for HEK293 / D9 / mfCD40 cells, CellTrace® Yellow for HEK293 / D9 cells, and HEK293 / D9 / hCD40 cells were not stained). The CellTrace® labeling reaction was stopped by adding FBS to 1x PBS to a final concentration of 25%, followed by incubation at room temperature for 5 minutes to quench the unbound dye in the solution. The cells were washed with 1x PBS, and each of the four cell lines (stained and unstained) was mixed in a 1:1:1:1 ratio for multiplexing. A 96-well Corning plate contains approximately 2 x 10⁶ wells per well. 5Cells were seeded and stained in 1x PBS with LIVE / DEAD® Fixable Near-IR (Thermo Fisher Scientific, Waltham, MA) at 4°C for 20 minutes according to the manufacturer's recommended procedure to distinguish between live and dead cells. The cells were washed with 2% FBS (w / v) (flow cytometry staining buffer) in 1x PBS by centrifugation using a benchtop centrifuge 5810R (Eppendorf®, Hamburg, Germany). The cells were incubated in flow cytometry staining buffer with serial dilutions of the following antibodies in the range of 1.7 pM to 100 nM at 4°C for 30 minutes. (1) CD40×CD40 bispecific antibody, or (2) parental CD40 antibody (a bivalent antibody containing two fragment antigen-binding [Fab] arms identical to one of the two anti-CD40 Fabs of the bispecific CD40×CD40 antibody), or (3) CD40× unrelated antibody (a bivalent antibody incorporating one Fab arm identical to one of the anti-CD40 Fabs of the CD40×CD40 antibody and another Fab arm specific to the unrelated antigen (i.e., Bet v1, the major allergen of birch pollen)), or (4) human IgG4 isotype control antibody with an Fc mutation. After washing, antibodies bound to cells were detected at 4°C for 30 minutes using 2.5 μg / ml allophycocyanin (APC)-conjugated goat anti-human IgG antibody (Jackson Immunoresearch, West Grove, PA) specific to the Fcγ fragment. The cells were washed and then fixed at room temperature for 20 minutes in a 50% solution of Cytofix® fixation buffer (BD, Franklin Lakes, NJ) in flow cytometry staining buffer. The cells were washed, resuspended in flow cytometry staining buffer, and stored at 4°C for downstream flow cytometry analysis.

[0233] Fluorescence signals were acquired and recorded on a ZE5 cell analyzer (Bio-Rad, Hercules, CA) according to the manufacturer's recommended procedure. Flow cytometry data analysis was performed using the open-source R packages flowCore, flowStats, flowDensity, ggCyto, and flowAI. Multiplexed samples were deconvoluted, and individual cell populations were identified based on their unique CellTrace® fluorescence signatures. Median APC fluorescence intensity (MFI) was recorded to indicate the binding strength of each antibody across various concentrations. Antibodies with an MFI exceeding 1500 at the highest test concentration (100 nM) were classified as specific binders. Furthermore, direct binding signals (APC MFI) were analyzed as a function of antibody concentration, and the data were fitted to a sigmoid (4-parameter logistic) dose-response model using GraphPad Prism® software. EC is defined as the antibody concentration that yields 50% of maximum binding. 50 The values ​​were measured and used as an indicator of antibody binding efficacy. EC 50 The values ​​were reported only for specific binders.

[0234] result The ability of CD40×CD40 bispecific antibodies to specifically bind to human or monkey CD40-expressing cells was evaluated by flow cytometry. The experimental results are summarized in Table 13 below.

[0235] Four CD40×CD40 bispecific antibodies (REGN16431, REGN16432, REGN16334, and REGN16335) showed concentration-dependent specific binding to hCD40 expressed on HEK293 / D9 / hCD40 or Ramos 2G6.4C10 cells. In HEK293 / D9 / hCD40 cells, the MFI value at the highest concentration tested (100 nM) ranged from 28,932 to 30,315. 50 The values ​​were 1.5nM to 2.0nM, and in Ramos 2G6.4C10 cells, the MFI values ​​were 3,748 to 3,852, and EC 50The values ​​were 1.0 nM to 2.0 nM. The four bispecific antibodies similarly showed specific binding to mfCD40 expressed on HEK293 / D9 / mfCD40 cells, with MFI values ​​ranging from 41,714 to 48,653. 50 The values ​​ranged from 2.1 nM to 9.1 nM.

[0236] Six parental CD40 antibodies (H4sH21519P2, H4sH21520P2, H4sH30027P2, REGN17288, REGN17544, and REGN17289) showed specific binding to HEK293 / D9 / hCD40 cells or Ramos 2G6.4C10 cells, with MFI values ​​in the HEK293 / D9 / hCD40 cells ranging from 20,213 to 20,911, and EC 50 The value is 0.72nM to 1.3nM, or in Ramos 2G6.4C10 cells, the MFI value is in the range of 2,408 to 2,664, and EC 50 The values ​​ranged from 0.45 nM to 1.5 nM. All parental CD40 antibodies showed specific binding to HEK293 / D9 / mfCD40 cells, with MFI values ​​ranging from 11,539 to 33,930. 50 The values ​​ranged from 1.0 nM to 10 nM.

[0237] Six CD40× unrelated antibodies (REGN17551, REGN17552, REGN17553, REGN17548, REGN17549, and REGN17550) showed specific binding to HEK293 / D9 / hCD40 cells or Ramos 2G6.4C10 cells, with MFI values ​​in HEK293 / D9 / hCD40 cells ranging from 21,394 to 24,591. 50The values ​​ranged from 1.1 nM to 8.2 nM, or in Ramos 2G6.4C10 cells, the MFI values ​​ranged from 1,833 to 3,140. Four CD40x unrelated antibodies (REGN17552, REGN17553, REGN17549, and REGN17550) showed specific binding to HEK293 / D9 / mfCD40 cells, with MFI values ​​ranging from 4,719 to 39,448; however, two CD40x unrelated antibodies (REGN17551 and REGN17548) did not specifically bind to mfCD40 on HEK293 / D9 / mfCD40 cells.

[0238] All CD40 antibodies tested did not bind to negative control HEK293 / D9 cells, and the binding signal was less than 950 MFI. As expected, no detectable cell surface binding was observed with the two hIgG4 isotype control antibodies with Fc mutations (REGN7540 and REGN4513) up to the highest antibody concentration tested (100 nM). [Table 13-1] [Table 13-2] MFI: Median fluorescence intensity, INC: Undetermined, ND: Not determined. Concentration-dependent binding was detected, but did not reach a plateau in the tested concentration range. 50 If it cannot be calculated, INC is applied. If the MFI value is 1500 or less and no specific binding is detected, EC is applied. 50 It was not calculated, and ND was applied.

[0239] Example 6: Bioassay to evaluate the regulation of CD40 signaling by a CD40 × CD40 bispecific antibody CD40 is a member of the tumor necrosis factor receptor superfamily (TNFRSF) and activates the immune system in response to the binding of its ligand, CD40L. To evaluate the regulation of CD40 signaling, we developed a bioassay to quantitatively assess receptor activation by measuring gene expression downstream of NFκB (nuclear factor κB) nuclear translocation. A luciferase-based reporter assay was designed in three different human cell lines (Ramos.2G6.4C10, Raji, and THP-1) that endogenously express CD40. Cells were transduced with an NFκB luciferase reporter lentivirus (QIAGEN CLS-013L-8), and stable reporter cell lines were selected and maintained in a medium containing 1 μg / ml puromycin.

[0240] In the bioassay, cells were seeded at a rate of 20,000 cells / well in 96-well plates in assay medium (RPMI-1640 containing 10% FBS, penicillin / streptomycin / glutamine). Antibodies were then serially diluted 1:3 in assay medium to a final concentration ranging from 100 nM to 1.7 pM (without the additional condition of no test molecule) and added to the cells with or without a constant concentration of 40L of human CD (500 pM, 900 pM, or 10 nM for Ramos, Raji, or THP1 reporter cells, respectively). To obtain the activation range, 40L of hCD was serially diluted 1:3 to a final concentration ranging from 100 nM to 1.7 pM (without the additional condition of no ligand) and added to the cells. After incubation at 37°C / 5% CO2 for 5 hours, ONE-Glo® (Promega) reagent was added, and luciferase activity was detected on an Envision multi-label plate reader (PerkinElmer). All conditions were tested in double-row configurations.

[0241] Using nonlinear regression (4-parameter logistics), EC is performed using GraphPad Prism™ software. 50 Value or IC 50 The following was determined. The inhibition rate was calculated based on the relative luminescence (RLU) value using the following formula.

number

number

[0242] "RLU" 抗体最大値 " and "RLU 抗体最小値 "RLU" represents the maximum and minimum luminescence values ​​obtained from the antibody. リガント最大 " and "RLU リガンド一定 " is the value obtained at the maximum and constant concentrations of CD40L. バックグラウンド The value shown is for the absence of any CD40L. For antibodies that showed enhancement in the presence of CD40L, the value shown is "RLU". 抗体最大値 The inhibition rate was calculated using [this method], and a negative inhibition value was obtained.

[0243] result As shown in Table 14, the CD40×CD40 bispecific antibodies REGN16431, REGN16432, REGN16334, and REGN16335 showed minimal activation of 4-6% in Ramos.2G6.4C10 / NFκB-luc cells without CD40L, and 90-94% inhibition in the presence of 500 pM CD40L. IC 50 The concentration ranged from 38.6 to 85.4 pM. CD40 bivalent antibodies and CD40x-unrelated (unrelated, non-CD40-targeted) antibodies showed 4–49% activation in the absence of CD40L and 28–95% inhibition in the presence of 500 pM CD40L. Four antibodies showed -110–142% inhibition of CD40L in the Ramos bioassay.

[0244] As shown in Table 15, the CD40×CD40 bispecific antibodies REGN16431, REGN16432, REGN16334, and REGN16335 showed minimal activation of 5–13% in Raji / NFκB-luc cells without CD40L, and inhibition of 68–113% in the presence of 900 pM CD40L. IC 50The concentration ranged from 49.4 to 107 pM. In the Raji bioassay, CD40 bivalent antibodies and CD40x-unrelated antibodies showed 7–25% activation in the absence of CD40L and 33–92% inhibition in the presence of 900 pM CD40L.

[0245] As shown in Table 16, the CD40×CD40 bispecific antibodies REGN16431, REGN16432, REGN16334, and REGN16335 showed minimal activation of 1-6% in THP-1 / NFκB-luc cells without CD40L, and 97-98% inhibition in the presence of 10nM CD40L. 50 The concentration was 148-553 pM. CD40 bivalent antibodies and CD40x-unrelated antibodies showed 0-5% activation in the absence of CD40L and 54-101% inhibition in the presence of 10 nM CD40L. In the THP-1 bioassay, the four antibodies showed enhancement of signal transduction and inhibition of -45-92% in the presence of CD40L.

[0246] Unrelated human IgG antibodies, control mAb1, control mAb2, and control mAb3, showed little to no activation (0-13%) in all cells without CD40L, and inhibition (3-23%) in the presence of CD40L. CD40L showed activation of signaling in Ramos.2G6.4C10 / NFκB-luc, Raji / / NFκB-luc, and THP-1 / NFκB-luc cells, and EC 50 These values ​​were 436 pM, 691 pM, and 1.47 nM, respectively. [Table 14-1] [Table 14-2] *Values ​​are EC in the presence of CD40L 50 This represents the calculated value. All other values ​​are IC. 50 That is the case. #I C 50 The values ​​were obtained by excluding the RLU values ​​from the condition containing the three highest concentrations of antibody due to the hook effect. [Table 15] [Table 16] *Values ​​are EC in the presence of CD40L 50 This represents the calculated value. If no best fit is found, the EC (Equivalent Value) is used. 50 The value was not determined. All other values ​​were IC. 50 That is the case.

[0247] Example 7: In vitro CD40L blocking activity by CD40×CD40 bispecific antibody B-cell assay #1 To determine the efficacy of the CD40×CD40 bispecific antibody in blocking CD40L-induced stimulation, IL-6, IL-10, and TNFα cytokine production was quantified in primary human B cell cultures treated with the antibody in the presence of soluble CD40L. B cells were placed in 96U bottom plates in RPMI 1640 medium containing 15% FBS and 1x penicillin-streptomycin, with 2×10⁶ cells per well. 5 Cells were seeded. CD40×CD40 bispecific antibodies were simultaneously added to the cells in the presence of a fixed dose of IL-4 (10 pM) and CD40L (500 nM). A dose-response of CD40L with a fixed dose of IL-4 (10 pM) was included as a control. The cells were cultured at 37°C for 3 days, and the cell culture supernatant was collected for analysis.

[0248] Cytokine levels in the supernatant were analyzed using the MSD V-PLEX pro-inflammatory panel 1. The MSD V-PELX assay was performed using cell culture supernatant diluted 1:2 according to the manufacturer's instructions. MSD plates were read using a MESO QuickPlex Sq 120MM instrument and MSD Discovery Workbench software. Data analysis was performed using Graphpad Prism software. The obtained data points were transformed using the X=Log(X) equation, and the transformed data were subjected to linear regression analysis and fitted to a sigmoid dose-response curve. IC 50 This was obtained from this analysis.

[0249] result All four antibodies tested (REGN16334, REGN16335, REGN16431, and REGN16432) blocked the production of IL-6 (Figures 1A, 1B, and 1C), IL-10 (Figures 2A, 2B, and 2C), and TNFα (Figures 3A, 3B, and 3C) from primary human B cells derived from three donors in response to CD40L stimulation. IC of the antibodies and COMP11209 (comparative CD40 antibody) 50 The values ​​are shown in Tables 17 (IL-6), 18 (IL-10), and 19 (TNFα). The maximum blockade percentage was calculated for the highest doses of REGN16334, REGN16335, REGN16431, and REGN16432 compared to the mean cytokine levels of cells treated without antibody. [Table 17] [Table 18] [Table 19]

[0250] B-cell assay #2 To determine the efficacy of the CD40×CD40 bispecific antibody in blocking CD40L-induced stimulation, IL-6, IL-10, and TNFα cytokine production was quantified in primary human B cell cultures treated with the antibody in the presence of soluble CD40L. B cells were placed in 96U bottom plates in RPMI 1640 medium containing 15% FBS and 1x penicillin-streptomycin, with 2×10⁶ cells per well. 5Cells were seeded and incubated with CD40×CD40 bispecific antibody at 37°C for 30 minutes. After antibody incubation, a fixed dose of IL-4 (10 pM) and CD40L (500 nM) was added to the B cell culture in the presence of CD40×CD40 bispecific antibody. A dose-response of CD40L with a fixed dose of IL-4 (10 pM) was included as a control. In parallel, to quantify the agonist activity of the CD40×CD40 bispecific antibody, B cells were incubated with the antibody in the presence of a fixed dose of IL-4 (10 pM) without CD40L. Cells were cultured at 37°C for 3 days, and the cell culture supernatant was collected for analysis.

[0251] Cytokine levels in the supernatant were analyzed using the MSD V-PLEX pro-inflammatory panel 1. The MSD V-PELX assay was performed using cell culture supernatant diluted 1:2 according to the manufacturer's instructions. MSD plates were read using a MESO QuickPlex Sq 120MM instrument and MSD Discovery Workbench software. Data analysis was performed using Graphpad Prism software. The obtained data points were transformed using the X=Log(X) equation, and the transformed data were subjected to linear regression analysis and fitted to a sigmoid dose-response curve. IC 50 This was obtained from this analysis.

[0252] result All three CD40×CD40 bispecific antibodies tested (REGN16334, REGN16335, and REGN20484) blocked the production of IL6 (Figure 1D and Figure 1E), IL10 (Figure 2D and Figure 2E), and TNFα (Figure 3D and Figure 3E) cytokines from primary human B cells derived from two donors in response to CD40L stimulation. IC10 of the antibodies and COMP11209 50The values ​​are shown in Tables 20-22. The maximum blockade percentage was calculated for the highest doses of REGN16334, REGN16335, and REGN20484 compared to the mean cytokine levels of cells treated without antibody. Analysis of the agonist activity of antibodies REGN16334, REGN16335, and REGN20484 showed that cytokine IL6 was induced in both donors within a range comparable to that of cells treated with IL4 alone (no antibody) (Figures 3F and 3G). [Table 20] [Table 21] [Table 22]

[0253] Dendritic cell assay To evaluate the efficacy of a CD40×CD40 bispecific antibody in blocking CD40L-mediated stimulation, IL-12 / IL-23p40 cytokine production was quantified in primary human monocyte-derived dendritic cell (MDDC) cultures treated with the antibody in the presence of soluble CD40L. To generate MDDCs, peripheral blood mononuclear cells (PBMCs) from healthy human donors were isolated from Leukopack obtained from the New York Blood Center by Ficoll-Paque density gradient centrifugation. CD14+ cells were purified from PBMCs by positive selection using CD14 human microbeads. The purified CD14+ cells were placed in 6-well plates in RPMI 1640 medium containing 10% FBS, 1x penicillin-streptomycin, 800 U / mL GM-CSF, and 500 U / mL IL-4, at a rate of 3 × 10⁶ cells per well. 6 Cells were seeded. The culture medium was replenished, and the entire amount of GM-CSF and IL-4 was returned to the cells on days 3 and 5. MDDC was collected on day 6 and placed in 96-U bottom plates at a rate of 1 × 10⁶ per well. 6Cells were seeded. A CD40×CD40 bispecific antibody was simultaneously added to the cells along with a fixed dose of CD40L (20 nM). A dose-response of CD40L was included as a control. The cells were cultured at 37°C for 4 days, and the cell culture supernatant was collected for analysis.

[0254] Cytokine levels in the supernatant were analyzed using the MSD V-PLEX cytokine panel 1. The MSD V-PLEX assay was performed using cell culture supernatant diluted 1:2 or 1:20 according to the manufacturer's instructions. MSD plates were read using a MESO QuickPlex Sq 120MM instrument and MSD Discovery Workbench software. Data analysis was performed using Graphpad Prism software. The obtained data points were transformed using the X=Log(X) equation, and the transformed data were subjected to linear regression analysis and fitted to a sigmoid dose-response curve. IC 50 This was obtained from this analysis.

[0255] result All four antibodies tested (REGN16334, REGN16335, REGN16431, and REGN16432) blocked IL-12 / IL-23p40 production from human primary monocyte-derived dendritic cells from two donors in response to CD40L stimulation (Figures 4A-4B). IC of antibodies and COMP11209 50 The values ​​are shown in Table 23. [Table 23]

[0256] Example 8: In vitro agonist activity in human B cells by CD40 × CD40 bispecific antibody To quantify the agonist activity of the CD40×CD40 bispecific antibody, B cells were cultured with the antibody in the presence of a constant dose of IL-4 (10 pM) without CD40L. Cells were cultured at 37°C for 3 days, and the cell culture supernatant was collected for analysis. Cytokine levels in the supernatant were analyzed using the MSD V-PLEX pro-inflammatory panel 1. The MSD V-PELX assay was performed using cell culture supernatant diluted 1:2 according to the manufacturer's instructions. MSD plates were read using a MESO QuickPlex Sq 120MM instrument and MSD Discovery Workbench software. Data analysis was performed using Graphpad Prism software. The obtained data points were transformed using the X=Log(X) equation, and the transformed data were subjected to linear regression analysis and fitted to a sigmoid dose-response curve. IC 50 This was obtained from this analysis.

[0257] result Analysis of the agonist activity of antibodies REGN16334, REGN16335, REGN16431, and REGN16432 showed that cytokines IL-6 (Figures 5A-5B) and IL-10 (Figures 6A-6B) were induced in both donors within a range comparable to that of cells treated without antibodies (IL-4 only).

[0258] Example 9: Effect of CD40×CD40 bispecific antibody in mouse NP-KLH immunization model To determine the effect of CD40 blockade on antigen-specific antibody responses, the widely used T-dependent immunization model, the NP-KLH immunization model, was used in mice homozygous for human CD40 instead of mouse CD40. Mice were immunized with 25 μg of NP-KLH by subcutaneous injection into the flank of 100 μL of a 1:1 mixture of NP-KLH and alum, emulsified by shaking for 30 minutes. The alum-only group received 100 μL of a 1:1 mixture of PBS and alum. As shown in Figure 7, CD40 × CD40 bispecific antibodies (REGN16334, REGN16335, REGN16431, or REGN16432) or isotype controls (REGN4439 or REGN4460) were administered to mice 3 days prior to immunization, followed by administration at a dose of 1 mg / kg twice weekly for a total of 2 weeks after immunization. Following the immunization and antibody treatment protocols, the mice were euthanized and blood and inguinal lymph node samples were collected. Blood was collected from all mouse groups by cardiac puncture and transferred to BD microtainer tubes (catalog number 365967) for serum isolation.

[0259] For tissue processing, lymph nodes were ground on a 74-micron cell strainer in 2 mL of RPMI medium + 10% FBS using the posterior end of a 3 mL syringe, and the single-cell suspension was filtered through a Millipore plate filter (100 μm) into a 2 mL deep-well plate. The cells were centrifuged at 400 g for 4 minutes and resuspended in 200 μL of PBS. The cells were transferred to a 96-well u-bottom plate, centrifuged at 400 g for 4 minutes, and stained with live / dead markers at room temperature for 15 minutes. The cells were washed and incubated in an Fc block at 4°C for 15 minutes, followed by antibody staining with the antibody mixture shown in Table 24 at 4°C for 30 minutes. After staining, the cells were washed twice with MACS buffer, fixed in 1:4 diluted BD Cytofix (catalog no. 554655) in PBS for 15 minutes, and then resuspended in MACS buffer and stored at 4°C. On the day of collection, cells were washed with BD Perm / wash, incubated in BD Perm / wash buffer (catalog number 554723) for 20 minutes, and stained with intracellular antibodies [Table 24] for 30 minutes. Cells were washed twice, fixed with BD Cytofix, and then resuspended in MACS buffer. Subsequently, cells were acquired using a FACSymphony A5 instrument and analyzed using OMIQ software. NP+ germinal center B cells were identified as live cells → dump- (dump containing TCRb, CD200R3, Ly6G, CD49b, and CD11b) → non-marginal zone B cells (CD1d-) → CD19+B220+ → CD38-IgD- → GC-positive (GL7+CD95+) → NP+. Statistical significance was determined in GraphPad Prism by the Shapiro-Wilk test to assess normality and the Kruskal-Wallis test with Dunn's post-hoc multiple comparison test. [Table 24-1] [Table 24-2]

[0260] Experiment #1 - Methods and Results In one experiment, NP-specific IgG1 levels in the final serum were quantified by ELISA. NP-2-BSA was diluted to 4 μg / mL in PBS, and a 384-well plate was coated overnight at 4°C with 25 μL / well of the solution. The plate was washed four times with wash buffer and blocked at room temperature for 1 hour with 50 μL / well of 0.5% BSA-containing PBS. The plate was washed four times with wash buffer, and mouse serum was diluted 1:100 for group A and 1:10,000 for groups B-G. The serum was further serially diluted eight times 3-fold using 0.5% BSA-containing PBS and added to the plate at 12.5 μL / well. The diluted serum was incubated on the plate at room temperature for 1 hour, followed by four washes. For detection, 25 μL of rat anti-mouse IgG1 HRP-binding antibody, diluted 1:1000 in 0.5% BSA-containing PBS, was added to the plate at room temperature for 1 hour. The plates were washed seven times, and color development was induced by adding 25 μL of OptEIA® TMB substrate solution. After 20 minutes, the reaction was stopped by adding 25 μL of 2N sulfuric acid. Absorbance at 450 nm (OD450) was measured on a Molecular Devices SpectraMax M5 plate reader. The relative level of circulating NP-specific IgG1 in serum was expressed in titer units, defined as the dilution factor required to achieve an OD450 reading equal to twice the background OD450. Graph analysis was performed using GraphPad Prism software (version 7.0). Statistical significance was determined in GraphPad Prism by the Shapiro-Wilk test to assess normality and the Kruskal-Wallis test with Dunn's post-hoc multiple comparison test.

[0261] In this NP-KLH immunization model, flow cytometry quantification revealed that prophylactic treatment with CD40×CD40 bispecific antibodies blocked the formation of antigen-specific germinal center B cells in the influx region lymph nodes. All CD40×CD40 bispecific antibodies reduced the frequency of NP-specific germinal center B cells at 1 mg / kg compared to their associated isotype controls (Figure 8A and Table 25). Prophylactic treatment with CD40×CD40 bispecific antibodies at 1 mg / kg also blocked the generation of a high-affinity NP-IgG1 antibody response, as measured by serum NP-specific IgG1 titers, with most samples below the lower limit of quantification in the assay (Figure 8B and Table 26). [Table 25] SD = Standard Deviation, N / A = Not Available, ns = Not Statistically Significant. * p<0.05. [Table 26] SD = Standard Deviation, N / A = Not Available, ns = Not Statistically Significant. * p<0.05, ** p<0.005.

[0262] Experiment #2 - Methods and Results In another experiment, NP-specific IgG1 levels in the final serum were quantified by ELISA. NP-2-BSA was diluted to 4 μg / mL in PBS, and 384-well plates were coated overnight at 4°C with 25 μL / well of the solution. The plates were washed five times with wash buffer and blocked at room temperature for 1 hour with 50 μL / well of 0.5% BSA-containing PBS. The plates were washed five times with wash buffer, and mouse serum was diluted 1:100 for groups A-B and 1:10,000 for groups C-L. The serum was further serially diluted eight times 3-fold using 0.5% BSA-containing PBS and added to the plates at 12.5 μL / well. The diluted serum was incubated on the plates at room temperature for 1 hour, followed by four washes. For detection, 25 μL of rat anti-mouse IgG1 HRP-binding antibody, diluted 1:1000 in 0.5% BSA-containing PBS, was added to the plates at room temperature for 1 hour. The plates were washed seven times, and color development was induced by adding 25 μL of OptEIA® TMB substrate solution. After 20 minutes, the reaction was stopped by adding 25 μL of 2N sulfuric acid. Absorbance at 450 nm (OD450) was measured on a Molecular Devices SpectraMax M5 plate reader. The relative level of circulating NP-specific IgG1 in serum was expressed in titer units, defined as the dilution factor required to achieve an OD450 reading equal to twice the background OD450. Data analysis was performed using GraphPad Prism. Statistical significance was determined in GraphPad Prism by the Shapiro-Wilk test to assess normality and the Kruskal-Wallis test with Dunn's post-hoc multiple comparison test.

[0263] In this experiment, as quantified by flow cytometry, prophylactic treatment with CD40×CD40 bispecific antibodies blocked the formation of antigen-specific germinal center B cells in influx region lymph nodes. All CD40×CD40 bispecific antibodies tested reduced the frequency of NP-specific germinal center B cells at doses of 1 mg / kg or higher compared to isotype controls (Figure 9A and Table 27). Prophylactic treatment with CD40×CD40 bispecific antibodies at 1 mg / kg also blocked the generation of a high-affinity NP-IgG1 antibody response, as measured by serum NP-specific IgG1 titers, with most samples falling below the lower limit of quantification in the assay (Figure 9B and Table 28). [Table 27] SD = Standard Deviation, N / A = Not Available, ns = Not Statistically Significant. * p<0.05. [Table 28] SD = Standard Deviation, N / A = Not Available, ns = Not Statistically Significant. * p<0.05.

[0264] Example 10: Effects of prophylactic administration with CD40×CD40 bispecific antibody in a mouse model of experimental autoimmune encephalomyelitis (EAE) To investigate the effects of CD40 blockade in autoimmune diseases, mice were created in which the entire mouse CD40 gene was replaced with the full-length human CD40 gene (CD40 hu / huAn experimental autoimmune encephalomyelitis (EAE) model was used in mice. Mice were immunized by subcutaneous injection of 0.1 mL of MOG35-55 emulsified with complete Freund's adjuvant into the upper and lower back (total 0.2 mL / mouse). Two hours after immunization, mice were intraperitoneally administered 150 ng of pertussis toxin (PTX) at a dose of 0.1 mL / dose. PTX administration was repeated 24 hours later. CD40×CD40 bispecific antibody or isotype control was administered to mice at 25 mg / kg three days before immunization, and then twice a week for a total of 5 weeks after immunization. See, for example, Figure 10. Mice were monitored and their body weight measured twice a week for the first two weeks after immunization, and daily from weeks 3 to 5. Mice were assigned scores according to the scoring guidelines outlined in Table 29. Mice were euthanized if their symptom score reached 4 for two consecutive days, or if their score reached 4.5 or 5, or if their body weight decreased by 30% from their baseline. [Table 29-1] [Table 29-2]

[0265] Experiment #1 - Results In one experiment using an experimental autoimmune encephalomyelitis (EAE) model, prophylactic blockade of CD40 with CD40×CD40 bispecific antibodies REGN16334, REGN16335, REGN16431, and REGN16432 reduced the severity of EAE disease symptom scores compared to groups treated without antibodies or with isotype controls (Figures 11A-11B and Table 30). In addition to the reduction in symptom severity, all CD40 blocking antibodies reduced the rate of weight loss in mice compared to groups treated without antibodies or with isotype controls (Figures 11C-11D and Table 31). The frequency of mice with disease score progression was also reduced in the groups treated with CD40 blocking antibodies (Table 32). [Table 30] [Table 31] [Table 32]

[0266] Experiment #2 - Results In another experiment, prophylactic blockade of CD40 with the CD40×CD40 bispecific antibodies REGN16335 or REGN20484 reduced the severity of EAE disease symptom scores compared to groups treated without antibodies or with isotype controls (Figure 12A and Table 33). In addition to the reduction in symptom severity, all CD40 blocking antibodies reduced the rate of weight loss in mice compared to groups treated without antibodies or with isotype controls (Figure 12B and Table 34). The frequency of mice with disease score progression was also reduced in the groups treated with CD40 blocking antibodies (Table 35). [Table 33] [Table 34] [Table 35]

[0267] Example 11: CryoEM analysis of CD40×CD40 bispecific antibody To better understand the binding of REGN16335 and REGN16334 to CD40, structural analysis was performed using cryo-electron microscopy (cryo-EM). Fab fragments were enzymatically prepared. Fab fragment 30027P2 corresponds to arm 1 of REGN16335, Fab fragment 21519P2 corresponds to arm 1 of REGN16634, and Fab fragment 21520P2 corresponds to arm 2 of both REGN16335 and REGN16334.

[0268] A 3D reconstruction map (resolution 3 Å) of the complex of CD40 and three Fab arms (30027P2, 21519P2, and 21520P2) shows that the three arms bind to non-overlapping epitopes across the CRD1, CRD2, and CRD3 domains (Figure 13). The 30027P2 Fab primarily binds to the CRD1 domain and a small portion of the CRD2 domain, the 21520P2 Fab binds across the CRD1 and CRD2 domains, and the 21519P2 Fab binds across the CRD2 and CRD3 domains. [Table 36-1] [Table 36-2] [Table 36-3] [Table 36-4] [Table 36-5] [Table 36-6] [Table 36-7] [Table 36-8] [Table 36-9] [Table 36-10] [Table 36-11] [Table 36-12] [Table 36-13] [Table 36-14] [Table 36-15] [Table 36-16]

[0269] The scope of the present invention is not limited by the specific embodiments described herein. In fact, various modifications of the present invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to be included within the scope of the accompanying claims. All disclosures of patents and non-patent documents cited herein are expressly incorporated herein in their entirety by reference.

Claims

1. It is a bispecific antigen-binding molecule, (a) A first antigen-binding domain (D1) that binds to the first epitope of human CD40, (b) A bispecific antigen-binding molecule comprising a second antigen-binding domain (D2) that binds to a second epitope of human CD40.

2. The aforementioned bispecific antigen-binding molecule (i) When measured by surface plasmon resonance at 25°C, K is less than 25 nM. Ｄ It binds to human CD40. (ii) When measured by surface plasmon resonance at 37°C, K is less than 70 nM. Ｄ It binds to human CD40. (iii) When measured by surface plasmon resonance at 25°C, the dissociation half-life (t) exceeds 75 minutes. １／２ ) binds to human CD40, (iv) EC of approximately 10 nM or less ５０ It binds to human CD40-expressing cells based on its value. (v) Inhibit the binding of human CD40 monomers to CD40L, (vi) inhibiting CD40 ligand (CD40L)-induced activation, and / or (vii) The bispecific antigen-binding molecule according to claim 1, which does not significantly activate CD40 in the absence of CD40L.

3. The bispecific antigen-binding molecule according to claim 1 or 2, wherein the D1 domain and the D2 domain each comprise a heavy chain immunoglobulin variable region comprising a set of three heavy chain complementarity-determining region sequences HCDR1, HCDR2, and HCDR3 independently selected from the group consisting of: (a) HCDR1 containing the amino acid sequence of SEQ ID NO: 4, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, and HCDR3 containing the amino acid sequence of SEQ ID NO: 8 (b) HCDR1 containing the amino acid sequence of SEQ ID NO: 24, HCDR2 containing the amino acid sequence of SEQ ID NO: 26, and HCDR3 containing the amino acid sequence of SEQ ID NO: 28, (c) HCDR1 containing the amino acid sequence of SEQ ID NO: 34, HCDR2 containing the amino acid sequence of SEQ ID NO: 36, and HCDR3 containing the amino acid sequence of SEQ ID NO:

38.

4. The bispecific antigen-binding molecule according to any one of claims 1 to 3, wherein the D1 domain and the D2 domain each comprise a light chain immunoglobulin variable region comprising a set of three light chain complementarity-determining region sequences LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 12, LCDR2 comprises the amino acid sequence AAS, and LCDR3 comprises the amino acid sequence of SEQ ID NO:

16.

5. The D1 domain comprises the following, a bispecific antigen-binding molecule according to any one of claims 1 to 4: (a) HCDR1 containing the amino acid sequence of SEQ ID NO: 4, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, HCDR3 containing the amino acid sequence of SEQ ID NO: 8, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence of SEQ ID NO: 16, or (b) HCDR1 containing the amino acid sequence of SEQ ID NO: 24, HCDR2 containing the amino acid sequence of SEQ ID NO: 26, HCDR3 containing the amino acid sequence of SEQ ID NO: 28, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence of SEQ ID NO:

16.

6. The bispecific antigen-binding molecule according to claim 5, wherein the D1 domain comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 4, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, HCDR3 containing the amino acid sequence of SEQ ID NO: 8, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence of SEQ ID NO:

16.

7. The bispecific antigen-binding molecule according to claim 6, wherein the D1 domain includes a heavy chain variable region (HCVR) containing the amino acid sequence of SEQ ID NO:

2.

8. The bispecific antigen-binding molecule according to claim 5, wherein the D1 domain comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 24, HCDR2 containing the amino acid sequence of SEQ ID NO: 26, HCDR3 containing the amino acid sequence of SEQ ID NO: 28, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence of SEQ ID NO:

16.

9. The bispecific antigen-binding molecule according to claim 8, wherein the D1 domain comprises an HCVR containing the amino acid sequence of SEQ ID NO:

22.

10. The bispecific antigen-binding molecule according to any one of claims 1 to 9, wherein the D1 domain includes a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO:

10.

11. The bispecific antigen-binding molecule according to any one of claims 1 to 10, wherein the D2 domain comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 34, HCDR2 containing the amino acid sequence of SEQ ID NO: 36, HCDR3 containing the amino acid sequence of SEQ ID NO: 38, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence of SEQ ID NO:

16.

12. The bispecific antigen-binding molecule according to claim 11, wherein the D2 domain comprises an HCVR containing the amino acid sequence of SEQ ID NO:

32.

13. The bispecific antigen-binding molecule according to claim 11 or 12, wherein the D2 domain comprises an LCVR containing the amino acid sequence of SEQ ID NO:

10.

14. A bispecific antigen-binding molecule according to any one of claims 1 to 13, comprising the following: HCDR1 containing the amino acid sequence of SEQ ID NO: 4, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, HCDR3 containing the amino acid sequence of SEQ ID NO: 8, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and D1 containing LCDR3 containing the amino acid sequence of SEQ ID NO: 16, and HCDR1 containing the amino acid sequence of SEQ ID NO: 34, HCDR2 containing the amino acid sequence of SEQ ID NO: 36, HCDR3 containing the amino acid sequence of SEQ ID NO: 38, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and D2 containing LCDR3 containing the amino acid sequence of SEQ ID NO:

16.

15. The bispecific antigen-binding molecule according to claim 14, wherein D1 comprises an HCVR containing the amino acid sequence of SEQ ID NO: 2 and an LCVR containing the amino acid sequence of SEQ ID NO: 10, and D2 comprises an HCVR containing the amino acid sequence of SEQ ID NO: 32 and an LCVR containing the amino acid sequence of SEQ ID NO:

10.

16. A bispecific antigen-binding molecule according to any one of claims 1 to 15, comprising the following: HCDR1 containing the amino acid sequence of SEQ ID NO: 24, HCDR2 containing the amino acid sequence of SEQ ID NO: 26, HCDR3 containing the amino acid sequence of SEQ ID NO: 28, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and D1 containing LCDR3 containing the amino acid sequence of SEQ ID NO: 16, and HCDR1 containing the amino acid sequence of SEQ ID NO: 34, HCDR2 containing the amino acid sequence of SEQ ID NO: 36, HCDR3 containing the amino acid sequence of SEQ ID NO: 38, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and D2 containing LCDR3 containing the amino acid sequence of SEQ ID NO:

16.

17. The bispecific antigen-binding molecule according to claim 16, wherein D1 comprises an HCVR containing the amino acid sequence of SEQ ID NO: 22 and an LCVR containing the amino acid sequence of SEQ ID NO: 10, and D2 comprises an HCVR containing the amino acid sequence of SEQ ID NO: 32 and an LCVR containing the amino acid sequence of SEQ ID NO:

10.

18. The bispecific antigen-binding molecule according to any one of claims 1 to 4, wherein the D1 domain comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 34, HCDR2 containing the amino acid sequence of SEQ ID NO: 36, HCDR3 containing the amino acid sequence of SEQ ID NO: 38, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence of SEQ ID NO:

16.

19. The bispecific antigen-binding molecule according to claim 18, wherein the D1 domain comprises an HCVR containing the amino acid sequence of SEQ ID NO:

32.

20. The bispecific antigen-binding molecule according to claim 18 or 19, wherein the D1 domain comprises an LCVR containing the amino acid sequence of SEQ ID NO:

10.

21. A bispecific antigen-binding molecule according to any one of claims 1 to 4 and 18 to 20, wherein the D2 domain comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 4, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, HCDR3 containing the amino acid sequence of SEQ ID NO: 8, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence of SEQ ID NO:

16.

22. The bispecific antigen-binding molecule according to claim 21, wherein the D2 domain comprises an HCVR containing the amino acid sequence of SEQ ID NO:

2.

23. The bispecific antigen-binding molecule according to claim 21, wherein the D2 domain comprises an LCVR containing the amino acid sequence of SEQ ID NO:

10.

24. A bispecific antigen-binding molecule according to any one of claims 1 to 4 and 18 to 23, comprising the following: HCDR1 containing the amino acid sequence of SEQ ID NO: 34, HCDR2 containing the amino acid sequence of SEQ ID NO: 36, HCDR3 containing the amino acid sequence of SEQ ID NO: 38, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and D1 containing LCDR3 containing the amino acid sequence of SEQ ID NO: 16, and D2 includes HCDR1 containing the amino acid sequence of SEQ ID NO: 4, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, HCDR3 containing the amino acid sequence of SEQ ID NO: 8, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence of SEQ ID NO:

16.

25. The bispecific antigen-binding molecule according to claim 24, wherein D1 comprises an HCVR containing the amino acid sequence of SEQ ID NO: 32 and an LCVR containing the amino acid sequence of SEQ ID NO: 10, and D2 comprises an HCVR containing the amino acid sequence of SEQ ID NO: 2 and an LCVR containing the amino acid sequence of SEQ ID NO:

10.

26. A bispecific antigen-binding molecule according to any one of claims 1 to 25, which is a bispecific antibody.

27. The bispecific antigen-binding molecule according to claim 26, wherein the bispecific antibody includes the constant region of the human IgG heavy chain.

28. The bispecific antigen-binding molecule according to claim 27, wherein the constant region of the human IgG heavy chain is isotype IgG4 or IgG1.

29. The bispecific antigen-binding molecule according to claim 27 or 28, wherein the constant region of the human IgG heavy chain includes one or more modifications that reduce binding to the Fc receptor.

30. The bispecific antigen-binding molecule according to any one of claims 1 to 17 and 26 to 29, wherein D1 comprises the following: A heavy chain containing the amino acid sequence of SEQ ID NO: 42 and a light chain containing the amino acid sequence of SEQ ID NO: 20, or A heavy chain containing the amino acid sequence of SEQ ID NO: 46 and a light chain containing the amino acid sequence of SEQ ID NO: 20, or A heavy chain containing the amino acid sequence of SEQ ID NO: 48 and a light chain containing the amino acid sequence of SEQ ID NO: 20, or A heavy chain containing the amino acid sequence of SEQ ID NO: 52 and a light chain containing the amino acid sequence of SEQ ID NO:

20.

31. The bispecific antigen-binding molecule according to any one of claims 1 to 17 and 26 to 30, wherein D2 comprises the following: A heavy chain containing the amino acid sequence of SEQ ID NO: 44 and a light chain containing the amino acid sequence of SEQ ID NO: 20, or A heavy chain containing the amino acid sequence of SEQ ID NO: 50 and a light chain containing the amino acid sequence of SEQ ID NO:

20.

32. A bispecific antigen-binding molecule according to any one of claims 1 to 17 and 26 to 31, comprising (i) D1 comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 42 and a light chain comprising the amino acid sequence of SEQ ID NO: 20, and (ii) D2 comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 44 and a light chain comprising the amino acid sequence of SEQ ID NO:

20.

33. A bispecific antigen-binding molecule according to any one of claims 1 to 17 and 26 to 31, comprising (i) D1 comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 46 and a light chain comprising the amino acid sequence of SEQ ID NO: 20, and (ii) D2 comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 44 and a light chain comprising the amino acid sequence of SEQ ID NO:

20.

34. A bispecific antigen-binding molecule according to any one of claims 1 to 17 and 26 to 31, comprising (i) D1 comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 48 and a light chain comprising the amino acid sequence of SEQ ID NO: 20, and (ii) D2 comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 50 and a light chain comprising the amino acid sequence of SEQ ID NO:

20.

35. A bispecific antigen-binding molecule according to any one of claims 1 to 17 and 26 to 31, comprising (i) D1 comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 52 and a light chain comprising the amino acid sequence of SEQ ID NO: 20, and (ii) D2 comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 50 and a light chain comprising the amino acid sequence of SEQ ID NO:

20.

36. The bispecific antigen-binding molecule according to any one of claims 1 to 4 and 18 to 29, wherein D1 comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 58 and a light chain containing the amino acid sequence of SEQ ID NO:

20.

37. The bispecific antigen-binding molecule according to any one of claims 1 to 4, 18 to 29, and 36, wherein D2 comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 60 and a light chain containing the amino acid sequence of SEQ ID NO:

20.

38. The bispecific antigen-binding molecule according to claim 36 or 37, comprising (i) D1 comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 58 and a light chain containing the amino acid sequence of SEQ ID NO: 20, and (ii) D2 comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 60 and a light chain containing the amino acid sequence of SEQ ID NO:

20.

39. A pharmaceutical composition comprising a bispecific antigen-binding molecule according to any one of claims 1 to 38 and a pharmaceutically acceptable carrier.

40. A nucleic acid molecule comprising a nucleotide sequence encoding a bispecific antigen-binding molecule according to any one of claims 1 to 38.

41. A nucleic acid molecule containing one or more nucleotide sequences as described in Table 36.

42. An expression vector comprising the nucleic acid molecule according to claim 40 or 41.

43. A host cell comprising the expression vector described in claim 42.

44. A method for producing a bispecific antigen-binding molecule, the method comprising culturing the host cells described in claim 43 under conditions that enable the production of the bispecific antigen-binding molecule, and recovering the bispecific antigen-binding molecule thus produced.

45. A method for inhibiting CD40L-induced signaling, the method comprising contacting a cell expressing CD40 with a bispecific antigen-binding molecule according to any one of claims 1 to 38 or the pharmaceutical composition according to claim 39.

46. A method for treating, improving, or preventing a CD40-mediated disease or condition in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of a bispecific antigen-binding molecule according to any one of claims 1 to 38 or a pharmaceutical composition according to claim 39.

47. The method according to claim 46, wherein the CD40-mediated disease or condition is an autoimmune disease or condition, an inflammatory disease or condition, a cardiovascular disease or condition, or an organ transplant.

48. The aforementioned CD40-mediated diseases or conditions include autoimmune thyroid disease, autoimmune hemolytic anemia, Crohn's disease, diabetes, experimental autoimmune encephalomyelitis (EAE), focal segmental glomerulosclerosis (FSGS), glomerulonephritis, graft-versus-host disease (GVHD), hidradenitis suppurativa (HS), immune thrombocytopenia, inflammatory bowel disease, inflammatory neuropathy (e.g., acute inflammatory demyelinating polyneuropathy (AIDP) or chronic inflammatory demyelinating polyneuropathy (CIDP)), Kawasaki disease, The method according to claim 46 or 47, wherein the condition is lupus nephritis, mixed connective tissue disease (MCTD), multiple sclerosis, myasthenia gravis, neuromyelitis optica spectrum disorder, organ transplantation, pemphigus, primary biliary cirrhosis, psoriasis, psoriatic arthritis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Sjögren's syndrome, skin grafting, systemic lupus erythematosus, systemic scleroderma, transplant rejection, vasculitis, ANCA-associated vasculitis, ulcerative colitis, or Wegener's granulomatosis.

49. The method according to any one of claims 46 to 48, further comprising administering a second therapeutic agent to the subject.

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