Cd40 x cd40 bispecific antigen-binding molecules and uses thereof

Abstract

The present 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 do not significantly agonize CD40 in the absence of CD40L. Methods for using the antigen-binding molecules for treating CD40-mediated diseases are also provided.

Description

CD40 X CD40 BISPECIFIC ANTIGEN-BINDING MOLECULES AND USES THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Patent Application No. 63 / 521 ,408, filed June 16, 2023, the entire contents of which are incorporated by reference herein.REFERENCE TO SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on June 13, 2024, is named 118003-50020.XML and is 99,202 bytes in size.FIELD OF THE INVENTION

[0003] The present disclosure relates to antigen-binding molecules that bind to CD40 and inhibit CD40L-induced activation, and methods of use thereof, e.g., for treating autoimmune diseases.BACKGROUND

[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 some non-immune cells and tumors (Dakal et al, Immunobiology 2020, 225:151899). The interaction of CD40 with its ligand CD40L provides a co-stimulatory signal that is essential to the survival of many cell types and is required for functions of immune response such as germinal center formation, antibody responses to T-dependent antigens, and "licensing" dendritic cells to mature and become potent to trigger 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 implicated in autoimmune conditions that are largely driven by autoantibodies, such as systemic rheumatic diseases where autoantibodies play an important role in disease progression (such as multiple sclerosis, autoimmune nephritis, rheumatoid arthritis, Sjogren's syndrome, and systemic lupus erythematosus) as well as non-rheumatic conditions having an autoantibody component (such as myasthenia gravis, Grave's disease, and neuromyelitis optica) (see, Karnell et al., Adv Drug Delivery Rev. 2019, 141 :92-103). Additionally, because CD40-CD40L signaling is essential for activation of antigen-presenting cells, blocking antigen presentation by dendritic cells or B cells may impact CD8+ T cell responses in some diseases, such as multiple sclerosis (see, e.g., Denic et al., Expert Opin Ther Targets 2013, 17:1053-1066). Altered CD40-CD40L signaling is alsoimplicated in other diseases and conditions such as cardiovascular disease and transplantation (see, e.g., Dakal et al, Immunobiology 2020, 225:151899; Pamukcu et al., Ann. Med. 2011 , 43:331 ; 340; Pinelli et al., Immunotherapy 2015, 7:399-410).

[0006] Early clinical trials using a monoclonal antibody targeting CD40L provided biological validation for inhibiting the CD40-CD40L signaling pathway, but also revealed unexpected safety concerns due to thromboembolism complications (see, Karnell et al., Adv Drug Delivery Rev. 2019, 141 :92-103). Several monoclonal antibodies targeting CD40 are in clinical development; although no significant safety events have been reported with these antibodies, in several Phase 2a trials CD40 antibodies have failed to meet the primary endpoint (Kumar et al., Biopharm. Drug Dispos. 2018, 39:245-255; Visvanathan et al., Ann Rheum Dis. 2019, 78:754-760).SUMMARY

[0007] In one aspect, the present disclosure provides antigen-binding molecules, such as monospecific, bispecific, or multispecific antibodies, that bind to CD40. 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 comprises:(a) a first antigen-binding domain (D1 ) that binds a first epitope of human CD40; and(b) a second antigen-binding domain (D2) that binds a second epitope of human CD40.

[0008] In some embodiments, the bispecific antigen-binding molecule comprises D1 and D2 domains that do not compete with one another for binding to human CD40.

[0009] In some embodiments, the bispecific antigen-binding molecule exhibits one or more of the following characteristics:(i) binds human CD40 with a KD of less than 25 nM as measured by surface plasmon resonance at 25°C;(ii) binds human CD40 with a KD of less than 70 nM as measured by surface plasmon resonance at 37°C;(iii) binds human CD40 with a dissociative half-life (ti / 2) of greater than 75 minutes as measured by surface plasmon resonance at 25°C;(iv) binds a human CD40-expressing cell with an EC50 value of about 10 nM or less;(v) inhibits binding of human CD40 monomer to CD40L;(vi) inhibits CD40 ligand (CD40L)-induced activation; and / or(vii) does not significantly agonize CD40 in the absence of CD40L.

[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 does not significantly agonize CD40 in the absence of CD40L.

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

[0012] In some embodiments, 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 the LCDR1 comprises the amino acid sequence of SEQ ID NO:12, the LCDR2 comprises the amino acid sequence AAS, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:16. In some embodiments, the D1 domain comprises a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NQ:10.

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

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

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

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

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

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

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

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

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

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

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

[0024] In some embodiments, the D1 comprises an HCVR comprising the amino acid sequence of SEQ ID NO:32 and an LCVR comprising the amino acid sequence of SEQ ID NQ:10, and the D2 comprises an HCVR comprising the amino acid sequence of SEQ ID NO:2 and an LCVR comprising the amino acid sequence of SEQ ID NQ: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 lgG4 or lgG1. In some embodiments, the human IgG heavy chain constant region comprises one or more modifications that reduces binding to an Fc receptor (e.g., one or more modifications in a hinge region and / or CH region).

[0026] In some embodiments, the bispecific antigen-binding molecule comprises a D1 that comprises: 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 NQ:20; or 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 NQ:20; or 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 NQ:20; or 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 NQ:20; or a heavy chain comprising the amino acid sequence of SEQ ID NO:58 and a light chain comprising the amino acid sequence of SEQ ID NQ:20.

[0027] In some embodiments, the bispecific antigen-binding molecule comprises a D2 that comprises: 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 NQ:20; or a heavy chain comprising the amino acid sequence of SEQ ID NQ:50 and a light chain comprising the amino acid sequence of SEQ ID NQ:20; or a heavy chain comprising the amino acid sequence of SEQ ID NQ:60 and a light chain comprising the amino acid sequence of SEQ ID NQ:20.

[0028] In some embodiments, the bispecific antigen-binding molecule is a bispecific antibody comprising (i) a 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) a 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 NQ:20.

[0029] In some embodiments, the bispecific antigen-binding molecule is a bispecific antibody comprising (i) a 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 NQ:20; and (ii) a 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 NQ:20.

[0030] In some embodiments, the bispecific antigen-binding molecule is a bispecific antibody comprising (i) a 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 NQ:20; and (ii) a D2 comprising a heavy chain comprising the amino acid sequence of SEQ ID NQ:50 and a light chain comprising the amino acid sequence of SEQ ID NQ:20.

[0031] In some embodiments, the bispecific antigen-binding molecule is a bispecific antibody comprising (i) a 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 NQ:20; and (ii) a D2 comprising a heavy chain comprising the amino acid sequence of SEQ ID NQ:50 and a light chain comprising the amino acid sequence of SEQ ID NQ:20.

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

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

[0034] In another aspect, pharmaceutical compositions comprising antigen-binding molecules that bind to CD40 are provided. In some embodiments, the pharmaceutical composition comprises a bispecific antigen-binding molecule as disclosed herein and a pharmaceutically acceptable carrier.

[0035] In another aspect, the present disclosure provides nucleic acid molecules comprising a nucleotide sequence encoding an antigen-binding molecule that binds to CD40 (e.g., a monospecific or bispecific antigen-binding molecule as disclosed herein). In some embodiments, the nucleic acid molecule comprises one or more nucleotide sequences encoding a bispecific antigen-binding molecule as disclosed herein. In some embodiments, the nucleic acid molecule comprises one or more nucleotide sequences set forth in Table 36.

[0036] In another aspect, the present disclosure provides expression vectors and host cells comprising one or more nucleic acid molecule(s) comprising a nucleotide sequence encoding an antigen-binding molecule that binds to CD40 (e.g., a monospecific or bispecific antigen-binding molecule as disclosed herein). In some embodiments, the expression vectors and / or host cells comprise a nucleic acid molecule that comprises one or more nucleotide sequences set forth in Table 36.

[0037] In another aspect, the present disclosure provides methods of producing an antigen-binding molecule that binds to CD40 (e.g., a monospecific or bispecific antigenbinding molecule as disclosed herein). In some embodiments, the method comprises culturing a host cell as disclosed herein under conditions permitting production of the antigen-binding molecule, and recovering the antigen-binding molecule so produced.

[0038] In another aspect, the present disclosure provides methods of inhibiting CD40L- induced signaling. In some embodiments, the method comprises contacting a cell that expresses CD40 with an antigen-binding molecule or pharmaceutical composition as disclosed herein (e.g., a bispecific antigen-binding molecule comprising the sequences set forth in any of Tables 1-4 or 36, or a pharmaceutical composition comprising a bispecific antigen-binding molecule comprising the sequences set forth in any of Tables 1 -4 or 36).

[0039] In another aspect, the present disclosure provides methods of treating, ameliorating, or preventing a CD40-mediated disease or condition in a subject. In some embodiments, the method comprises administering the subject a therapeutically effective amount of an antigen-binding molecule or pharmaceutical composition as disclosed herein (e.g., a bispecific antigen-binding molecule comprising the sequences set forth in any of Tables 1-4 or 36, or a pharmaceutical composition comprising a bispecific antigen-binding molecule comprising the sequences set forth 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 organ transplant. In some embodiments, the CD40-mediated disease or condition is an 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, 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, Sjogren's syndrome, skin graft, systemic lupus erythematosus, systemic sclerosis, transplant rejection, vasculitis, ANCA- associated vasculitis, ulcerative colitis, or Wegener granulomatosis. In some embodiments, the method further comprises administering to the subject a second therapeutic agent.

[0040] In another aspect, the present disclosure provides antigen-binding molecules (e.g., bispecific antigen-binding molecules) as disclosed herein, or pharmaceutical compositions comprising said antigen-binding molecules, for use in inhibiting CD40L-induced signaling. In some embodiments, the antigen-binding molecule is a bispecific antigen-binding molecule (e.g., bispecific antibody) comprising the sequences set forth in any of Tables 1-4 or 36.

[0041] In another aspect, the present disclosure provides antigen-binding molecules (e.g., bispecific antigen-binding molecules) as disclosed herein, or pharmaceutical compositions comprising said antigen-binding molecules, for use in treating, ameliorating, or preventing a CD40-mediated disease or condition in a subject. In some embodiments, the antigen-binding molecule is a bispecific antigen-binding molecule (e.g., bispecific antibody) comprising the sequences set forth 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 organ transplant. In some embodiments, the CD40-mediated disease or condition is an 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, 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, Sjogren's syndrome, skin graft, systemic lupus erythematosus, systemic sclerosis, transplant rejection, vasculitis, ANCA-associated vasculitis, ulcerative colitis, or Wegener granulomatosis.

[0042] In another aspect, the present disclosure provides antigen-binding molecules (e.g., bispecific antigen-binding molecules) as disclosed herein for use in the manufacture of a medicament for treating, ameliorating, or preventing a CD40-mediated disease or condition in a subject. In some embodiments, the antigen-binding molecule is a bispecific antigen-binding molecule (e.g., bispecific antibody) comprising the sequences set forth 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 organ transplant. In some embodiments, the CD40-mediated disease or condition is an 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, 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, Sjogren's syndrome, skin graft, systemic lupus erythematosus, systemic sclerosis, transplant rejection, vasculitis, ANCA-associated vasculitis, ulcerative colitis, or Wegener granulomatosis.

[0043] Other embodiments will be apparent from a review of the ensuing detailed description.BRIEF DESCRIPTION OF THE FIGURES

[0044] FIGS. 1A-1 C show the effect of CD40xCD40 bispecific antibodies on IL6 production in the presence of constant CD40L in human B cells from three different donors.

[0045] FIGS. 1 D-1 E show the effect of CD40xCD40 bispecific antibodies on IL6 production in the presence of constant CD40L in human B cells from two different donors.

[0046] FIGS. 2A-2C show the effect of CD40xCD40 bispecific antibodies on IL10 production in the presence of constant CD40L in human B cells from three different donors.

[0047] FIGS. 2D-2E show the effect of CD40xCD40 bispecific antibodies on IL10 production in the presence of constant CD40L in human B cells from two different donors.

[0048] FIGS. 3A-3C show the effect of CD40xCD40 bispecific antibodies on TNFa production in the presence of constant CD40L in human B cells from three different donors.

[0049] FIGS. 3D-3E show the effect of CD40xCD40 bispecific antibodies on TNFa production in the presence of constant CD40L in human B cells from two different donors.

[0050] FIGS. 3F-3G show analysis of agonist activity of CD40xCD40 bispecific antibodies as measured by IL6 production in human B cells from two different donors.

[0051] FIGS. 4A-4B show the effect of CD40xCD40 bispecific antibodies on IL-12 / IL-23p40 production in the presence of constant CD40L from human monocyte derived dendritic cells from two different donors.

[0052] FIGS. 5A-5B show analysis of agonist activity of CD40xCD40 bispecific antibodies as measured by IL6 production in human B cells from two different donors.

[0053] FIGS. 6A-6B show analysis of agonist activity of CD40xCD40 bispecific antibodies as measured by IL10 production in human B cells from two different donors.

[0054] FIG. 7 shows an experiment timeline using a NP-KLH immunization model as disclosed in Example 9.

[0055] FIG. 8A shows the effect of CD40xCD40 bispecific antibodies on the frequency of NP positive germinal center B cells. * p<0.05.

[0056] FIG. 8B shows the effect of CD40xCD40 bispecific antibodies on the frequency of NP lgG1 titers in mouse serum. * p<0.05; ** p<0.005.

[0057] FIG. 9A shows the effect of CD40xCD40 bispecific antibodies on the frequency of NP positive germinal center B cells.

[0058] FIG. 9B shows the effect of CD40xCD40 bispecific antibodies on the frequency of NP lgG1 titers in mouse serum.

[0059] FIG. 10 shows an experiment timeline using a mouse EAE model as disclosed in Example 10.

[0060] FIG. 11A shows mean EAE symptom scores for REGN16431- or REGN16432- treated mice.

[0061] FIG. 11 B shows mean EAE symptom scores for REGN16334- or REGN16335- treated mice.

[0062] FIG. 11 C shows percent of initial body weight in REGN16431 - or REGN16432- treated mice.

[0063] FIG. 11 D shows percent of initial body weight in REGN16334- or REGN16335- treated mice.

[0064] FIG. 12A shows mean EAE symptom scores for CD40xCD40 bispecific antibody- treated mice.

[0065] FIG. 12B shows percent of initial body weight in CD40xCD40 bispecific antibody- treated mice.

[0066] FIG. 13 shows a cryoEM reconstruction of CD40 in complex with the Fab arms 30027P2, 21519P2, and 21520P2.DETAILED DESCRIPTIONDefinitions

[0067] Before the present invention is described, it is to be understood that the invention is not limited to particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0068] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0069] As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1 , 99.2, 99.3, 99.4, etc.).

[0070] The term "CD40," as used herein, refers to cluster of differentiation 40 (CD40 / TNFRSF5), a co-stimulatory cell surface receptor that is part of the tumor necrosis factor (TNF) receptor superfamily. In some embodiments, the CD40 is a human CD40. In some embodiments, the CD40 protein comprises the amino acid sequence of human CD40 set forth in UniProt Accession No. Q09LL4.

[0071] The term "antigen-binding molecule" includes antibodies and antigen-binding fragments of antibodies, including multispecific antibodies, e.g., bispecific antibodies.

[0072] The term "antibody," as used herein, refers to an antigen-binding molecule or molecular complex comprising a set of complementarity determining regions (CDRs) that specifically bind to or interact with a particular antigen (e.g., CD40). The term “antibody,” as used herein, includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof e.g., IgM). In a typical antibody, each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1 , CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or V ) and a light chain constant region. The light chain constant region comprises one domain (Ci_1 )- The V and V regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. In some embodiments, the FRs of the antibody(or antigen-binding portion thereof) may be identical to the human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0073] Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition (enhanced Chothia or Martin), the IMGT definition, and the Honneger definition (AHo). In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of the structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., 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); see also, Dondelinger et al., Front. Immunol. (2018), 9:2278, doi:10.3389 / fimmu.2018.02278. Public databases are also available for identifying CDR sequences within an antibody.

[0074] The term “antibody,” as used herein, also includes antigen-binding fragments of full antibody molecules. The terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, "antigen-binding domain," and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigenbinding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and / or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

[0075] 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) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3- CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, singledomain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment," as used herein.

[0076] An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a V domain associated with a V domain, the V and V domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH, VH-V or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.

[0077] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody include: (i) V -CH1 ; (ii) V -CH2; (iii) V -CH3; (iv) V - CH1 -CH2; (V) VH-CH1 -CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1 ; (ix) VL-CH2; (X) VL- CH3; (xi) V -CH1 -CH2; (xii) V -CH1 -CH2-CH3; (xiii) V -CH2-CH3; and (xiv) V -CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and / or with one or more monomeric V or V domain (e.g., by disulfide bond(s)).

[0078] The term "antibody," as used herein, also includes multispecific (e.g., bispecific) antibodies. A multispecific antibody or antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding 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 an antigen and an arm that binds to a second epitope of the same antigen.

[0079] Any multispecific antibody format may be adapted for use in the context of an antibody or antigen-binding fragment of an antibody of the present disclosure using routinetechniques available in the art. For example, the present disclosure includes bispecific antibodies wherein one arm of an immunoglobulin is specific for a first epitope of CD40, and the other arm of the immunoglobulin is specific for a second epitope of CD40. Exemplary bispecific formats that can be used in the context of the present disclosure include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-lg, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, lgG1 / lgG2, dual acting Fab (DAF)-lgG, and Mab2bispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6, 1 -11 , and references cited therein, for a review of the foregoing formats). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugation, e.g., wherein unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates which then self-assemble into multimeric complexes with defined composition, valency and geometry. (See, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]).

[0080] The term “human antibody,” as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the disclosure may nonetheless include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term “human antibody,” as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0081] The term “recombinant antibody,” as used herein, is intended to include all antibodies that are prepared, expressed, created or isolated by recombinant means. The term includes, but is not limited to, antibodies expressed using a recombinant expression vector transfected into a host cell (e.g., Chinese hamster ovary (CHO) cell) or cellular expression system, antibodies isolated from a recombinant, combinatorial human antibody library, and antibodies isolated from a non-human animal (e.g., a mouse, such as a mouse that is transgenic for human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295). In some embodiments, the recombinant antibody is a recombinant human antibody. In some embodiments, recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the V and V regions of the recombinant antibodies are sequences that, while derived from and related to humangermline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0082] An "isolated antibody" refers to an antibody that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally exists or is naturally produced, is an "isolated antibody." An isolated antibody also includes an antibody in situ within a recombinant cell. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0083] The term “specifically binds,” or the like, means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiologic conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1 x106M or less, e.g., 10-7M, 10-8M, 10-9M, 10-1° M, 10-11M, or 10-12M (a smaller KDdenotes a tighter 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™), bio-layer interferometry assay (e.g., Octet® HTX biosensor), solution-affinity ELISA, and the like. In some embodiments, specific binding is measured in a surface plasmon resonance assay, e.g., at 25°C or 37°C. An antibody or antigen-binding fragment that specifically binds an antigen from one species may or may not have cross-reactivity to other antigens, such as an orthologous antigen from another species.

[0084] The term "KD," as used herein, refers to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0085] The term "surface plasmon resonance," as used herein, refers to an optical phenomenon that allows for the analysis of real-time biomolecular interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIACORE™ system (Cytiva, Marlborough, MA).

[0086] The term "epitope," as used herein, refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. The term "epitope" also refers to a site on an antigen to which B and / or T cells respond. It also refers to a region of an antigen that is bound by an antibody. Epitopes may be either linear or discontinuous (e.g., conformational). A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. In certainembodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics, and / or specific charge characteristics. Epitopes may also be defined as structural or functional. Functional epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction. An epitope typically includes at least 3, and more usually, at least 5 or at least 8-10 amino acids in a unique spatial conformation.

[0087] Methods for determining the epitope of an antigen-binding protein, e.g., an antibody or antigen-binding fragment, include alanine scanning mutational analysis, peptide blot analysis (Reineke, Methods Mol Biol 2004, 248:443-463), peptide cleavage analysis, crystallographic studies, and NMR analysis. In addition, methods such as epitope exclusion, epitope extraction, and chemical modification of antigens can be employed (Tomer, Prot Sci 2000, 9:487-496). Another method that can be used to identify the amino acids within a polypeptide with which an antigen-binding protein (e.g., an antibody or antigen-binding fragment) interacts is hydrogen / deuterium exchange detected by mass spectrometry (HDX). See, e.g., Ehring, Analytical Biochemistry 1999, 267:252-259; Engen and Smith, Anal Chem 2001 , 73:256A-265A.

[0088] The term "competes," as used in reference to competing for binding, refers to an antigen-binding protein (e.g., antibody or antigen-binding fragment) that binds to an antigen and inhibits or blocks the binding of another antigen-binding protein (e.g., antibody or antigen-binding fragment) to the antigen. Unless otherwise stated, the term also includes competition between two antigen-binding proteins (e.g., antibodies) in both orientations, i.e., a first antigen that binds antigen and blocks binding by a second antibody and vice versa. Thus, in some embodiments, competition occurs in one such orientation. In some embodiments, the first antigen-binding protein (e.g., antibody) and second antigen-binding protein (e.g., 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 be overlapping or non-overlapping, wherein binding of one antigen-binding protein inhibits or blocks the binding of the second antigen-binding protein, e.g., via steric hindrance. Competition between antigen-binding proteins may be measured by methods known in the art, e.g., by a real-time, label-free bio-layer interferometry assay.

[0089] The terms "substantial identity" and "substantially identical," as used with reference to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 90%, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or GAP, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0090] As applied to polypeptides, the terms "substantial identity" and "substantially identical" mean that two peptide sequences, when optimally aligned, 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, residue positions that are not identical differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein.

[0091] Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG software contains 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 of organisms or between a wild-type protein and a mutein thereof. See, e.g., GCG Version 6.1 . Polypeptide sequences also can be compared using FASTA with default or recommended parameters; a program in GCG Version 6.1 . FASTA (e.g., FASTA2 and FAST A3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, 2000 supra). Another preferred algorithm when comparing a sequence of the disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. (See, e.g., Altschul et al., 1990, J. Mol. Biol. 215: 403-410 and 1997 Nucleic Acids Res. 25:3389-3402).

[0092] A "variant" of a polypeptide, such an immunoglobulin, VH, VL, heavy chain, light chain, or CDR comprising an amino acid sequence specifically set forth herein, refers to a polypeptide comprising an amino acid sequence that is at least about 70%-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 the reference polypeptide sequence (e.g., as set forth in the sequence listing below), when the comparison is performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences. In some embodiments, a variant of a polypeptide includes a polypeptide having the amino acid sequence of a reference polypeptide sequence(e.g., as set forth in the sequence listing below) but for 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.CD40 Antigen-Binding Molecules

[0093] In one aspect, the present disclosure relates to antigen-binding molecules, including monospecific, bispecific, 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. Multispecific antibodies may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, e.g., Tutt et al., 1991 , J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238- 244. In some embodiments, the CD40 antibodies of the present disclosure can be linked to or co-expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment to produce a bispecific or a multispecific antibody with a second binding specificity. In some embodiments, the multispecific antibody contains an antigen-binding domain that is specific for CD40 and an antigen-binding domain that is specific for another antigen (i.e., not CD40). In some embodiments, the multispecific antibody contains an antigen-binding domain that is specific for a first epitope of CD40 and an antigen-binding domain that is specific for a second epitope of CD40.Monospecific CD40 Antibodies

[0094] In some embodiments, the present disclosure provides monospecific CD40 antibodies or antigen-binding fragments thereof that specifically bind CD40 (e.g., human CD40). In some embodiments, the CD40 antibody or antigen-binding fragment thereof comprises the HCDR1 -HCDR2-HCDR3-LCDR1 -LCDR2-LCDR3 amino acid sequences 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.

[0095] In some embodiments, the CD40 antibody or antigen-binding fragment thereof comprises:(a) an HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:4, 24, and 34;(b) an HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:6, 26, and 36;(c) an HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:8, 28, and 38;(d) an LCDR1 comprising the amino acid sequence of SEQ ID NO:12;(e) an LCDR2 comprising the amino acid sequence AAS; and(f) an LCDR3 comprising the amino acid sequence of SEQ ID NO:16.

[0096] In some embodiments, the CD40 antibody or antigen-binding fragment comprises:(a) an HCDR1 comprising the amino acid sequence of SEQ ID NO:4, an HCDR2 comprising the amino acid sequence of SEQ ID NO:6, an HCDR3 comprising the amino acid sequence of SEQ ID NO:8, an LCDR1 comprising the amino acid sequence of SEQ ID NO:12, an LCDR2 comprising the amino acid sequence AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:16; or(b) an HCDR1 comprising the amino acid sequence of SEQ ID NO:24, an HCDR2 comprising the amino acid sequence of SEQ ID NO:26, an HCDR3 comprising the amino acid sequence of SEQ ID NO:28, an LCDR1 comprising the amino acid sequence of SEQ ID NO:12, an LCDR2 comprising the amino acid sequence AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:16; or(c) an HCDR1 comprising the amino acid sequence of SEQ ID NO:34, an HCDR2 comprising the amino acid sequence of SEQ ID NO:36, an HCDR3 comprising the amino acid sequence of SEQ ID NO:38, an LCDR1 comprising the amino acid sequence of SEQ ID NO:12, an LCDR2 comprising the amino acid sequence AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:16.

[0097] In some embodiments, the CD40 antibody comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:4, an HCDR2 comprising the amino acid sequence of SEQ ID NO:6, an HCDR3 comprising the amino acid sequence of SEQ ID NO:8, an LCDR1 comprising the amino acid sequence of SEQ ID NO:12, an LCDR2 comprising the amino acid sequence AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:16. In some embodiments, the CD40 antibody comprises an HCDR1 consisting of the amino acid sequence of SEQ ID NO:4, an HCDR2 consisting of the amino acid sequence of SEQ ID NO:6, an HCDR3 consisting of the amino acid sequence of SEQ ID NO:8, an LCDR1 consisting of the amino acid sequence of SEQ ID NO:12, an LCDR2 consisting of the aminoacid sequence AAS, and an LCDR3 consisting of the amino acid sequence of SEQ ID NO:16.

[0098] 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 NQ:10. In some embodiments, the CD40 antibody comprises an HCVR comprising or consisting of the amino acid sequence of SEQ ID NO:2. In some embodiments, the CD40 antibody comprises an LCVR comprising or consisting of the amino acid sequence of SEQ ID NQ:10.

[0099] In some embodiments, the CD40 antibody comprises: a heavy chain comprising an amino acid sequence that has 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:18; and / or a light chain comprising an amino acid sequence that has 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 NQ:20. In some embodiments, the CD40 antibody comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO:18. In some embodiments, the CD40 antibody comprises a light chain comprising or consisting of the amino acid sequence of SEQ ID NQ:20.

[0100] In some embodiments, the CD40 antibody comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:24, an HCDR2 comprising the amino acid sequence of SEQ ID NO:26, an HCDR3 comprising the amino acid sequence of SEQ ID NO:28, an LCDR1 comprising the amino acid sequence of SEQ ID NO:12, an LCDR2 comprising the amino acid sequence AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:16. In some embodiments, the CD40 antibody comprises an HCDR1 consisting of the amino acid sequence of SEQ ID NO:24, an HCDR2 consisting of the amino acid sequence of SEQ ID NO:26, an HCDR3 consisting of the amino acid sequence of SEQ ID NO:28, an LCDR1 consisting of the amino acid sequence of SEQ ID NO:12, an LCDR2 consisting of the amino acid sequence AAS, and an LCDR3 consisting of the amino acid sequence of SEQ ID NO:16.

[0101] 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 comprising or consisting of the amino acid sequence of SEQ ID NO:22. In some embodiments, the CD40 antibody comprises an LCVR comprising or consisting of the amino acid sequence of SEQ ID NQ:10.

[0102] In some embodiments, the CD40 antibody comprises: a heavy chain comprising an amino acid sequence that has 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:28; and / or a light chain comprising an amino acid sequence that has 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 NQ:20. In some embodiments, the CD40 antibody comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO:28. In some embodiments, the CD40 antibody comprises a light chain comprising or consisting of the amino acid sequence of SEQ ID NQ:20.

[0103] In some embodiments, the CD40 antibody comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:34, an HCDR2 comprising the amino acid sequence of SEQ ID NO:36, an HCDR3 comprising the amino acid sequence of SEQ ID NO:38, an LCDR1 comprising the amino acid sequence of SEQ ID NO:12, an LCDR2 comprising the amino acid sequence AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:16. In some embodiments, the CD40 antibody comprises an HCDR1 consisting of the amino acid sequence of SEQ ID NO:34, an HCDR2 consisting of the amino acid sequence of SEQ ID NO:36, an HCDR3 consisting of the amino acid sequence of SEQ ID NO:38, an LCDR1 consisting of the amino acid sequence of SEQ ID NO:12, an LCDR2 consisting of the amino acid sequence AAS, and an LCDR3 consisting of the amino acid sequence of SEQ ID NO:16.

[0104] 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 NQ:10. In some embodiments, the CD40 antibody comprises an HCVR comprising or consisting of the amino acid sequence of SEQ ID NO:32. In some embodiments, the CD40 antibody comprises an LCVR comprising or consisting of the amino acid sequence of SEQ ID NQ:10.

[0105] In some embodiments, the CD40 antibody comprises: a heavy chain comprising an amino acid sequence that has 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:40; and / or a light chain comprising an amino acid sequence that has 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:20. In some embodiments, the CD40 antibody comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NQ:40. In some embodiments, the CD40 antibody comprises a light chain comprising or consisting of the amino acid sequence of SEQ ID NQ:20.

[0106] In some embodiments, the CD40 antibody has at least 85% sequence identity, 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% sequence identity, to 30027P2. In some embodiments, the CD40 antibody has the amino acid sequence of 30027P2.

[0107] In some embodiments, the CD40 antibody has at least 85% sequence identity, 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% sequence identity, to 21519P2. In some embodiments, the CD40 antibody has the amino acid sequence of 21519P2.

[0108] In some embodiments, the CD40 antibody has at least 85% sequence identity, 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% sequence identity, to 21520P2. In some embodiments, the CD40 antibody comprises or consists of the amino acid sequence of 21520P2.CD40xCD40 Bispecific Antigen-Binding Molecules

[0109] The present disclosure also provides multispecific antigen-binding molecules that specifically bind CD40. In some embodiments, the antigen-binding molecule is a bispecific antigen-binding molecule, e.g., bispecific antibody. In some embodiments, the bispecific antigen-binding molecule comprises a first antigen-binding domain (D1 ) that binds a first epitope of CD40 (e.g., human CD40), and a second antigen-binding domain (D2) that binds a second epitope of CD40 (e.g., human CD40). In some embodiments, D1 and D2 do not compete with one another for binding to CD40 (e.g., human CD40). In some embodiments, D1 and D2 compete with one another for binding to CD40 (e.g., human CD40).

[0110] In some embodiments, the bispecific antigen-binding molecule comprises two different heavy chain immunoglobulin variable regions, wherein at least one heavy chain immunoglobulin variable region comprises an HCDR1 -HCDR2-HCDR3 amino acid sequences 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 sequences set contained within an HCVR amino acid sequence selected from the groupconsisting 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.

[0111] In some embodiments, one or more of the heavy chain immunoglobulin variable regions comprises a set of HCDR sequences selected from the group consisting of:(a) an HCDR1 comprising the amino acid sequence of SEQ ID NO:4, an HCDR2 comprising the amino acid sequence of SEQ ID NO:6, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:8;(b) an HCDR1 comprising the amino acid sequence of SEQ ID NO:24, an HCDR2 comprising the amino acid sequence of SEQ ID NO:26, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:28; and(c) an HCDR1 comprising the amino acid sequence of SEQ ID NO:34, an HCDR2 comprising the amino acid sequence of SEQ ID NO:36, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:38.

[0112] In some embodiments, the bispecific antigen-binding molecule comprises (i) an antigen-binding domain that comprises a heavy chain immunoglobulin variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO:4, an HCDR2 comprising the amino acid sequence of SEQ ID NO:6, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:8; and (ii) an antigen-binding domain that comprises a heavy chain immunoglobulin variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO:24, an HCDR2 comprising the amino acid sequence of SEQ ID NO:26, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:28.

[0113] In some embodiments, the bispecific antigen-binding molecule comprises (i) an antigen-binding domain that comprises a heavy chain immunoglobulin variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO:4, an HCDR2 comprising the amino acid sequence of SEQ ID NO:6, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:8; and (ii) an antigen-binding domain that comprises a heavy chain immunoglobulin variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO:34, an HCDR2 comprising the amino acid sequence of SEQ ID NO:36, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:38.

[0114] In some embodiments, the bispecific antigen-binding molecule comprises (i) an antigen-binding domain that comprises a heavy chain immunoglobulin variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO:24, an HCDR2 comprising the amino acid sequence of SEQ ID NO:26, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:28; and (ii) an antigen-binding domain that comprises aheavy chain immunoglobulin variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO:34, an HCDR2 comprising the amino acid sequence of SEQ ID NO:36, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:38.

[0115] In some embodiments, the bispecific antigen-binding molecule comprises a common light chain variable region. In some embodiments, the light chain variable region comprises an HCDR1-HCDR2-HCDR3 amino acid sequences set contained within the LCVR amino acid sequence of SEQ ID NQ:10. In some embodiments, the light chain variable region comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO:12, an LCDR2 comprising the amino acid sequence AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:16.

[0116] In some embodiments, the bispecific antigen-binding molecule comprises a D1 that binds a first epitope of human CD40, wherein the D1 domain comprises a heavy chain immunoglobulin chain comprising:(a) an HCDR1 comprising the amino acid sequence of SEQ ID NO:4, an HCDR2 comprising the amino acid sequence of SEQ ID NO:6, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:8; or(b) an HCDR1 comprising the amino acid sequence of SEQ ID NO:24, an HCDR2 comprising the amino acid sequence of SEQ ID NO:26, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:28.

[0117] In some embodiments, the D1 domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:4, an HCDR2 comprising the amino acid sequence of SEQ ID NO:6, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:8. In some embodiments, the D1 domain comprises an HCDR1 consisting of the amino acid sequence of SEQ ID NO:4, an HCDR2 consisting of the amino acid sequence of SEQ ID NO:6, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:8. In some embodiments, the D1 domain comprises an HCVR comprising an amino acid sequence that has 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. In some embodiments, the D1 domain comprises an HCVR comprising or consisting of the amino acid sequence of SEQ ID NO:2.

[0118] In some embodiments, the D1 domain comprises a heavy chain comprising an amino acid sequence that has 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:46. In some embodiments, the D1 domain comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO:46.

[0119] In some embodiments, the D1 domain comprises a heavy chain comprising an amino acid sequence that has at least 85%, at least 90%, at least 91%, at least 92%, at least93%, 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:52. In some embodiments, the D1 domain comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO:52.

[0120] In some embodiments, the D1 domain further comprises a light chain immunoglobulin chain comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO:12, an LCDR2 comprising the amino acid sequence AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:16.

[0121] In some embodiments, the D1 domain comprises a LCVR comprising an amino acid sequence that has 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 NQ:10. In some embodiments, the D1 domain comprises an LCVR comprising the amino acid sequence of SEQ ID NQ:10.

[0122] In some embodiments, the D1 domain comprises a light chain comprising an amino acid sequence that has 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 NQ:20. In some embodiments, the D1 domain comprises a light chain comprising the amino acid sequence of SEQ ID NQ:20.

[0123] In some embodiments, the D1 domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:24, an HCDR2 comprising the amino acid sequence of SEQ ID NO:26, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:28. In some embodiments, the D1 domain comprises an HCDR1 consisting of the amino acid sequence of SEQ ID NO:24, an HCDR2 consisting of the amino acid sequence of SEQ ID NO:26, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:28. In some embodiments, the D1 domain comprises an HCVR comprising an amino acid sequence that has 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. In some embodiments, the D1 domain comprises an HCVR comprising or consisting of the amino acid sequence of SEQ ID NO:22.

[0124] In some embodiments, the D1 domain comprises a heavy chain comprising an amino acid sequence that has 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:42. In some embodiments, the D1 domain comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO:42.

[0125] In some embodiments, the D1 domain comprises a heavy chain comprising an amino acid sequence that has 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:46. In some embodiments, the D1 domain comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO:46.

[0126] In some embodiments, the D1 domain further comprises a light chain immunoglobulin chain comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO:12, an LCDR2 comprising the amino acid sequence AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:16.

[0127] In some embodiments, the D1 domain comprises a LCVR comprising an amino acid sequence that has 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 NQ:10. In some embodiments, the D1 domain comprises an LCVR comprising the amino acid sequence of SEQ ID NQ:10.

[0128] In some embodiments, the D1 domain comprises a light chain comprising an amino acid sequence that has 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 NQ:20. In some embodiments, the D1 domain comprises a light chain comprising the amino acid sequence of SEQ ID NQ:20.

[0129] In some embodiments, the bispecific antigen-binding molecule comprises a D2 that binds a second epitope of human CD40, wherein the D2 domain comprises a heavy chain immunoglobulin chain comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO:34, an HCDR2 comprising the amino acid sequence of SEQ ID NO:36, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:38. In some embodiments, the D2 domain comprises an HCDR1 consisting of the amino acid sequence of SEQ ID NO:34, an HCDR2 consisting of the amino acid sequence of SEQ ID NO:36, and an 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 that has 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. In some embodiments, the D2 domain comprises an HCVR comprising or consisting of the amino acid sequence of SEQ ID NO:32.

[0130] In some embodiments, the D2 domain comprises a heavy chain comprising an amino acid sequence that has 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:44. In some embodiments, the D2 domain comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO:44.

[0131] In some embodiments, the D2 domain comprises a heavy chain comprising an amino acid sequence that has 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:50. In some embodiments, the D2 domain comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO:50.

[0132] In some embodiments, the D2 domain further comprises a light chain immunoglobulin chain comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO:12, an LCDR2 comprising the amino acid sequence AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:16.

[0133] In some embodiments, the D2 domain comprises an LCVR comprising an amino acid sequence that has 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 NQ:10. In some embodiments, the D2 domain comprises an LCVR comprising the amino acid sequence of SEQ ID NQ:10.

[0134] In some embodiments, the D2 domain comprises a light chain comprising an amino acid sequence that has 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 NQ:20. In some embodiments, the D2 domain comprises a light chain comprising the amino acid sequence of SEQ ID NQ:20.

[0135] In some embodiments, the bispecific antigen-binding molecule comprises a D1 domain that binds a first epitope of human CD40 and a D2 domain that binds a second epitope of human CD40, wherein the D1 domain comprises a heavy chain immunoglobulin chain comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO:34, an HCDR2 comprising the amino acid sequence of SEQ ID NO:36, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:38. In some embodiments, the D1 domain comprises an HCDR1 consisting of the amino acid sequence of SEQ ID NO:34, an HCDR2 consisting of the amino acid sequence of SEQ ID NO:36, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:38. In some embodiments, the D1 domain comprises an HCVR comprising an amino acid sequence that has 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. In some embodiments, the D1 domain comprises an HCVR comprising or consisting of the amino acid sequence of SEQ ID NO:32.

[0136] In some embodiments, the D1 domain comprises a heavy chain comprising an amino acid sequence that has 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:58. In some embodiments, the D1 domain comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO:58.

[0137] In some embodiments, the D2 domain comprises a heavy chain immunoglobulin chain comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO:4, anHCDR2 comprising the amino acid sequence of SEQ ID NO:6, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:8. In some embodiments, the D2 domain comprises an HCDR1 consisting of the amino acid sequence of SEQ ID NO:4, an HCDR2 consisting of the amino acid sequence of SEQ ID NO:6, and an HCDR3 consisting of the amino acid sequence of SEQ ID NO:8. In some embodiments, the D2 domain comprises an HCVR comprising an amino acid sequence that has 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. In some embodiments, the D2 domain comprises an HCVR comprising or consisting of the amino acid sequence of SEQ ID NO:2.

[0138] In some embodiments, the D2 domain comprises a heavy chain comprising an amino acid sequence that has 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 NQ:60. In some embodiments, the D2 domain comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NQ:60.

[0139] In some embodiments, the D1 and / or D2 domain further comprises a light chain immunoglobulin chain comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO:12, an LCDR2 comprising the amino acid sequence AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:16. In some embodiments, the D1 and / or D2 domain comprises a LCVR comprising an amino acid sequence that has 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 NQ:10. In some embodiments, the D1 and / or D2 domain comprises an LCVR comprising the amino acid sequence of SEQ ID NQ:10. In some embodiments, the D1 and / or D2 domain comprises a light chain comprising an amino acid sequence that has 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 NQ:20. In some embodiments, the D1 and / or D2 domain comprises a light chain comprising the amino acid sequence of SEQ ID NQ:20.

[0140] In some embodiments, the bispecific antigen-binding molecule comprises: a D1 domain that binds a first epitope of human CD40, wherein the D1 domain comprises an immunoglobulin chain comprising an HCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO:4, an HCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO:6, an HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO:8, an LCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO:12, an LCDR2 comprising or consisting of the amino acid sequence AAS, and an LCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO:16; anda D2 domain that binds a second epitope of human CD40, wherein the D2 domain comprises an immunoglobulin chain comprising an HCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO:34, an HCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO:36, an HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO:38, an LCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO:12, an LCDR2 comprising or consisting of the amino acid sequence AAS, and an LCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO:16.

[0141] In some embodiments, the bispecific antigen-binding molecule comprises: a D1 domain that comprises an HCVR comprising an amino acid sequence that has at least 85% (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%) sequence identity to SEQ ID NO:2, and an LCVR comprising an amino acid sequence that has at least 85% (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%) sequence identity to SEQ ID NQ:10; and a D2 domain that comprises an HCVR comprising an amino acid sequence that has at least 85% (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%) sequence identity to SEQ ID NO:32, and an LCVR comprising an amino acid sequence that has at least 85% (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%) sequence identity to SEQ ID NQ:10.

[0142] In some embodiments, the bispecific antigen-binding molecule comprises: a D1 domain that comprises an HCVR comprising or consisting of the amino acid sequence of SEQ ID NO:2 and an LCVR comprising or consisting of the amino acid sequence of SEQ ID NQ:10; and a D2 domain that comprises an HCVR comprising or consisting of the amino acid sequence of SEQ ID NO:32 and an LCVR comprising or consisting of the amino acid sequence of SEQ ID NQ:10.

[0143] In some embodiments, the bispecific antigen-binding molecule comprises: a D1 domain that comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO:46 and a light chain comprising or consisting of the amino acid sequence of SEQ ID NQ:20; and a D2 domain that comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO:44 and a light chain comprising or consisting of the amino acid sequence of SEQ ID NQ:20.

[0144] In some embodiments, the bispecific antigen-binding molecule comprises: a D1 domain that comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO:52 and a light chain comprising or consisting of the amino acid sequence of SEQ ID NQ:20; and a D2 domain that comprises a heavy chain comprising or consisting ofthe amino acid sequence of SEQ ID NO:50 and a light chain comprising or consisting of the amino acid sequence of SEQ ID NO:20.

[0145] In some embodiments, the bispecific antigen-binding molecule comprises: a D1 domain that binds a first epitope of human CD40, wherein the D1 domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:24, an HCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO:26, an HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO:28, an LCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO:12, an LCDR2 comprising or consisting of the amino acid sequence AAS, and an LCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO:16; and a D2 domain that binds a second epitope of human CD40, wherein the D2 domain comprises an HCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO:34, an HCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO:36, an HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO:38, an LCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO:12, an LCDR2 comprising or consisting of the amino acid sequence AAS, and an LCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO:16.

[0146] In some embodiments, the bispecific antigen-binding molecule comprises: a D1 domain that comprises an HCVR comprising an amino acid sequence that has at least 85% (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%) sequence identity to SEQ ID NO:2, and an LCVR comprising an amino acid sequence that has at least 85% (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%) sequence identity to SEQ ID NQ:10; and a D2 domain that comprises an HCVR comprising an amino acid sequence that has at least 85% (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%) sequence identity to SEQ ID NO:32, and an LCVR comprising an amino acid sequence that has at least 85% (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%) sequence identity to SEQ ID NQ:10.

[0147] In some embodiments, the bispecific antigen-binding molecule comprises: a D1 domain that comprises an HCVR comprising or consisting of the amino acid sequence of SEQ ID NO:2 and an LCVR comprising or consisting of the amino acid sequence of SEQ ID NQ:10; and a D2 domain that comprises an HCVR comprising or consisting of the amino acid sequence of SEQ ID NO:32 and an LCVR comprising or consisting of the amino acid sequence of SEQ ID NQ:10.

[0148] In some embodiments, the bispecific antigen-binding molecule comprises: a D1 domain that comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO:42 and a light chain comprising or consisting of the amino acid sequence of SEQ ID NO:20; and a D2 domain that comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO:44 and a light chain comprising or consisting of the amino acid sequence of SEQ ID NQ:20.

[0149] In some embodiments, the bispecific antigen-binding molecule comprises: a D1 domain that comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO:48 and a light chain comprising or consisting of the amino acid sequence of SEQ ID NQ:20; and a D2 domain that comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NQ:50 and a light chain comprising or consisting of the amino acid sequence of SEQ ID NQ:20.

[0150] In some embodiments, the bispecific antigen-binding molecule comprises: a D1 domain that binds a first epitope of human CD40, wherein the D1 domain comprises an immunoglobulin chain comprising an HCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO:34, an HCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO:36, an HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO:38, an LCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO:12, an LCDR2 comprising or consisting of the amino acid sequence AAS, and an LCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO:16; and a D2 domain that binds a second epitope of human CD40, wherein the D2 domain comprises an immunoglobulin chain comprising an HCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO:4, an HCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO:6, an HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO:8, an LCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO:12, an LCDR2 comprising or consisting of the amino acid sequence AAS, and an LCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO:16.

[0151] In some embodiments, the bispecific antigen-binding molecule comprises: a D1 domain that comprises an HCVR comprising an amino acid sequence that has at least 85% (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%) sequence identity to SEQ ID NO:32, and an LCVR comprising an amino acid sequence that has at least 85% (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%) sequence identity to SEQ ID NQ:10; and a D2 domain that comprises an HCVR comprising an amino acid sequence that has at least 85% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:2, and an LCVR comprising an amino acid sequence that has at least 85% (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%) sequence identity to SEQ ID NO:10.

[0152] In some embodiments, the bispecific antigen-binding molecule comprises: a D1 domain that comprises an HCVR comprising or consisting of the amino acid sequence of SEQ ID NO:32 and an LCVR comprising or consisting of the amino acid sequence of SEQ ID NQ:10; and a D2 domain that comprises an HCVR comprising or consisting of the amino acid sequence of SEQ ID NO:2 and an LCVR comprising or consisting of the amino acid sequence of SEQ ID NQ:10.

[0153] In some embodiments, the bispecific antigen-binding molecule comprises: a D1 domain that comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO:58 and a light chain comprising or consisting of the amino acid sequence of SEQ ID NQ:20; and a D2 domain that comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NQ:60 and a light chain comprising or consisting of the amino acid sequence of SEQ ID NQ:20.

[0154] The bispecific antigen-binding molecules disclosed herein may be bispecific antibodies. In some cases, the bispecific antibody comprises a human IgG heavy chain constant region. In some cases, the human IgG heavy chain constant region is isotype lgG1. In some cases, the human IgG heavy chain constant region is isotype lgG4.

[0155] The first antigen-binding domain and the second antigen-binding domain may be directly or indirectly connected to one another to form a bispecific antigen-binding molecule of the present invention. Alternatively, the first antigen-binding domain and the second antigen-binding domain may each be connected to a separate multimerizing domain. The association of one multimerizing domain with another multimerizing domain facilitates the association between the two antigen-binding domains, thereby forming a bispecific antigenbinding molecule. As used herein, a "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 constitution. For example, a multimerizing domain may be a polypeptide comprising an immunoglobulin CH3 domain. A non-limiting example of a multimerizing component is an Fc portion of an immunoglobulin (comprising a CH2-CH3 domain), e.g., an Fc domain of an IgG selected from the isotypes IgG 1 , lgG2, lgG3, and lgG4, as well as any allotype within each isotype group.

[0156] In some embodiments, a bispecific antigen-binding molecules of the present disclosure comprises two multimerizing domains, e.g., two Fc domains that are each individually part of a separate antibody heavy chain. The first and second multimerizing domains may be of the same IgG isotype such as, e.g., IgG 1 / IgG 1 , lgG2 / lgG2, lgG4 / lgG4.Alternatively, the first and second multimerizing domains may be of different IgG isotypes such as, e.g., lgG1 / lgG2, lgG1 / lgG4, lgG2 / lgG4, etc.

[0157] In some embodiments, the multimerizing domain is an Fc fragment or an amino acid sequence of from 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 comprising or consisting of a leucine zipper, a helix-loop motif, or a coiled-coil motif.

[0158] In some embodiments, the bispecific antigen-binding molecule is a CD40xCD40 bispecific antibody that has at least 85% sequence identity, 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% sequence identity, to REGN16334. In some embodiments, the bispecific antigen-binding molecule is a CD40xCD40 bispecific antibody having the amino acid sequences of REGN16334.

[0159] In some embodiments, the bispecific antigen-binding molecule is a CD40xCD40 bispecific antibody that has at least 85% sequence identity, 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% sequence identity, to REGN16335. In some embodiments, the bispecific antigen-binding molecule is a bispecific antibody having the amino acid sequences of REGN16335.

[0160] In some embodiments, the bispecific antigen-binding molecule is a CD40xCD40 bispecific antibody that has at least 85% sequence identity, 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% sequence identity, to REGN16431 . In some embodiments, the bispecific antigen-binding molecule is a CD40xCD40 bispecific antibody having the amino acid sequences of REGN16431 .

[0161] In some embodiments, the bispecific antigen-binding molecule is a CD40xCD40 bispecific antibody that has at least 85% sequence identity, 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% sequence identity, to REGN16432. In some embodiments, the bispecific antigen-binding molecule is a CD40xCD40 bispecific antibody having the amino acid sequences of REGN16432.

[0162] In some embodiments, the bispecific antigen-binding molecule is a CD40xCD40 bispecific antibody that has at least 85% sequence identity, 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% sequence identity, to REGN20484. In some embodiments, the bispecific antigen-binding molecule is a CD40xCD40 bispecific antibody having the amino acid sequences of REGN20484.Sequence Variants

[0163] The antigen-binding molecules of the present disclosure may comprise one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and / or light chain variable domains as compared to the corresponding germline sequences from which the individual antigen-binding domains were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germ line sequences available from, for example, public antibody sequence databases. The antigen-binding molecules of the present disclosure may comprise antigen binding fragments which are derived from any of the exemplary amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and / or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as "germline mutations"). A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and / or CDR residues within the V and / or V domains are mutated back to the residues found in the original germline sequence from which the antigen-binding domain was originally derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1 , CDR2 or CDR3. In other embodiments, one or more of the framework and / or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germ line sequence from which the antigen-binding domain was originally derived). Furthermore, the antigen-binding domains may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germ line sequence while certain other residues that differ from the original germ line sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antigen-binding domains that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties, reduced immunogenicity, etc. Bispecific antigen-binding molecules comprisingone or more antigen-binding domains obtained in this general manner are encompassed within the present disclosure.

[0164] The present disclosure also includes antigen-binding molecules wherein one or both antigen-binding domains comprise variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present disclosure includes antigen-binding molecules comprising an antigenbinding domain having HCVR, LCVR, and / or CDR amino acid sequences with, e.g., 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 conservative amino acid substitution(s) relative to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. Examples of groups of amino acids that have 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: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445. A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

[0165] The present disclosure also includes antigen-binding molecules comprising an antigen binding domain with an HCVR, LCVR, and / or CDR amino acid sequence that is substantially identical to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. In some embodiments, an antigen-binding molecule comprises HCVR, LCVR, and / or CDR amino acid sequence having at least 85% sequence identity, 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% sequence identity, to a sequence disclosed in Table 36. In some embodiments, an antigen-binding molecule comprises HCVR, LCVR, and / or CDR amino acid sequence having at least 85% sequence identity, 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% sequence identity, to a sequence disclosed in Table 36,wherein the differences in the amino acid residue(s) relative to the sequence disclosed in Table 36 are conservative substitutions or moderately conservative substitutions.Antigen-Binding Proteins Comprising Fc Modifications

[0166] In some embodiments, a CD40 antigen-binding molecule as disclosed herein (e.g., a CD40xCD40 bispecific antigen-binding molecule, e.g., as disclosed in any one of Tables 1 - 4) comprises an Fc domain comprising one or more modifications or mutations that enhance or diminish antibody binding to the FcRn receptor. For example, the present disclosure includes antigen-binding molecules comprising one or more mutations in the CH2 and / or CH3 region of the Fc domain, wherein the mutation(s) increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Such mutations may result in an increase in serum half-life of the antibody when administered to an animal.

[0167] Non-limiting examples of such Fc modifications include, e.g., a modification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or a modification 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 a modification at position 250 and / or 428; or a modification at position 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modification comprises a 428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Q and M428L); and a 307 and / or 308 modification (e.g., 308F or 308P). See, e.g.., Ko et al., BioDrugs 2021 , 35:147-157.

[0168] In certain embodiments, a CD40xCD40 bispecific antigen-binding molecule comprises an Fc domain comprising one or more pairs or groups of mutations 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).

[0169] In some embodiments, the CD40xCD40 bispecific antigen-binding molecules of the present disclosure comprise a modified Fc domain having reduced effector function. As used herein, a "modified Fc domain having reduced effector function" means any Fc portion of an immunoglobulin that has been modified, mutated, truncated, etc., relative to a wild-type, naturally occurring Fc domain such that a molecule comprising the modified Fc exhibits a reduction in the severity or extent of at least one effect selected from the group consisting of cell killing (e.g., ADCC and / or CDC), complement activation, phagocytosis and opsonization, relative to a comparator molecule comprising the wild-type, naturally occurring version of theFc portion. In certain embodiments, a "modified Fc domain having reduced effector function" is an Fc domain with reduced or attenuated binding to an Fc receptor (e.g., FcyR).

[0170] In certain embodiments, a modified Fc domain having reduced binding to an Fc receptor (e.g., Fey receptor, e.g., FcyRI, FcyRIIA, FcyRIIB, or FcyRIIIA) is a variant lgG1 Fc or a variant lgG4 Fc comprising one or more substitutions or modifications in the hinge region and / or a CH region (e.g., CH2). For example, a modified Fc domain may comprise a variant IgG 1 Fc wherein at least one amino acid of an IgG 1 Fc hinge region and / or CH region is replaced with the corresponding amino acid from an lgG2 Fc hinge region and / or CH region. Alternatively, a modified Fc domain may comprise a variant lgG4 Fc wherein at least one amino acid of an lgG4 Fc hinge region and / or CH region is replaced with the corresponding amino acid from an lgG2 Fc hinge region and / or CH region. In some embodiments, a modified Fc domain comprises modifications in which each of positions 233- 236 by Ell numbering is occupied by G or is unoccupied. Non-limiting, exemplary modified Fc regions that can be used in the context of the present disclosure are set forth in US Patent No. 11 ,518,807, the disclosure of which is hereby incorporated by reference in its entirety, as well as any functionally equivalent variants of the modified Fc regions set forth therein. Other modified Fc domains and Fc modifications that can be used in the context of the present disclosure include any of the modifications as set forth in US 8,697,396, US 10,988,537, US 2014 / 0171623, US 2014 / 0134162, US 2014 / 0243504, and WO 2014 / 043361 , the disclosures of each of which are incorporated by reference herein.

[0171] In certain embodiments, a bispecific antigen-binding molecule as disclosed herein comprises immunoglobulin heavy chains that are heterodimeric (i.e., differing by at least one amino acid) and have differential affinity toward an affinity reagent, such as Protein A. In some embodiments, one of the heavy chains comprises one or more modifications in the Fc domain that reduces or eliminates binding of the Fc domain to Protein A. In some embodiments, one of the heavy chains comprises H435R / Y436F (by EU numbering system) substitutions in the CH3 region. Non-limiting, exemplary modified Fc regions that can be used in the context of the present disclosure are set forth in US Patent No. 8,586,713, the disclosure of which is hereby incorporated by reference in its entirety.

[0172] All possible combinations of the foregoing Fc domain mutations, and other mutations within the antibody variable domains disclosed herein, are contemplated within the scope of the present disclosure.Polynucleotides, Vectors, and Host Cells

[0173] In another aspect, the present disclosure provides nucleic acid molecules comprising one or more polynucleotide sequences encoding the antigen-binding moleculesdisclosed herein, as well as vectors (e.g., expression vectors) encoding such polynucleotide sequences and host cells into which such vectors have been introduced.

[0174] Polynucleotides, as disclosed herein, may encode all or a portion of an antigenbinding molecule, antibody, or antigen-binding fragment as disclosed throughout the present disclosure. In some cases, a single polynucleotide may encode both a HCVR and a LCVR (e.g., defined with reference to the CDRs contained within the respective amino acid sequence-defined HCVR and LCVR, defined with reference to the amino acid sequences of the CDRs of the HCVR and LCVR, respectively, or defined with reference to the amino acid sequences of the HCVR and LCVR, respectively) of an antibody or antigen-binding fragment, or the HCVR and LCVR may be encoded by separate polynucleotides (i.e., a pair of polynucleotides). In the latter case, in which the HCVR and LCVR are encoded by separate polynucleotides, the polynucleotides may be combined in a single vector or may be contained in separate vectors (i.e., a pair of vectors). In any case, a host cell used to express the polynucleotide(s) or vector(s) may contain the full complement of component parts to generate the antibody or antigen-binding fragment thereof. For example, a host cell may comprise separate vectors, each encoding a HCVR and a LCVR, respectively, of an antibody or antigen-binding fragment thereof as discussed above or herein. Similarly, the polynucleotide or polynucleotides, and the vector or vectors, may be used to express the full- length heavy chain and full-length light chain of an antibody as discussed above or herein. For example, a host cell may comprise a single vector with polynucleotides encoding both a heavy chain and a light chain of an antibody, or the host cell may comprise separate vectors with polynucleotides encoding, respectively, a heavy chain and a light chain of an antibody as disclosed above or herein.

[0175] In some embodiments, the nucleic acid molecule comprises one or more polynucleotide sequences encoding an antigen-binding molecule disclosed in any of Tables 1-4. In some embodiments, the nucleic acid molecule comprises one or more polynucleotide sequences set forth in Table 36.

[0176] In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence that encodes an CD40 HCVR comprising the HCDR1 , HCDR2, and HCDR3 of SEQ ID NOs: 4, 6, and 8, respectively, of SEQ ID NOs: 24, 26, and 28, respectively, or of SEQ ID NOs: 34, 36, and 38, respectively. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence that encodes an CD40 HCVR comprising or consisting of the sequence of SEQ ID NO:2, SEQ ID NO:22, or SEQ ID NO:32.

[0177] In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence that encodes an LCVR comprising an LCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO:12, an LCDR2 comprising the amino acid sequence AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:16. In someembodiments, the nucleic acid molecule comprises a polynucleotide sequence that encodes an LCVR comprising the sequence of SEQ ID NO:10.

[0178] In some embodiments, compositions are provided comprising one or more nucleic acid molecules as disclosed herein. For example, in some embodiments, a composition comprises a first nucleic acid molecule comprising a polynucleotide sequence encoding an HCVR and / or LCVR of a first antigen-binding molecule that binds a first epitope of CD40, and a second nucleic acid molecule comprising a polynucleotide sequence encoding an HCVR and / or LCVR of a second antigen-binding molecule that binds a second epitope of CD40. In some embodiments, a composition comprises a first nucleic acid molecule comprising a polynucleotide sequence encoding an HCVR of a first antigen-binding molecule that binds a first epitope of CD40, a second nucleic acid molecule comprising a polynucleotide sequence encoding an LCVR of a first antigen-binding molecule that binds a first epitope of CD40, a third nucleic acid molecule comprising a polynucleotide sequence encoding an HCVR of a second antigen-binding molecule that binds a second epitope of CD40, and a fourth nucleic acid molecule comprising a polynucleotide sequence encoding an LCVR of a second antigen-binding molecule that binds a second epitope of CD40. In some embodiments, the HCVR sequences of the first and second antigen-binding molecules are selected from: an CD40 HCVR comprising the HCDR1 , HCDR2, and HCDR3 of SEQ ID NOs: 4, 6, and 8, respectively, an CD40 HCVR comprising the HCDR1 , HCDR2, and HCDR3 of SEQ ID NOs: 24, 26, and 28, respectively, and an CD40 HCVR comprising the 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 NO:2, SEQ ID NO:22, and SEQ ID NO:32. In some embodiments, the LCVR sequences of first and second antigen-binding molecules each comprise an LCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO:12, an LCDR2 comprising the amino acid sequence AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:16. In some embodiments, the LCVR sequences of first and second antigen-binding molecules each comprise the sequence of SEQ ID NQ:10.

[0179] In another aspect, the present disclosure also provides recombinant expression vectors carrying one or more nucleic acid molecules as 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., E. coli). In some embodiments, the host cell is a eukaryotic cell, such as a non-human mammalian cell (e.g., a Chinese Hamster Ovary (CHO) cell). Also provided herein are methods of producing the antigen-binding molecules of the disclosure by culturing the host cells under conditions permitting production of the antigen-binding molecules, and recovering the antigen-binding molecules so produced.Characterization of CD40xCD40 Bispecific Antigen-Binding Molecules

[0180] The present disclosure includes antibodies and antigen-binding fragments thereof that bind to human CD40 with high affinity, e.g., bispecific antigen-binding molecules that bind to two different epitopes of CD40. In some embodiments, the antibodies and antigen binding fragments thereof (e.g., bispecific antigen-binding molecules) bind to CD40 and inhibit CD40L-induced activation but do not have agonist activity and / or cytotoxic effector functions.

[0181] In some embodiments, the present disclosure includes bispecific antigen-binding molecules (e.g., bispecific antibodies as disclosed herein, such as a bispecific antibody having the amino acid sequences of REGN16334, REGN16335, REGN16431 , REGN16432, or REGN20484) that bind human CD40 (e.g., at 25°C or 37°C) with a KDof less than about 75 nM as measured by surface plasmon resonance, e.g., using an assay format as described in Example 2 herein. In certain embodiments, the antigen-binding molecules of the present disclosure bind human CD40 with a KDof less than about 75 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 25 nM, less than about 20 nM, less than about 15 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 90 pM, less than about 80 pM, less than about 70 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 10 pM, less than about 5 pM, less than about 4 pM, less than about 2 pM, less than about 1 pM, less than about 0.5 pM, less than about 0.2 pM, less than about 0.1 pM, or less than about 0.05 pM, as measured by surface plasmon resonance, e.g., using an assay format as defined in Example 2 herein, or a substantially similar assay.

[0182] In some embodiments, the present disclosure includes bispecific antigen-binding molecules (e.g., bispecific antibodies as disclosed herein, such as a bispecific antibody having the amino acid sequences of REGN16334, REGN16335, REGN16431 , REGN16432, or REGN20484) having an improved half-life as compared to monospecific antibodies (e.g., parental CD40 antibodies). In some embodiments, the present disclosure includes bispecific antigen-binding molecules (e.g., bispecific antibodies as disclosed herein) that bind human CD40 with a dissociative half-life (t / 2) of greater than about 70 minutes as measured by surface plasmon resonance at 25°C, e.g., using an assay format as defined in Example 2 herein, or a substantially similar assay. In certain embodiments, the antibodies or antigenbinding fragments of the present disclosure bind human CD40 with a t / 2of greater than about 75 minutes, greater than about 80 minutes, greater than about 85 minutes, or greater thanabout 90 minutes, as measured by surface plasmon resonance at 25°C, e.g., using an assay format as defined in Example 2 herein, or a substantially similar assay.

[0183] In some embodiments, the present disclosure includes bispecific antigen-binding molecules (e.g., bispecific antibodies as disclosed herein, such as a bispecific antibody having the amino acid sequences of REGN16334, REGN16335, REGN16431 , REGN16432, or REGN20484) that inhibit binding of human CD40 (e.g., a monomeric form of hCD40) to human CD40L. In some embodiments, inhibition of CD40 binding to CD40L is measured using an ELISA-based blocking assay as described in Example 4 herein. In some embodiments, a bispecific antigen-binding molecule inhibits binding of human CD40 (e.g., 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, e.g., using an assay format as defined in Example 4 herein, or a substantially similar assay.

[0184] In some embodiments, the present disclosure includes bispecific antigen-binding molecules (e.g., bispecific antibodies as disclosed herein, such as a bispecific antibody having the amino acid sequences of REGN16334, REGN16335, REGN16431 , REGN16432, or REGN20484) that specifically interact (e.g., bind with) cells that express CD40. The extent to which an antigen-binding molecule binds cells that express CD40 can be assessed by flow cytometry, as illustrated in Example 5 below. For example, in some embodiments, the present disclosure provides CD40xCD40 bispecific antibodies that specifically bind cells that express CD40 on the cell surface (e.g., primary human B cells, or a human B cell line such as Ramos 2G6.4C10). In some embodiments, the disclosure provides CD40xCD40 bispecific antibodies that bind CD40-expressing cells or cell lines with an EC50 value of about 10 nM or less, e.g., from about 0.5 nM to about 10 nM, e.g., an EC50 value of about 1 nM, about 1 .5 nM, about 2 nM, about 2.5 nM, about 3 nM, about 3.5 nM, about 4 nM, about 4.5 nM, about 5 nM, about 5.5 nM, about 6 nM, about 6.5 nM, about 7 nM, about 7.5 nM, about 8 nM, about 8.5 nM, about 9 nM, about 9.5 nM, or about 10 nM, as determined by flow cytometry as set forth in Example 5 or a substantially similar assay.

[0185] In some embodiments, the present disclosure includes bispecific antigen-binding molecules (e.g., bispecific antibodies as disclosed herein, such as a bispecific antibody having the amino acid sequences of REGN16334, REGN16335, REGN16431 , REGN16432, or REGN20484) that inhibit CD40L-induced activation. In some embodiments, CD40L- induced activation is measured using a reporter assay, such as a luciferase-based reporter assay that quantitatively assesses receptor activation in a CD40-expressing cell by measuring downstream gene expression. In some embodiments, the reporter assay is an 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 TNFa) in the presence of CD40L. In some embodiments, the cytokine secretion assay is performed inprimary cells that express CD40 (e.g., human B cells). In some embodiments, the cytokine secretion assay is performed in a stable cell line, e.g., a cell line that expresses CD40 and a reporter gene such as luciferase. In some embodiments, the cytokine secretion assay is an assay described in Example 7 herein. In some embodiments, the CD40xCD40 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, relative to a control or a reference value.

[0186] In some embodiments, the present disclosure includes bispecific antigen-binding molecules (e.g., bispecific antibodies as disclosed herein, such as a bispecific antibody having the amino acid sequences of REGN16334, REGN16335, REGN16431 , REGN16432, or REGN20484) that do not significantly agonize CD40 in the absence of CD40L. As used herein, "do[es] not significantly agonize CD40 in the absence of CD40L" means that in the presence of the bispecific antigen-binding molecule and absence of CD40L, the level of activation of CD40 is less than 15%, e.g., less than 13%, less than 10%, less than 8%, or less than 6%, e.g., as measured by downstream gene expression or cytokine secretion. In some embodiments, agonism of CD40 is measured by a reporter assay, such as a luciferase-based reporter assay that quantitatively assesses receptor activation in a CD40- expressing cell by measuring downstream gene expression. In some embodiments, agonism of CD40 is measured by a cytokine secretion assay (e.g., secretion of IL-6, IL-10, IL-23, or TNFa) in the absence of CD40L. In some embodiments, the assay is an assay described in Example 6 or Example 8 herein.Epitope Mapping and Related Technologies

[0187] In some embodiments, the epitope on CD40 to which the antigen-binding molecules of the present disclosure bind (e.g., a first epitope of human CD40 to which a first antigenbinding domain (D1 ) binds, or a second epitope of human CD40 to which a second antigenbinding domain (D2) binds) may consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of a CD40 protein. Alternatively, the epitope may consist of a plurality of non-contiguous 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 in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacentamino acid residues in a polypeptide chain. In certain circumstance, an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.

[0188] Various techniques known to persons of ordinary skill in the art can be used to determine whether an 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 an epitope or binding domain of a particular antibody or antigen-binding domain include, e.g., routine crossblocking assay such as that described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), point mutagenesis (e.g., alanine scanning mutagenesis, arginine scanning mutagenesis, etc.), peptide blots analysis (Reineke, 2004, Methods Mol 8 / 0 / 248:443-463), protease protection, and peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (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. In general terms, the hydrogen / deuterium exchange method involves deuterium-labeling the protein of interest, followed by binding the antibody to the deuterium-labeled protein. Next, the protein / antibody complex is transferred to water to allow hydrogen-deuterium exchange to occur at all residues except for the residues protected by the antibody (which remain deuterium-labeled). After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterium-labeled residues which correspond to the specific amino acids with which the antibody interacts.See, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 7325QA-2Q5A. X-ray crystal structure analysis can also be used to identify the amino acids within a polypeptide with which an antibody interacts.

[0189] In some embodiments, the present disclosure includes CD40 antibodies and CD40xCD40 bispecific antibodies that bind to the same epitope or epitopes as any of the specific exemplary antibodies described herein (e.g., antibodies comprising any of the amino acid sequences set forth in Table 36 below). In some embodiments, the present disclosure includes CD40 antibodies and CD40xCD40 bispecific antibodies that compete for binding with the exemplary antibodies described herein (e.g., antibodies comprising any of the amino acid sequences set forth in Table 36 below).

[0190] In some embodiments, the present disclosure provides CD40xCD40 bispecific antibodies comprising a first antigen-binding domain (D1 ) that binds a first epitope of human CD40 and a second antigen-binding domain (D1 ) that binds a second epitope of human CD40, wherein the first epitope and the second epitope are different epitopes of CD40. In some embodiments, the first epitope and the second epitope are non-overlapping epitopes.

[0191] In some embodiments, the present disclosure provides CD40xCD40 bispecific antibodies comprising a first antigen-binding domain (D1 ) that binds a first epitope of human CD40 and a second antigen-binding domain (D1) that binds a second epitope of human CD40, wherein D1 and D2 do not compete with one another for binding to human CD40.

[0192] One skilled in the art can determine whether or not a particular antigen-binding molecule (e.g., antibody) or antigen-binding domain thereof binds to the same epitope as, or competes for binding with, a reference antigen-binding molecule of the present disclosure by using routine methods known in the art. For example, to determine if a test antibody binds to the same epitope on CD40 as a reference bispecific antigen-binding molecule of the present disclosure, the reference bispecific molecule is first allowed to bind to a CD40 protein. Next, the ability of a test antibody to bind to the CD40 molecule is assessed. If the test antibody is able to bind to CD40 following saturation binding with the reference bispecific antigenbinding molecule, it can be concluded that the test antibody binds to a different epitope of CD40 than the reference bispecific antigen-binding molecule. On the other hand, if the test antibody is not able to bind to the CD40 molecule following saturation binding with the reference bispecific antigen-binding molecule, then the test antibody may bind to the same epitope of CD40 as the epitope bound by the reference bispecific antigen-binding molecule of the disclosure. Additional routine experimentation (e.g., peptide mutation and binding analyses) can then be carried out to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference bispecific antigenbinding molecule or if steric blocking (or another phenomenon) is responsible for the lack of observed binding. Experiments of this sort can be performed using ELISA, RIA, Biacore, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art. In accordance with certain embodiments of the present disclosure, two antigen-binding proteins bind to the same (or overlapping) epitope if, e.g., a 1-, 2-, 5-, 10-, 20- or 100-fold excess of one antigen-binding protein inhibits binding of the other by at least 50% but preferably 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502). Alternatively, two antigen-binding proteins are deemed to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antigen-binding protein reduce or eliminate binding of the other. Two antigen-binding proteins are deemed to have "overlapping epitopes" if only a subset of the amino acid mutations that reduce or eliminate binding of one antigen-binding protein reduce or eliminate binding of the other.

[0193] To determine if an antibody or antigen-binding domain thereof competes for binding with a reference antigen-binding molecule, the above-described binding methodology is performed in two orientations: In a first orientation, the reference antigen-binding molecule is allowed to bind to a CD40 protein under saturating conditions followed by assessment ofbinding of the test antibody to the CD40 molecule. In a second orientation, the test antibody is allowed to bind to a CD40 molecule under saturating conditions followed by assessment of binding of the reference antigen-binding molecule to the CD40 molecule. If, in both orientations, only the first (saturating) antigen-binding molecule is capable of binding to the CD40 molecule, then it is concluded that the test antibody and the reference antigen-binding molecule compete for binding to CD40. As will be appreciated by a person of ordinary skill in the art, an antibody that competes for binding with a reference antigen-binding molecule may not necessarily bind to the same epitope as the reference antibody, but may sterically block binding of the reference antibody by binding an overlapping or adjacent epitope.Preparation of Antigen-Binding Domains and Construction of Multispecific Antigen- Binding Molecules

[0194] Antigen-binding domains specific for particular antigens can be prepared by any antibody generating technology known in the art. Once obtained, two different antigenbinding domains can be appropriately arranged relative to one another to produce a bispecific antigen-binding molecule of the present disclosure using routine methods. (A discussion of exemplary bispecific antibody formats that can be used to construct the bispecific antigen-binding molecules of the present disclosure is provided elsewhere herein). In certain embodiments, one or more of the individual components (e.g., heavy and light chains) of the multispecific antigen-binding molecules are derived from chimeric, humanized or fully human antibodies. Methods for making such antibodies are well known in the art. For example, one or more of the heavy and / or light chains of the bispecific antigen-binding molecules of the present disclosure can be prepared using VELOCIMMUNE™ technology. Using VELOCIMMUNE™ technology (or any other human antibody generating technology), high affinity chimeric antibodies to a particular antigen (e.g., CD40) are initially isolated having a human variable region and a mouse constant region. The antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with a desired human constant region to generate fully human heavy and / or light chains that can be incorporated into the bispecific antigen-binding molecules.

[0195] In some embodiments, genetically engineered animals may be used to make human bispecific antigen binding molecules. For example, a genetically modified mouse can be used which is incapable of rearranging and expressing an endogenous mouse immunoglobulin light chain variable sequence, wherein the mouse expresses only one or two human light chain variable domains encoded by human immunoglobulin sequences operably linked to the mouse kappa constant gene at the endogenous mouse kappa locus. Such genetically modified mice can be used to produce fully human bispecific antigen-binding molecules comprising two different heavy chains that associate with an identical light chain that comprises a variable domain derived from one of two different human light chain variable region gene segments. (See, e.g., US 2011 / 0195454, the entire contents of which are incorporated herein by reference, for a detailed discussion of such engineered mice and the use thereof to produce bispecific antigen-binding molecules). As used herein, "fully human" refers to an antigen-binding molecule, e.g., an antibody, or antigen-binding fragment or immunoglobulin domain thereof, comprising an amino acid sequence encoded by a DNA derived from a human sequence over the entire length of each polypeptide of the antigenbinding molecule, antibody, antigen-binding fragment, or immunoglobulin domain thereof. In some instances, the fully human sequence is derived from a protein endogenous to a human. In other instances, the fully human protein or protein sequence comprises a chimeric sequence wherein each component sequence is derived from human sequence. While not being bound by any one theory, chimeric proteins or chimeric sequences are generally designed to minimize the creation of immunogenic epitopes in the junctions of component sequences, e.g., compared to any wild-type human immunoglobulin regions or domains.Bioequivalents

[0196] The present disclosure encompasses antigen-binding molecules having amino acid sequences that vary from those of the described antibodies but that retain the ability to bind CD40. Such variant molecules comprise one or more additions, deletions, or substitutions of amino acids when compared to the parent sequence, but exhibit biological activity that is essentially equivalent to that of the described antigen-binding molecules. Likewise, the nucleic acid sequences encoding the antigen-binding molecules of the present disclosure encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to the disclosed sequence, but that encode an antigen binding molecule that is essentially bioequivalent to the antigen-binding molecules disclosed herein.

[0197] The present disclosure includes antigen-binding molecules that are bioequivalent to any of the exemplary antigen-binding molecules set forth herein. Two antigen-binding proteins, e.g., bispecific antibodies, are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, either single does or multiple dose. Some antibodies will be considered equivalents or pharmaceutical alternatives if they are equivalent in the extent of their absorption but not in their rate of absorption and yet may be considered bioequivalent because such differences in the rate of absorption are intentional and are reflected in the labeling, are not essential to the attainment of effective body drug concentrations on, e.g., chronic use, and are considered medically insignificant for theparticular drug product studied.

[0198] In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.

[0199] In one embodiment, two antigen-binding proteins are bioequivalent if a patient can be switched one or more times between the first antigen-binding protein (e.g., reference product) and the second antigen-binding protein (e.g., biological product) without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity, or diminished effectiveness, as compared to continued therapy without such switching.

[0200] In one embodiment, two antigen-binding proteins are bioequivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known.

[0201] Bioequivalence may be demonstrated by in vivo and in vitro methods. Non-limiting examples of bioequivalence measures include, e.g., (a) an in vivo test in humans or other mammals, in which the concentration of the antibody or its metabolites is measured in blood, plasma, serum, or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably predictive of human in vivo bioavailability data; (c) an in vivo test in humans or other mammals in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) in a well-controlled clinical trial that establishes safety, efficacy, or bioavailability or bioequivalence of an antibody.

[0202] Bioequivalent variants of the exemplary bispecific antigen-binding molecules set forth herein may be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for biological activity. For example, cysteine residues not essential for biological activity can be deleted or replaced with other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation. In other embodiments, bioequivalent antibodies may include the exemplary bispecific antigen-binding molecules set forth herein comprising amino acid changes which modify the glycosylation characteristics of the antibodies, e.g., mutations which eliminate or remove glycosylation.Pharmaceutical Compositions

[0203] In another aspect, the present disclosure provides pharmaceutical compositions comprising the CD40xCD40 bispecific antigen-binding molecule (e.g., bispecific antibodies) disclosed herein. The pharmaceutical compositions are formulated with one or more pharmaceutically acceptable vehicle, carriers, and / or excipients. Various pharmaceutically acceptable carriers and excipients are well-known in the art. See, e.g., Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, PA. In some embodiments, the carrier is suitable for intravenous, intramuscular, oral, intraperitoneal, intrathecal, transdermal, topical, or subcutaneous administration.

[0204] In some embodiments, the pharmaceutical composition comprises an injectable preparation, such as a dosage form for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by known methods. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared can be filled in an appropriate ampoule.

[0205] The dose of antigen-binding molecule administered to a patient according to the present disclosure may vary depending upon the age and the size of the patient, symptoms, conditions, route of administration, and the like. The dose is typically calculated according to body weight or body surface area. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted. Effective dosages and schedules for administering pharmaceutical compositions as disclosed herein may be determined empirically; for example, patient progress can be monitored by periodic assessment, and the dose adjusted accordingly. Moreover, interspecies scaling of dosages can be performed using well-known methods in the art (e.g., Mordenti et al., 1991 , Pharmaceut. Res. 8:1351).

[0206] Various delivery systems are known and can be used to administer the pharmaceutical composition, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., 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 composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. In some embodiments, a pharmaceutical composition as disclosed herein is administered intravenously. In some embodiments, a pharmaceutical composition asdisclosed herein is administered subcutaneously.

[0207] In some embodiments, an antigen-binding molecule or a pharmaceutical composition comprising an antigen-binding molecule as disclosed herein is contained within a container. Thus, in another aspect, containers comprising an antigen-binding molecule or pharmaceutical composition as disclosed herein are provided. For example, in some embodiments, an antibody or pharmaceutical composition is contained within a container selected from the group consisting of a glass vial, a syringe, a pen delivery device, and an autoinjector.

[0208] In some embodiments, an antigen-binding molecule or pharmaceutical composition of the present disclosure is delivered, e.g., subcutaneously or intravenously, with a standard needle and syringe. In some embodiments, the syringe is a pre-filled syringe. In some embodiments, a pen delivery device or autoinjector is used to deliver a pharmaceutical composition of the present disclosure (e.g., for subcutaneous delivery). A pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0209] Examples of suitable pen and autoinjector delivery devices include, but are not limited to 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, IN), 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™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany). Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present invention include, but are not limited to the SOLOSTAR™ pen (sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, CA), the PENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), and the HUMIRA™ Pen (Abbott Labs, Abbott Park IL).

[0210] In some embodiments, the pharmaceutical composition is delivered using a controlled release system. In one embodiment, a pump may be used (see Langer, supra;Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201 ). In another embodiment, polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, a controlled release system can be placed in proximity of the composition’s target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0211] In some embodiments, pharmaceutical compositions for use as described herein are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. In some embodiments, the amount of the antigen-binding molecule contained in the dosage form is about 5 to about 1000 mg, e.g., from about 5 to about 500 mg, from about 5 to about 100 mg, or from about 10 to about 250 mg.Therapeutic Uses

[0212] In another aspect, the present disclosure provides for methods of using the antigenbinding molecules of the disclosure (e.g., CD40xCD40 bispecific antigen-binding molecules disclosed herein). In some embodiments, the present disclosure provides methods of inhibiting CD40L-induced activation of CD40, e.g., in or on a cell that expresses CD40 (e.g., a B cell, dendritic cell, monocyte, platelet, or macrophage). In some embodiments, the method comprises contacting a CD40-expressing cell with a CD40xCD40 bispecific antigenbinding molecule as disclosed herein.

[0213] In some embodiments, the present disclosure provides methods of treating, ameliorating, or preventing a CD40-mediated disease or condition by administering a therapeutically effective amount of a CD40 antigen-binding molecule, e.g., a CD40xCD40 bispecific antigen-binding molecule, to a subject in need thereof. A CD40-mediated disease or condition, as used herein, is any disease or condition that is caused or exacerbated by an activity of CD40 (e.g., activation of downstream signaling due to CD40 binding to its ligand CD40L). In some embodiments, a CD40-mediated disease or condition is due to a mutation in CD40 or CD40L, or in a gene in the CD40 signaling pathway.

[0214] In some embodiments, a CD40-mediated disease or condition is an autoimmune disease or condition, an inflammatory disease or condition, a cardiovascular disease or condition, or organ transplant. 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-Barre 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 or liver transplant), pemphigus, primary biliary cirrhosis, psoriasis, psoriatic arthritis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Sjogren's syndrome, skin graft, spondyloarthropathies, 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 granulomatosis.

[0215] In some embodiments, administration of a CD40 antigen-binding molecule, e.g., a CD40xCD40 bispecific antigen-binding molecule (e.g., as disclosed in any one of Tables 1- 4) prevents or delays the increase of disease symptoms or the progression of disease in a subject having a CD40-mediated disease or condition.Dosage and Administration Regimens

[0216] In some embodiments, an amount of CD40 antigen-binding molecule, e.g., a CD40xCD40 bispecific antigen-binding molecule (e.g., bispecific antibody), that is 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.

[0217] In some embodiments, the CD40 antigen-binding molecule (e.g., CD40xCD40 bispecific antibody) is administered to a subject as a weight-based dose. A "weight-based dose" (e.g., a dose in mg / kg) is a dose of the antigen-binding molecule that will change depending on the subject's weight.

[0218] In other embodiments, the CD40 antigen-binding molecule (e.g., CD40xCD40 bispecific antibody) is administered as a fixed dose. A "fixed dose" (e.g., a dose in mg) means that one dose of the antigen-binding molecule is used for all subjects regardless of any specific subject- related factors, such as weight. In one particular embodiment, a fixed dose of an antigen-binding molecule is based on a predetermined weight or age.

[0219] Typically, a suitable dose of the antigen-binding molecule can be in the range of about 0.001 to about 200.0 milligram per kilogram body weight of the recipient, generally in the range of about 1 to 50 mg per kilogram 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, about1 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. Values and ranges intermediate to the recited values are also intended to be part of this disclosure.

[0220] In some embodiments, the antigen-binding molecule is administered as a fixed dose of between about 5 mg to about 2500 mg. In some embodiments, the antigen-binding molecule is administered as a fixed dose of about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, about600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about750 mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg, about 875 mg, about900 mg, about 925 mg, about 950 mg, about 975 mg, about 1000 mg, about 1500 mg, about2000 mg, or about 2500 mg. Values and ranges intermediate to the recited values are also intended to be part of this disclosure.

[0221] In some embodiments, the CD40 antigen-binding molecule (e.g., CD40xCD40 bispecific antibody) is administered to a subject at a dosing frequency of about 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 frequently so long as a therapeutic response is achieved.

[0222] In some embodiments, multiple doses of a CD40 antigen-binding molecule (e.g., CD40xCD40 bispecific antibody) are administered to a subject over a defined time course. In some embodiments, the methods of the present disclosure comprise sequentially administering to a subject multiple doses of the antigen-binding molecule. As used herein, "sequentially administering" means that each dose of the antigen-binding molecule is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). In some embodiments, the methods of the disclosure comprise sequentially administering to the patient 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.

[0223] The terms "initial dose," "secondary dose(s)," and "tertiary dose(s)" refer to the temporal sequence of administration of the antigen-binding molecule. Thus, the "initial dose" is the dose which is administered at the beginning of the treatment regimen (also referred to as the "loading dose"); the "secondary doses" are the doses which are administered after the initial dose; and the "tertiary doses" are the doses which are administered after thesecondary doses. In some embodiments, the initial, secondary, and tertiary doses may all contain the same amount of the antigen-binding molecule, but may differ from one another in terms of frequency of administration. In some embodiments, the amount of the antigenbinding molecule contained in the initial, secondary and / or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In certain embodiments, one or more (e.g., 1 , 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as "loading doses" followed by subsequent doses that are administered on a less frequent basis (e.g., "maintenance doses"). In some embodiments, the initial dose and the one or more secondary doses each contain the same amount of the antigen-binding molecule. In other embodiments, the initial dose comprises a first amount of the antigen-binding molecule, and the one or more secondary doses each comprise a second amount of the antigen-binding molecule. For example, the first amount of the antigen-binding molecule can be 1 ,5x, 2x, 2.5x, 3x, 3.5x, 4x, 5x or more than the second amount of the antigen-binding molecule.

[0224] In some embodiments, each secondary and / or tertiary dose is administered 1 to 14 (e.g., 1 , 11 / 2, 2, 21 / 2, 3, 31 / 2, 4, 41 / 2, 5, 51 / 2, 6, 61 / 2, 7, 71 / 2, 8, 81 / 2, 9, 91 / 2, 10, 101 / 2, 11 , 1 11 / 2, 12, 121 / 2, 13, 131 / 2, 14, 141 / 2, or more) weeks after the immediately preceding dose. The phrase "the immediately preceding dose," as used herein, means, in a sequence of multiple administrations, the dose of the antigen-binding molecule that is administered to a patient prior to the administration of the very next dose in the sequence with no intervening doses.

[0225] The methods of the disclosure may comprise administering to a patient any number of secondary and / or tertiary doses of an antigen-binding molecule. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Likewise, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.

[0226] 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 1 , 2, 3, or 4 weeks after the immediately preceding dose. Similarly, in some embodiments involving multiple tertiary doses, each tertiary dose is administered at the same frequency as the other tertiary doses. Alternatively, the frequency at which the secondary and / or tertiary doses are administered to a patient can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination.Combination Therapies

[0227] In yet another aspect, the present disclosure includes compositions and therapeutic formulations comprising any of the exemplary antibodies and bispecific antigen-binding molecules described herein in combination with one or more additional therapeutic agents, and methods of treatment comprising administering such combinations to subjects in need thereof. In some embodiments, the additional therapeutic agent(s) is an immunomodulatory agent or anti-inflammatory agent. In some embodiments, the additional therapeutic agent(s) is immunosuppressive therapy. In some embodiments, the additional therapeutic agent(s) is a surgical procedure.

[0228] Exemplary additional therapeutic agents that may be combined with or administered in combination with an antigen-binding molecule of the present disclosure include, e.g., another CD40-CD40L inhibitor (e.g., an CD40 antibody such as iscalimab [CFZ-533], belselumab [ASKP1240], Bl 655064, cd5D12, or FFP104; or an CD40L antibody or antigen-binding protein such as dapirolizumab pegol, dazodalibep [VIB4920], frexalimab [INX-021], letolizumab [BMS-986004], MR-1 , ruplizumab [BG9588], tegoprubart [AT-1501], or toralizumab [IDEC-131 ]; a B cell depleting agent (e.g., a direct agent such as a CD19 antibody, a CD20 antibody, a CD22 antibody) or an indirect agent such as a BLyS inhibitor or an APRIL inhibitor); a plasma cell depleting agent (e.g., a B-cell maturation antigen (BCMA) targeting agent, an BCMAxCD3 bispecific antibody, a CD38 antibody (e.g., daratumumab), a proteasome inhibitor, a histone deacetylase inhibitor, a B-cell activating factor (BAFF) inhibitor, an APRIL inhibitor); a steroid (e.g., corticosteroids such as topical, systemic, oral, or inhaled corticosteroids, including but not limited to betamethasone, clobetasol, dexamethasone, fluocinolone, fluocinonide, halobetasol, hydrocortisone, methylprednisolone, prednisone, prednisolone, or triamcinolone); a non-steroidal topical medication such as but not limited to a PDE4 inhibitor or a calcineurin inhibitor; a nonsteroidal anti-inflammatory drug (NSAID) such as but not limited to celecoxib, diclofenac, etodolac, fenprofen, flurbiprofen, ibuprofen, ketoprofen, meclofamate, meloxicam, nabumetone, naproxen, oxaprozin, piroxicam, rofecoxib, salicylates, sulfasalazinem, sulindac, or tolmetin; an anti-inflammatory antibody or biologic (e.g., a TNFa antibody or biologic such as but not limited to adalimumab, certolizumab, etanercept, golimumab, or infliximab; an IL-1 antibody or biologic such as but not limited to LY2189102, anakinra, canakinumab, gerokizumab, or rilonacept; an IL-6 / IL-6R antibody or biologic such as but not limited to sarilumab, siltuximab, or tocilizumab; an IL-17A / IL-17R antibody or biologic such as but not limited to bimekizumab, brodalumab, ixekizumab, or secukinumab; or an IL-12 / IL- 23 antibody or biologic such as but not limited to AMG139, BI655066, brazikumab, briankizumab, guselkumab, mirikizumab, risankizumab, tildrakizumab, or ustekinumab); aJAK inhibitor such as but not limited to abrocitinib, baricitinib, fedratinib, filgotinib, ruxolitinib, tofacitinib, or upadacitinib; an immunosuppressive agent (e.g., a systemic immunosuppressant such as but not limited to methotrexate, cyclophosphamide, mizoribine, chlorambucil, cyclosporine, mycophenolate mofetil, or azathioprine); a disease-modifying antirheumatic drug (DMARD) such as but not limited to apremilast, azathioprine, baricitinib, cyclophosphamide, cyclosporine, hydroxychloroquine, leflunomide, methotrexate, mycophenolate mofetil, sulfasalazine, or tofacitinib; an exogenously administered therapeutic protein such as an enzyme (e.g., enzyme replacement therapy); radiation therapy; chemotherapy; intravenous immunoglobulin therapy; or a surgery or a surgical procedure (such as but not limited to splenectomy, lymphadenectomy, thyroidectomy, plasmaphoresis, leukophoresis, or cell, tissue, or organ transplantation).

[0229] In some embodiments, a CD40 antigen-binding molecule as disclosed herein (e.g., a CD40xCD40 bispecific antigen-binding molecule, e.g., as disclosed in any one of Tables 1 - 4) may be combined with or administered in combination with a B cell depleting agent and / or a plasma cell depleting agent. In some embodiments, the depleting agent is an agent that directly targets a B cell, e.g., an agent that binds to a B cell surface molecule. In some embodiments, the B cell depleting agent is a CD19 antibody (e.g., MEDI-551 ), a CD20 antibody (e.g., rituximab, ocrelizumab, or ofatumumab), or a CD22 antibody (e.g., epratzumab). In some embodiments, the depleting agent is an agent that indirectly targets a B cell and / or plasma cell, e.g., by targeting a B cell or plasma cell survival factor. In some embodiments, the B cell depleting 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., anti-BR3). In some embodiments, the depleting agent is a BCMA targeting agent, such as a BCMAxCD3 bispecific antibody, a chimeric antigen receptor against BCMA, or a BCMA antibody conjugated to a cytotoxic drug (e.g., linvoseltemab, REGN5459, pacanalotamab, or teclistamab).

[0230] The additional therapeutically active component(s) may be administered just prior to, concurrent with, or shortly after the administration of an antigen-binding molecule of the present disclosure. For the purposes of the present disclosure, such administration regimens are considered the administration of an antigen-binding molecule "in combination with" an additional therapeutically active component.

[0231] The present disclosure includes pharmaceutical compositions in which an antigenbinding molecule of the present invention is co-formulated with one or more of the additional therapeutically active component(s) as described elsewhere herein.EXAMPLES

[0232] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the 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 numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.Example 1 : Construction of CD40xCD40 Bispecific AntibodiesGeneration of Parental CD40 Antibodies

[0233] Antibodies against CD40 were obtained by immunizing a VELOCIMMUNE® mouse (i.e., an engineered mouse comprising DNA encoding human Immunoglobulin heavy and kappa chain variable regions) with a human CD40 antigen (human CD40 extracellular domain with C-terminal MMH tag; SEQ ID NO:53).

[0234] Following immunization, antibodies were isolated directly from antigen-positive mouse B cells, e.g., as described in U.S. Pat. No. 7,582,298, incorporated by reference herein. Using this method, fully human CD40 antibodies (i.e., antibodies possessing human variable domains and human constant domains) were obtained. Antibodies generated using this method were characterized and selected for desirable characteristics, including affinity, selectivity, etc.

[0235] CD40 antibodies generated using this method include antibodies designated 30027P2, 21519P2, and 21520P2. Certain biological properties of the exemplary CD40 antibodies generated in accordance with the methods of this Example are described in detail in the Examples set forth below.Table 1 : Amino Acid Sequence Identifiers for Parental CD40 Monoclonal AntibodiesTable 2: Nucleic Acid Sequence Identifiers for Parental CD40 Monoclonal AntibodiesGeneration of CD40xCD40 Bispecific Antibodies

[0236] 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 methodologies, wherein the two CD40 binding arms comprise distinct HCVRs paired with a common light chain. Two different heavy chain constant regions, e.g., as described in US Patent No. 11 ,518,807, were used for each CD40 binding arm. Exemplary CD40xCD40 bispecific antibodies were generated in accordance with the present Example and comprise the sequences shown below in Tables 3-4.

[0237] As shown in Table 3, for REGN16334 and REGN16431 , the first CD40-binding arm ("D1") comprises the HCVR sequences of parental antibody 21519P2 and the second CD40- binding arm ("D2") comprises the HCVR sequences of parental antibody 21520P2;REGN16334 and REGN16431 have different constant region modifications for reducing Fc receptor binding and effector function.

[0238] For REGN16335 and REGN16432, the first CD40-binding arm ("D1 ") comprises the HCVR sequences of parental antibody 30027P2 and the second CD40-binding arm ("D2") comprises the HCVR sequences of parental antibody 21520P2; REGN16335 and REGN16432 have different constant region modifications for reducing Fc receptor binding and effector function.

[0239] For REGN20484, the first CD40-binding arm ("D1 ") comprises the HCVR sequences of parental antibody 21520P2 and the second CD40-binding arm ("D2") comprises the HCVR sequences of parental antibody 30027P2.

[0240] REGN16334, REGN16335, REGN16431 , REGN16432, and REGN20484 all comprise a common light chain sequence.118003-50020Table 3: Amino Acid Sequence Identifiers for CD40xCD40 Bispecific AntibodiesMEI 48749815v.l -59-118003-50020Table 4: Nucleic Acid Sequence Identifiers for CD40xCD40 Bispecific AntibodiesMEI 48749815v.l -60-Example 2: Biacore Binding Kinetics of CD40 Bivalent Parental and CD40xCD40 Bispecific Antibodies

[0241] The equilibrium dissociation constants (KD) for CD40 bivalent and bispecific monoclonal antibodies (mAbs) were determined using a real-time surface plasmon resonance (SPR)-based Biacore 4000 biosensor. All binding studies were performed in 10 mM HEPES, 150 mM NaCI, 3 mM EDTA, and 0.05% v / v surfactant Tween-20, pH 7.4 (HBS- ET) running buffer at 25°C and 37°C. The Biacore CM5 sensor surface was first derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (REGN2567) to capture CD40 bivalent parental and CD40xCD40 bispecific antibodies. Different concentrations of CD40 reagents, 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), 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 running buffer were injected at a flow rate of 30 pL / min for 4 minutes or 50 pL / min for 5 minutes. The dissociation of different CD40 reagents bound to CD40 bivalent parental and CD40xCD40 bispecific antibodies was monitored for 10 minutes in HBS-ET running buffer. At the end of each cycle, the CD40 bivalent parental and CD40xCD40 bispecific antibodies capture surface was regenerated using a 12 sec injection of 20mM H3PO4.

[0242] The association rate (ka) and dissociation rate (kd) were determined by fitting the real-time binding sensorgrams to a 1 :1 binding model with mass transport limitation using Scrubber 2.0c curve-fitting software. Binding dissociation equilibrium constant (KD) and dissociative half-life (t1 / a) were calculated from the kinetic rates as: KD (M) = kd / ka, and t / 2(min) = [ln(2) / (60*kd)].

[0243] Binding kinetics parameters for different CD40 reagents to CD40 bivalent parental and CD40xCD40 bispecific antibodies at 25eC and 37°C are shown in Tables 5-10.

[0244] As shown in Table 5, at 25°C, CD40 bivalent parental and CD40xCD40 bispecific antibodies bound to hCD40-MMH with KD values ranging from 101 pM to 25.1 nM. At 25°C, CD40 bivalent parental antibodies bound to mfCD40-MMH with KD values ranging from 901 pM to 205 nM (Table 6). CD40 bivalent parental antibodies did not bind to mCD40-MMH at 25°C (Table 7).

[0245] As shown in Table 8, at 37°C, CD40 bivalent parental and CD40xCD40 bispecific antibodies bound to hCD40-MMH with KD values ranging from 101 pM to 69.9 nM. At 37°C,CD40 bivalent parental antibodies bound to mfCD40-MMH with KD value 3.16 nM (Table 9).CD40 bivalent parental antibodies did not bind to mCD40-MMH at 37°C (Table 10).Table 5: Kinetic binding parameters for the interaction of hCD40-MMH with CD40 bivalent parental and CD40xCD40 bispecific antibodies at 25°CTable 6: Kinetic binding parameters for the interaction of mfCD40-MMH with CD40 bivalent parental and CD40xCD40 bispecific antibodies at 25°CNT = not testedTable 7: Kinetic binding parameters for the interaction of mCD40-MMH with CD40 bivalent parental and CD40xCD40 bispecific antibodies at 25°CNB = no binding was observed under the experimental conditions; NT = not testedTable 8: Kinetic binding parameters for the interaction of hCD40-MMH with CD40 bivalent parental and CD40xCD40 bispecific antibodies at 37°CTable 9: Kinetic binding parameters for the interaction of mfCD40-MMH with CD40 bivalent parental and CD40xCD40 bispecific antibodies at 37°CIC = inconclusive; NT = not testedTable 10: Kinetic binding parameters for the interaction of mCD40-MMH with CD40 bivalent parental and CD40xCD40 bispecific antibodies at 37°CNB = no binding was observed under the experimental conditions; NT = not testedExample 3: Cross-Competition Between Different CD40 Monoclonal Antibodies

[0246] Binding competition between different CD40 monoclonal antibodies (mAbs) was determined using a real time, label-free bio-layer interferometry (BLI) assay on the Octet HTX biosensor platform (Pall ForteBio Corp.). In addition to parental antibodies 21519P2, 21520P2, and 30027P2, a comparator CD40 antibody (REGN11209) was also tested; this comparator has the heavy chain and light chain sequences of iscalimab (see, US 8,828,396). The entire experiment was performed at 25°C in 10 mM HEPES buffer containing 150 mM NaCI, 3 mM EDTA, 1 mg / mL BSA, 0.02% NaNs, and 0.05% v / v Surfactant Tween-20 at pH 7.4 (HBS-EP) with the plate shaking at a speed of OOrpm.

[0247] To assess the ability of one antibody to compete with another antibody for binding to CD40, around 0.47 nm - 0.54 nm of recombinant human CD40 extracellular domain expressed with a C-terminal myc-myc-hexahistidine (hCD40-MMH; SEQ ID NO:53) was first captured onto anti-Penta-His antibody coated Octet biosensor tips (Fortebio Inc, # 18-5122) by submerging the biosensor tips in wells containing 10 pg / mL solution of the hCD40-MMH for 90 seconds. The antigen captured biosensor tips were then saturated with a first CD40 monoclonal antibody (subsequently referred to as "mAb-1") by dipping into wells containing 50 pg / mL solution of mAb-1 for 4 minutes. The biosensor tips were then subsequently dipped into wells containing 50 pg / mL solution of a second CD40 monoclonal antibody (subsequently referred to as "mAb-2") for 3 minutes. The biosensor tips were washed in HBS-EBT buffer in between every step of the experiment. The real-time binding response was monitored and the binding response at the end of every step was recorded. The response of mAb-2 binding to hCD40-MMH pre-complexed with mAb-1 was compared to the binding response hCD40-MMH alone (sample of isotype control), and if pre-bound mAb-1reduced binding of mAb-2 by more than 50%, mAb-1 was considered to be a competitor to mAb-2.

[0248] Competitors of each antibody tested are summarized in Table 11 below. Parental antibodies 21519P2 and 21520P2 were found to compete with each other, but not with 30027P2, for binding to hCD40-MMH.Table 11 : Cross-competition between different CD40 monoclonal antibodies for binding to hCD40-MMHExample 4: ELISA Assay to Assess Blocking Activity of CD40xCD40 Bispecific Antibodies

[0249] An ELISA-based blocking assay was developed to determine the ability of C40xCD40 bispecific antibodies to block the binding of the hCD40 monomer to plate-coated hCD40L. The recombinant human CD40-mmH protein (hCD40-MMH; REGN3094; SEQ ID NO:53) used in the experiments comprises a portion of the human CD40 extracellular domain (amino acids P20-R193) fused to 2xMyc peptide and 6xhistadine at the C-terminus of human CD40, and the human CD40L (accession # NM 000074.2) with amino acids E108- L261 of the extracellular domain with 9x His-2x(SGGG)-IGER at the N-terminus (9His- hCD40L) was commercially obtained from Biolegend.

[0250] In the blocking assay, 9His-hCD40L was passively absorbed at a concentration of 5 pg / mL in PBS on a 96-well microtiter plate overnight at 4°C. Nonspecific binding sites were subsequently blocked using a 0.5% (w / v) solution of BSA in PBS. In a separate 96-well microtiter plate, a fixed amount of 40 nM hCD40-mmH was pre-mixed with one of the following antibodies, at concentrations ranging from 977 pM to 1 pM in PBS+0.5% BSA: (1 ) CD40xCD40 bispecific antibodies (REGN16431 , REGN16432, REGN16634, andREGN16335); (2) parental CD40 mAbs (bivalent antibodies comprising 2 fragment antigenbinding [Fab] arms identical to one of anti-CD40 Fab of CD40xCD40 antibodies); (3) CD40xlrrelevant antibodies (bivalent antibodies that incorporate one Fab arm identical to one of anti-CD40 Fab of CD40xCD40 antibodies and another Fab arm specific to an irrelevant antigen); and (4) human lgG4 with Fc mutation isotype control antibodies (REGN7540 and REGN4513). The fixed concentration of hCD40-mmH was selected to be near the concentration (EC50 value) that generated 50% of the maximal binding to the plate- adhered 9His-hCD40L. After one-hour incubation, the antibody-antigen complexes were transferred to the microtiter plate coated with 9His-hCD40L. After one hour incubation at room temperature, the plates were washed, and plate-bound hCD40-mmH protein was detected with horseradish peroxidase (HRP) conjugated goat anti-c-Myc antibody. The plates were then developed using TMB substrate solution (BD Biosciences) according to the manufacturer’s recommended procedure and the absorbance at 450nm (OD450) was measured on a SpectraMax i3x plate reader.

[0251] Binding data were analyzed using a sigmoidal (four-parameter logistic) doseresponse model with GraphPad Prism™ software. The IC50 value, defined as the concentration of antibody required to block 50% of 40 nM hCD40-mmH binding to plate- coated 9His-hCD40L, was determined to indicate blocking potency. The percent blocking of tested antibodies at the highest tested concentration (1 pM) was calculated based on the formula shown below:[Experimental Signal ooonM Ab) - Background Signal (buffer)]% Blocking = 100 — ( [Maximum Signal(inM hCD4o-mFc aione) - Background Signal (buffer)] )x 1 0° where the maximum signal was the interpolated binding signal for 40 nM hCD40-mmH from the hCD40-mmH concentration-response curve. Antibodies that blocked binding of hCD40 greater than 50% were classified as blockers. Antibodies that blocked binding equal or less than 50% were classified as non-blockers. IC50 values for nonblockers were not determined. Results

[0252] The ability of CD40xCD40 bispecific antibodies to block human CD40 monomer binding to plate-coated human CD40L was assessed using a sandwich ELISA-based blocking assay. The results are shown in Table 12. As shown in Table 12, each of the CD40xCD40 bispecific antibodies REGN16431 , REGN16432, REGN16334, and REGN16335 displayed concentration-dependent blocking of hCD40 binding to hCD40L with 93% to 98% blocking at the highest antibody concentration tested (1 pM). The IC50 values for these bispecific antibodies are similar around 32 nM. Four parental anti-CD40 antibodies (H4sH21519P2, H4sH21520P2, REGN17288, REGN17544) displayed maximum blocking ranging from 91% to 94% and IC50 values from 52 nM to 71 nM. Four CD40xlrrelevantantibodies (REGN17551 , REGN17552, REGN17548, RENG17549) also inhibited hCD40 binding to hCD40L with blocking ranging from 83% to 87%. Two parental CD40 antibodies (H4sH30027P2 and REGN17289) and two CD40xlrrelevant antibodies (REGN17553 and REGN17550) showed minimum blocking activity around 10% and were classified as nonblockers. In this experiment the two human lgG4 with Fc mutation isotype control antibodies (REGN7540, REGN4513) showed no blocking activity, as expected.Table 12: Summary of CD40 antibodies blocking human CD40 monomer binding to human CD40LNBL: Non-blocking (% blocking is 50% or less)ND: Not determined (no sigmoidal curve fit observed to calculate IC50 values)Example 5: CD40xCD40 Bispecific Antibody Binding to Cell-Surface CD40 as Measured by Flow Cytometry

[0253] Flow cytometry was used to assess the ability of CD40xCD40 bispecific antibodies to bind human CD40 (hCD40) or Macaca fascicularis CD40 (mfCD40) expressing cells.Human embryonic kidney 293 (HEK293) cells stably expressing luciferase reporter gene under the control of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF- kB) and enhanced green fluorescent protein (HEK293 / D9) were engineered to expresshCD40 (accession #P25942-1 ) or mfCD40 (accession #XP_005569274.1) by transfecting the cells with neomycin resistant pRG984 plasmid encoding full-length hCD40 (amino acids M1-Q277, HEK293 / D9 / hCD40), or neomycin resistant pRG984 plasmid encoding full-length mfCD40 (amino acids M1 -Q282, HEK293 / D9 / mfCD40). Ramos 2G6.4C10 cells were utilized to evaluate the binding of anti-CD40 antibodies to hCD40 endogenously expressed on the cell surface. CD40 negative HEK293 / D9 cells that showed no detectable expression of CD40 by flow cytometry with a commercial CD40 antibody were included as a background binding control.

[0254] Experiments were carried out according to the following procedure: HEK293 / D9 / hCD40, HEK293 / D9 / mfCD40 or HEK293 / D9 cells were rinsed once in 1xPBS buffer without Ca2+ / Mg2+and incubated for 10 minutes at 37°C with Enzyme Free Cell Dissociation Solution to detach cells from flask. The dissociated cells or Ramos 2G6.4G10 suspension cells were washed with 1xPBS and counted with Cellometer™ Auto T4 cell counter (Nexcelom Bioscience, Lawrence, MA). Cells were then resuspended to 1 x107per mL in 1xPBS and separately stained with 2.5 pM of CellTrace™ reagents (Invitrogen, Carlsbad, CA) for 20 minutes at room temperature (RT) to generate a unique fluorescence signature for each cell line (CellTrace™ CFSE for Ramos 2G6.4C10 cells, CellTrace™ Violet for HEK293 / D9 / mfCD40 cells, CellTrace™ Yellow for HEK293 / D9 cells, and HEK293 / D9 / hCD40 cells were unstained). CellTrace™ labeling reaction was stopped by adding FBS to a final concentration of 25% in 1xPBS followed by a 5-minute incubation at room temperature to quench unbound dye in solution. Cells were washed with 1xPBS and equal numbers of each of the four cell lines, stained and unstained, were mixed in a ratio of 1 :1 :1 :1 for multiplexing. Approximately 2x105cells per well were seeded onto 96-well Corning plates and stained with LIVE / DEAD™ Fixable Near-IR (Thermo Fisher Scientific, Waltham, MA) in 1xPBS for 20 minutes at 4°C following a manufacturer’s recommended procedure to discriminate live and dead cells. Cells were washed with 2% FBS (w / v) in 1xPBS (flow cytometry staining buffer) by centrifugation with benchtop Centrifuge 581 OR (Eppendorf®, Hamburg, Germany). Cells were incubated for 30 minutes at 4°C with serial dilutions of following antibodies ranging from 1.7 pM to 100 nM in flow cytometry staining buffer: (1) CD40xCD40 bispecific antibodies, or (2) parental CD40 antibodies (bivalent antibodies comprising 2 fragment antigen-binding [Fab] arms identical to one of the two anti- CD40 Fab of bispecific CD40xCD40 antibodies), or (3) CD40xlrrelevant antibodies (bivalent antibodies that incorporate one Fab arm identical to one of anti-CD40 Fab of CD40xCD40 antibodies and another Fab arm specific to an irrelevant antigen, i.c. birch pollen main allergen Bet v1 ), or (4) human lgG4 isotype control antibodies with Fc mutation. After washing, cell bound antibodies were detected with 2.5 pg / ml Allophycocyanin (APC)- conjugated goat anti-human IgG antibody specific for the Fey fragment (JacksonImmunoresearch, West Grove, PA) for 30 minutes at 4°C. Cells were washed and subsequently fixed with 50% solution of Cytofix™ Fixation Buffer (BD, Franklin Lakes, NJ) in flow cytometry staining buffer for 20 minutes at room temperature. Cells were washed and resuspended in flow cytometry staining buffer and stored at 4°C for downstream flow cytometry analysis.

[0255] Acquisition of fluorescence signals were recorded on the ZE5 Cell Analyzer (BioRad, Hercules, CA) according to the manufacturer’s recommended procedure and 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 signature. APC Median Fluorescence intensity (MFI) was recorded to indicate the binding intensity of each antibody over a range of concentrations. Antibodies with MFI greater than 1500 at the highest tested concentration (100 nM) were classified as specific binders. In addition, direct binding signals (APC MFI) were analyzed as a function of the antibody concentration and data were fitted with a sigmoidal (four-parameter logistic) dose-response model using GraphPad Prism™ software. The EC50 value, defined as the concentration of antibody that yields 50% of the maximal binding, was determined and used as an indicator of antibody binding potency. EC50 values were reported only for specific binders.Results

[0256] The ability of CD40xCD40 bispecific antibodies to bind specifically cells expressing human or monkey CD40 was assessed by flow cytometry. The experimental results are summarized in Table 13 below.

[0257] The four CD40xCD40 bispecific antibodies (REGN16431 , REGN16432, REGN16334, and REGN16335) displayed concentration-dependent specific binding to hCD40 expressed on HEK293 / D9 / hCD40 or Ramos 2G6.4C10 cells with MFI values ranging from 28,932 to 30,315 at the highest concentration tested (100 nM) and EC50 values of 1 .5 nM to 2.0 nM on HEK293 / D9 / hCD40 cells, or with MFI values from 3,748 to 3,852 and EC50 values of 1 .0 nM to 2.0 nM on Ramos 2G6.4C10 cells. The four bispecific antibodies similarly displayed specific binding to mfCD40 expressed on HEK293 / D9 / mfCD40 cells with MFI values ranging from 41 ,714 to 48,653 and EC50 values of 2.1 nM to 9.1 nM.

[0258] Six parental CD40 antibodies (H4sH21519P2, H4sH21520P2, H4sH30027P2, REGN17288, REGN17544, and REGN17289) displayed specific binding to HEK293 / D9 / hCD40 or Ramos 2G6.4C10 cells with MFI values ranging from 20,213 to 20,911 and EC50 values of 0.72 nM to 1 .3 nM on HEK293 / D9 / hCD40 cells, or with MFI values ranging from 2,408 to 2,664 and EC50 values of 0.45 nM to 1 .5 nM on Ramos 2G6.4C10 cells. All parental CD40 antibodies showed specific binding toHEK293 / D9 / mfCD40 cells with MFI values ranging from 1 1 ,539 to 33,930 and EC50 values of 1.0 nM to 10 nM.

[0259] Six CD40xlrrelevant antibodies (REGN17551 , REGN17552, REGN17553, REGN17548, REGN17549, and REGN17550) displayed specific binding to HEK293 / D9 / hCD40 or Ramos 2G6.4C10 cells with MFI values ranging from 21 ,394 to 24,591 and EC50 values of 1 .1 nM to 8.2 nM on HEK293 / D9 / hCD40 cells, or with MFI values ranging from 1 ,833 to 3,140 on Ramos 2G6.4C10 cells. Four CD40xlrrelevant antibodies (REGN17552, REGN17553, REGN17549, and REGN17550) displayed specific binding to HEK293 / D9 / mfCD40 cells with MFI values ranging from 4,719 to 39,448, but two CD40xlrrelevant antibodies (REGN17551 and REGN17548) did not specifically bind to mfCD40 on HEK293 / D9 / mfCD40 cells.

[0260] All tested CD40 antibodies did not bind to the negative control HEK293 / D9 cells: binding signals were below 950 MFI. As expected, no detectable cell-surface binding was observed for two hlgG4 with Fc mutation isotype control antibodies (REGN7540 and REGN4513) up to the highest antibody concentration tested (100 nM).118003-50020Table 13: Summary of CD40 antibody binding to human or monkey CD40 expressed on the cell surfaceMFI, median fluorescence intensity; INC, inconclusive; ND, not determined. INC is applied if concentration-dependent binding was detected, but no top plateau was reached in the concentraton range tested, and an EC50 therefore could not be calculated. If MFI values were less than or equal to 1500 and specifidc binding was not detected, EC50 was not calculated and ND was applied.MEI 48749815v.l -71-Example 6: Bioassay to Assess Regulation of CD40 Signaling by CD40xCD40 Bispecific Antibodies

[0261] CD40 is a member of the tumor necrosis factor receptor superfamily (TNFRSF) that activates the immune system in response to the binding of its ligand, CD40L. To evaluate regulation of CD40 signaling, a bioassay was developed to quantitatively assess receptor activation by measuring gene expression downstream of the nuclear translocation of NFKB (nuclear factor KB). The luciferase-based reporter assay was engineered in three different human cell lines (Ramos.2G6.4C10, Raji, and THP-1) that endogenously express CD40. Cells were transduced with NFKB-luciferase reporter lentivirus (QIAGEN CLS-013L-8) and stable reporter cell lines were selected and maintained in media containing 1 pg / ml of puromycin.

[0262] For the bioassay, cells were seeded at 20,000 cells / well into 96-well plates in assay media (RPMI-1640 with 10%FBS, pen / strep / glut). Antibodies were then serially diluted in assay media at 1 :3 to final concentrations ranging from 100 nM to 1 .7 pM (with an additional condition without test molecule) and added to the cells along with or without a constant concentration of human CD40L (500 pM, 900 pM, or 10 nM for Ramos, Raji, or THPI reporter cells, respectively). To obtain a range of activation, hCD40L was serially diluted 1 :3 to final concentrations ranging from 100 nM to 1.7 pM (with an additional condition without ligand) and added to cells. After 5 hours of incubation at 37°C / 5% CO2, luciferase activity was detected on an Envision multilabel plate reader (PerkinElmer) after the addition of ONE- Glo™ (Promega) reagent. All conditions were tested in duplicate.

[0263] The EC50 or IC50 values were determined with GraphPad Prism™ software using nonlinear regression (4-parameter logistics). The percentage of inhibition was calculated based on the relative luminescence unit (RLU) values using the equation:% Activation. . .. . .% Inhibition

[0264] "RLUAntibody Maximum" and "RLUAntibodyminimum" are the maximum and minimum luminescence value achieved with antibody. "RLUugand Maximum and RLU|_igand constant " are the values achieved by maximum and constant concentration of CD40L. "RLUsackground" is the value without any CD40L. For antibodies that showed potentiation in the presence of CD40L, "RLUAntibody Maximum" was used to calculate % inhibition leading to negative inhibition values.Results

[0265] As shown in Table 14, CD40xCD40 bispecific antibodies REGN16431 , REGN16432, REGN16334 and REGN16335 showed minimal activation ranging from 4 to 6% without CD40L and inhibition ranging from 90 to 94% with IC50S of 38.6 - 85.4 pM with 500 pM CD40L in Ramos.2G6.4C1 0 / NFKB-IUC cells. CD40 bivalent antibodies and CD40 x Irrel. (Irrelevant, Non-CD40 target) antibodies showed activation ranging from 4 to 49% without CD40L and inhibition ranging from 28 to 95% with 500 pM CD40L. Four antibodies showed potentiation of signaling, inhibition ranging from -1 10 to -142% in the presence of CD40L in Ramos bioassay.

[0266] As shown in Table 15, CD40xCD40 bispecific antibodies REGN16431 , REGN16432, REGN16334 and REGN16335 showed minimal activation ranging from 5 to 13% without CD40L and inhibition ranging from 68 to 1 13% with IC50S of 49.4 - 107 pM with 900 pM CD40L in Raji / NFKB-luc cells. CD40 bivalent antibodies and CD40 x Irrel, antibodies showed activation ranging from 7 to 25% without CD40L and inhibition ranging from 33 to 92% with 900 pM CD40L in Raji bioassay.

[0267] As shown in Table 16, CD40xCD40 bispecific antibodies REGN16431 , REGN16432, REGN16334 and REGN16335 showed minimal activation ranging from 1 to 6% without CD40L and inhibition ranging from 97 to 98% with IC50S of 148 - 553 pM with 10 nM CD40L in THP-1 / NFKB-IUC cells. CD40 bivalent antibodies and CD40 x Irrel, antibodies showed activation ranging from 0 to 5% without CD40L and inhibition ranging from 54 to 101% with 10 nM CD40L. Four antibodies showed potentiation of signaling, inhibition ranging from -45 to -92%, in the presence of CD40L in THP-1 bioassay.

[0268] Control mAb1 , Control mAb2 and Control mAb3, irrelevant human IgG antibodies, showed little to no activation (0 - 13%) without CD40L and inhibition (3 - 23%) with CD40L in all cells. CD40L showed activation of signaling with EC50S of 436 pM, 691 pM and 1 .47 nM in Ramos.2G6.4C1 0 / NFKB-IUC, Raji / / NFKB-luc, and THP-1 / NFKB-IUC cells, respectively.Table 14: Anti-CD40 x CD40 bispecific regulation of CD40 signaling in the presence or absence of human CD40L using Ramos.2G6.4C10 / NFKB-luc cells* Values represent EC50 calculations in the presence of CD40L. All other values are IC50S.# IC50 values was obtained by excluding RLU values from conditions containing three highest concentrations of antibody due to hook effect.Table 15: Anti-CD40 x CD40 bispecific regulation of CD40 signaling in the presence or absence of human CD40L using Raji / NFKB-luc cellsTable 16: Anti-CD40 x CD40 bispecific regulation of CD40 signaling in the presence or absence of human CD40L using THP-1 / NFKB-IUC cells* Values represent EC50 calculations in the presence of CD40L. EC50 value was not determined where best-fit value was not found. All other values are IC50S.Example 7: In Vitro CD40L Blocking Activity by CD40xCD40 Bispecific AntibodiesB Cell Assay #1

[0269] To determine the efficacy of CD40xCD40 bispecific antibodies to block stimulation by CD40L, IL-6, IL-10, and TNFa cytokine production was quantified in primary human B cell cultures treated with antibodies in the presence of soluble CD40L. B cells were plated at 2x105cells per well in a 96 U-bottom plate in RPMI 1640 media with 15% FBS and 1 x penicillin-streptomycin. CD40xCD40 bispecific antibodies were added to cells simultaneously in the presence of a constant dose of IL-4 (10 pM) and CD40L (500 nM). A dose response of CD40L with a constant dose of IL-4 (10 pM) was included as a control. Cells were cultured for 3 days at 37°C, and cell culture supernatant was collected for analysis.

[0270] Cytokine levels in supernatant were analyzed using the MSD V-PLEX proinflammatory panel 1 . MSD V-PELX assay was performed according to the manufacturer’s instruction using cell culture supernatant diluted 1 :2. MSD plates were read using an MESO QuickPlex Sq 120MM instrument and MSD Discovery Workbench software. Data analysis was performed using Graphpad Prism software. The data points obtained were transformed using an X=Log(X) equation, and the transformed data were subjected to a linear regression analysis and fitted into a sigmoidal dose response curve. IC50S were derived from this analysis.Results

[0271] All four antibodies tested (REGN16334, REGN16335, REGN16431 , and REGN16432) blocked IL-6 (Fig. 1A, 1 B, and 1 C), IL-10 (Fig. 2A, 2B, and 2C), and TNFa (Fig. 3A, 3B, and 3C) production from primary human B cells from three donors in responseto stimulation with CD40L. IC50 values of antibodies and C0MP11209 (a comparator CD40 antibody) are shown in Table 17 (IL-6), Table 18 (IL-10), and Table 19 (TNFa). The percent max blockade was calculated with the highest dose of REGN16334, REGN16335, REGN16431 , and REGN16432, relative to the average cytokine levels in cells treated with no antibody.Table 17: Effect of CD40xCD40 bispecific antibodies on human B cell IL-6 production in the presence of constant CD40LTable 18: Effect of CD40xCD40 bispecific antibodies on human B cell IL-10 production in the presence of constant CD40LTable 19: Effect of CD40xCD40 bispecific antibodies on human B cell TNFa production in the presence of constant CD40LB Cell Assay #2

[0272] To determine the efficacy of CD40xCD40 bispecific antibodies to block stimulation by CD40L, IL-6, IL-10, and TNFa cytokine production was quantified in primary human B cell cultures treated with antibodies in the presence of soluble CD40L. B cells were plated at 2x105cells per well in a 96 U-bottom plate in RPMI 1640 media with 15% FBS and 1 x penicillin-streptomycin and incubated with CD40xCD40 bispecific antibodies for 30 minutes at 37°C. Following antibody incubation, a constant dose of IL-4 (10 pM) and CD40L (500 nM) was added to the B cell cultures in the presence of the CD40xCD40 bispecific antibodies. A dose response of CD40L with a constant dose of IL-4 (10 pM) was included as a control. In parallel, to quantify the agonistic activity of CD40xCD40 bispecific antibodies, B cells were incubated with antibodies in the presence of a constant dose of IL4 (10 pM) without CD40L. Cells were cultured for 3 days at 37°C, and cell culture supernatant was collected for analysis.

[0273] Cytokine levels in supernatant were analyzed using the MSD V-PLEX proinflammatory panel 1 . MSD V-PELX assay was performed according to the manufacturer’s instruction using cell culture supernatant diluted 1 :2. MSD plates were read using an MESO QuickPlex Sq 120MM instrument and MSD Discovery Workbench software. Data analysis was performed using Graphpad Prism software. The data points obtained were transformed using an X=Log(X) equation, and the transformed data were subjected to a linear regression analysis and fitted into a sigmoidal dose response curve. IC50S were derived from this analysis.Results

[0274] All three CD40xCD40 bispecific antibodies tested (REGN16334, REGN16335, and REGN20484) blocked IL6 (Fig. 1 D and 1 E), IL10 (Fig. 2D and 2E), and TNFa (Fig. 3D and 3E) cytokine production from primary human B cells from two donors in response to stimulation with CD40L. IC50 values of antibodies and COMP11209 are shown in Tables 20- 22. The percent max blockade was calculated with the highest dose of REGN16334, REGN16335, and REGN20484, relative to the average cytokine levels in cells treated with no antibody. Analysis of agonistic activity of antibodies REGN16334, REGN16335, and REGN20484 showed induction of the cytokine IL6 within a range comparable to cells treated with IL4 only (no antibody) in both donors (Fig. 3F and 3G).Table 20: Effect of CD40xCD40 bispecific antibodies on human B cell IL-6 production in the presence of constant CD40LTable 21 : Effect of CD40xCD40 bispecific antibodies on human B cell IL-10 production in the presence of constant CD40LTable 22: Effect of CD40xCD40 bispecific antibodies on human B cell TNFa production in the presence of constant CD40LDendritic Cell Assay

[0275] To determine the efficacy of CD40xCD40 bispecific antibodies to block stimulation by CD40L, IL-12 / IL-23p40 cytokine production was quantified in primary human monocyte derived dendritic cell (MDDCs) cultures treated with antibodies in the presence of soluble CD40L. To generate MDDCs, peripheral blood mononuclear cells (PBMCs) from healthy human donors were isolated from leukopacks 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. Purified CD14+ cells were plated at 3x106cells per well in a 6-well plate in RPMI 1640 media with 10% FBS, 1x penicillin-streptomycin, 800 U / mL GM-CSF, and 500 U / mL of IL-4. Media was replenished and the complete amount of GM-CSF and IL-4 was added back to cells on day 3 and day 5. On day 6 MDDCs were collected and plated at 1x106cells per well in a 96-U bottom plate. CD40xCD40 bispecific antibodies were added to cells simultaneously with a constant dose of CD40L (20 nM). A dose response of CD40L was included as a control. Cells were cultured for 4 days at 37°C, and cell culture supernatant was collected for analysis.

[0276] Cytokine levels in supernatant were analyzed using the MSD V-PLEX cytokine panel 1 . MSD V-PLEX assay was performed according to the manufacturer’s instruction using cell culture supernatant diluted at 1 :2 or 1 :20. MSD plates were read using an MESO QuickPlex Sq 120MM instrument and MSD Discovery Workbench software. Data analysis was performed using Graphpad Prism software. The data points obtained were transformed using an X=Log(X) equation, and the transformed data were subjected to a linear regression analysis and fitted into a sigmoidal dose response curve. IC50S were derived from this analysis.Results

[0277] All four antibodies tested (REGN16334, REGN16335, REGN16431 , and REGN16432) blocked IL-12 / IL-23p40 production from primary human monocyte derived dendritic cells from two donors in response to stimulation with CD40L (Fig. 4A-4B). IC50 values of antibodies and COMP11209 are shown in Table 23.Table 23: Effect of CD40xCD40 bispecific antibodies on human dendritic cell IL-12 / IL-23p40 production in the presence of constant CD40LExample 8: In Vitro Agonist Activity in Human B Cells by CD40xCD40 Bispecific Antibodies

[0278] To quantify the agonistic activity of CD40xCD40 bispecific antibodies, B cells were incubated with antibodies in the presence of a constant dose of IL-4 (10 pm) without CD40L. Cells were cultured for 3 days at 37°C, and cell culture supernatant was collected for analysis. Cytokine levels in supernatant were analyzed using the MSD V-PLEX proinflammatory panel 1 . MSD V-PELX assay was performed according to themanufacturer’s instruction using cell culture supernatant diluted 1 :2. MSD plates were read using an MESO QuickPlex Sq 120MM instrument and MSD Discovery Workbench software. Data analysis was performed using Graphpad Prism software. The data points obtained were transformed using an X=Log(X) equation, and the transformed data were subjected to a linear regression analysis and fitted into a sigmoidal dose response curve. IC50S were derived from this analysis.Results

[0279] Analysis of the agonistic activity of antibodies REGN16334, REGN16335, REGN16431 , and REGN16432 showed induction of the cytokines IL-6 (Fig. 5A-5B) and IL- 10 (Fig. 6A-6B) within a range comparable to cells treated with no antibody (IL-4 only) in both donors.Example 9: Impact of CD40xCD40 Bispecific Antibodies in a Mouse NP-KLH Immunization Model

[0280] To determine the impact of CD40 blockade on an antigen specific antibody response, a widely used T-dependent immunization model, the NP-KLH immunization model, was utilized in mice homozygous for human CD40 in the place of mouse CD40. Mice were immunized with 25 pg of NP-KLH by subcutaneous injection on the flank with 100 pL of a 1 :1 mixture of NP-KLH and alum emulsified by shaking for 30 minutes. For the group receiving alum alone, 100 pL of a 1 :1 mixture of PBS and alum was administered. As shown in Fig. 7, CD40xCD40 bispecific antibody (REGN16334, REGN16335, REGN16431 , or REGN16432) or isotype control (REGN4439 or REGN4460) was administered to mice 3 days before immunization, and twice per week following immunization for a total of two weeks at a dose of 1 mg / kg. Following immunization and the antibody treatment protocol, the mice were sacrificed, and blood and inguinal lymph nodes were collected. The blood was collected from all groups of mice by cardiac puncture and transferred into BD microtainer tubes (cat# 365967) for serum isolation.For tissue processing, lymph nodes were mashed on a 74-micron cell strainer in 2 mL RPMI media + 10% FBS using the back end of a 3 mL syringe, and single cell suspensions were filtered through Millipore plate filter (100 pm) into a 2 mL deep well plate. Cells were centrifuged at 400g for 4 minutes and resuspended in in 200 pL of PBS. Cells were transferred to a 96 well u-bottom plate, centrifuged at 400g for 4 minutes and stained with a live / dead cell marker for 15 minutes at room temperature. Cells were washed and incubated in Fc block for 15 minutes at 4°C followed by antibody staining with antibody mixes as shown in Table 24 for 30 minutes at 4°C. After staining, the cells were washed twice with MACS buffer, fixed with BD Cytofix (cat# 554655) diluted 1 :4 in PBS for 15 minutes, then resuspended in MACS buffer and stored at 4°C. On the day of acquisition, the cells werewashed with BD Perm / wash, incubated in BD Perm / wash buffer (cat# 554723) for 20 minutes and stained with intracellular antibodies [Table 24] for 30 minutes. Cells were washed twice and fixed with BD Cytofix, then resuspended in MACS buffer. The cells were then acquired in an FACSymphony A5 instrument and analyzed using OMIQ software. NP+ germinal center B cells were identified as Live — > Dump- (dump includes TCRb, CD200R3, Ly6G, CD49b and CD11 b) — > non-marginal zone B cells (CD1d-) — > CD19+B220+ — > CD38- IgD- — > GC positive (GL7+CD95+)^ NP+. Statistical significance was determined by the Shaprio-Wilk test to assess normality and the Kruskal-Wallis test with Dunn’s post-hoc multicomparison test in GraphPad Prism.Table 24: Flow Cytometry PanelExperiment #1 - Methods and Results

[0281] In one experiment, levels of NP-specific lgG1 were qualified in terminal serum by ELISA. NP-2-BSA was diluted in to 4 pg / mL in PBS and 384 well plates were coated with 25 pL / well of solution overnight at 4°C. Plates were washed 4x with wash buffer and blocked with 50 pL / well of 0.5% BSA in PBS for 1 hour at room temperature. Plates were washed 4x with wash buffer and the mouse serum was diluted 1 :100 for group A and 1 :10,000 for groups B-G. Serum was further serially diluted 3-fold using 0.5% BSA in PBS 8 times and added to plates at 12.5 pL / well. Diluted serum was incubated on plates for 1 hour at room temperature followed by 4x wash. For detection, 25 pL of rat anti-mouse IgG 1 HRP conjugated antibody diluted 1 :1000 in 0.5% BSA in PBS was added to plates for 1 hour at room temperature. Plates were washed 7x and developed by adding 25 pL of usingOptEIA™ TMB Substrate solution. After 20 minutes the reaction was stopped by adding 25 pL 2N sulfuric acid. Absorbance at 450 nm (OD450) was measured on a Molecular Devices SpectraMax M5 plate reader. Relative levels of circulating NP-specific lgG1 in serum were represented as titer units which were defined as dilution factor required to achieve an OD450 reading that was equal to two times background OD450. Graphical analyses were performed using GraphPad Prism software (version 7.0). Statistical significance was determined by the Shaprio-Wilk test to assess normality and the Kruskal-Wallis test with Dunn’s post-hoc multicomparison test in GraphPad Prism.

[0282] In this NP-KLH immunization model, prophylactic treatment with CD40xCD40 bispecific antibody blocked antigen specific germinal center B cell formation in the draining lymph node as quantified by flow cytometry. All of the CD40xCD40 bispecific antibodies reduced the frequency of NP-specific germinal center B cells at 1 mg / kg compared to their relevant isotype controls (Fig. 8A and Table 25). Prophylactic treatment with CD40xCD40 bispecific antibody at 1 mg / kg also blocked the generation of high-affinity NP-lgG1 antibody responses as measured by NP-specific IgG 1 titers in serum, with most samples falling below the lower limit of quantification for the assay (Fig. 8B and Table 26).Table 25: Effect of CD40xCD40 Bispecific Antibodies on Frequency of NP+ Germinal CenterB CellsSD = standard deviation; N / A = not available; ns = not statistically significant. * p<0.05.Table 26: Effect of CD40xCD40 Bispecific Antibodies on Frequency of NP lqG1 Titers inMouse SerumSD = standard deviation; N / A = not available; ns = not statistically significant. * p<0.05; **p<0.005.Experiment #2 - Methods and Results

[0283] In another experiment, levels of NP-specific IgG 1 were qualified in terminal serum by ELISA. NP-2-BSA was diluted to 4 pg / mL in PBS and 384 well plates were coated with 25 pL / well of solution overnight at 4°C. Plates were washed 5x with wash buffer and blocked with 50 pL / well of 0.5% BSA in PBS for 1 hour at room temperature. Plates were washed 5x with wash buffer and the mouse serum was diluted 1 :100 for group A-B and 1 :10,000 for groups C-L. Serum was further serially diluted 3-fold using 0.5% BSA in PBS 8 times and added to plates at 12.5 pL / well. Diluted serum was incubated on plates for 1 hour at room temperature followed by 4x wash. For detection, 25 pL of rat anti-mouse IgG 1 HRP conjugated antibody diluted 1 :1 ,000 in 0.5% BSA in PBS was added to plates for 1 hour at room temperature. Plates were washed 7x and developed by adding 25 pL of using OptEIA™ TMB Substrate solution. After 20 minutes the reaction was stopped by adding 25 pL 2N sulfuric acid. Absorbance at 450nm (OD450) was measured on a Molecular Devices SpectraMax M5 plate reader. Relative levels of circulating NP-specific lgG1 in serum were represented as titer units which were defined as dilution factor required to achieve an OD450 reading that was equal to two times background OD450. Data analysis was performed using GraphPad Prism. Statistical significance was determined by the Shaprio-Wilk test to assess normality and the Kruskal-Wallis test with Dunn’s post-hoc multi-comparison test in GraphPad Prism.

[0284] In this experiment, prophylactic treatment with CD40xCD40 bispecific antibody blocked antigen specific germinal center B cell formation in the draining lymph node as quantified by flow cytometry. All of the tested CD40xCD40 bispecific antibodies reduced the frequency of NP-specific germinal center B cells at 1 mg / kg and higher compared to isotype control (Fig. 9A and Table 27). Prophylactic treatment with CD40xCD40 bispecific antibodyat 1 mg / kg also blocked the generation of high-affinity NP-lgG1 antibody responses as measured by NP-specific IgG 1 titers in serum, with most samples falling below the lower limit of quantification for the assay (Fig. 9B and Table 28).Table 27: Effect of CD40xCD40 Bispecific Antibodies on Frequency of NP+ Germinal CenterB CellsSD = standard deviation; N / A = not available; ns = not statistically significant. * p<0.05.Table 28: Effect of CD40xCD40 Bispecific Antibodies on Frequency of NP lqG1 Titers inMouse SerumSD = standard deviation; N / A = not available; ns = not statistically significant. * p<0.05.Example 10: Impact of Prophylactic Dosing with CD40xCD40 Bispecific Antibodies in a Mouse Model of Experimental Autoimmune Encephalomyelitis (EAE)

[0285] To determine the impact of CD40 blockade in an autoimmune disease, a model of experimental autoimmune encephalomyelitis (EAE) model was utilized in mice where the entire mouse CD40 gene was replaced by full-length human CD40 (CD40hu / humice). Mice were immunized by subcutaneous injection of 0.1 mL MOG35-55 in emulsion with complete Freund’s adjuvant on the upper back and lower back (0.2 mL / mouse total). Two hours following immunization, mice were administered 150 ng pertussis toxin (PTX) intraperitoneally at 0.1 mL / dose. The administration of PTX was repeated 24 hours later. CD40 x CD40 bispecific antibodies or isotype controls were administered to mice 3 days before immunization at 25 mg / kg, and twice a week following immunization for a total of five weeks. See, Fig. 10. Mice were monitored and weighed twice a week for the first two weeks following immunization and daily for weeks 3-5. Mice were assigned a score following the scoring guidelines outlined in Table 29. Mice were euthanized after receiving a symptom score of 4 for two consecutive days or upon a score of 4.5 or 5 or when they had dropped 30% of their starting body weight.Table 29: Mouse EAE Scoring GuidelinesExperiment #1 - Results

[0286] In one experiment using a model of experimental autoimmune encephalomyelitis (EAE), prophylactic blockade of CD40 by CD40xCD40 bispecific antibodies REGN16334, REGN16335, REGN16431 , and REGN16432 reduced the severity of EAE disease symptom score relative to no antibody or isotype control treated groups (Figs. 11 A-11 B and Table 30). In addition to reduction of symptom severity, all CD40 blocking antibodies reduced the percentage of mouse weight loss when compared to no antibody or isotype control treated groups (Fig. 11 C-11 D and Table 31). The frequency of mice with disease score development was also reduced in groups treated with CD40 blocking antibodies (Table 32).Table 30: Mean EAE Symptom ScoreTable 31 : Mean Weight LossTable 32: Number of Mice with Development of EAE SymptomsExperiment #2 - ResultsIn another experiment, prophylactic blockade of CD40 by CD40xCD40 bispecific antibodies REGN16335 or REGN20484 reduced the severity of EAE disease symptom score relative to no antibody or isotype control treated groups (Fig. 12A and Table 33). In addition to reduction of symptom severity, all CD40 blocking antibodies reduced the percentage of mouse weight loss when compared to no antibody or isotype control treated groups (Fig. 12B and Table 34). The frequency of mice with disease score development was also reduced in groups treated with CD40 blocking antibodies (Table 35).Table 33: Mean EAE Symptom ScoreTable 34: Mean Weight LossTable 35: Number of Mice with Development of EAE SymptomsExample 11 : CryoEM Analysis of CD40xCD40 Bispecific Antibodies

[0287] To better understand the binding of REGN16335 and REGN16334 to CD40, structural analysis was performed via cryo-electron microscopy (cryo-EM). Fab fragments were prepared enzymatically. The Fab fragment 30027P2 corresponds to arm 1 of REGN16335, the Fab fragment 21519P2 corresponds to arm 1 of REGN16634, and the Fab fragment 21520P2 corresponds to arm 2 for both REGN16335 and REGN16334.

[0288] A 3D reconstructed map of the complex of CD40 with the three Fab arms (30027P2, 21519P2, and 21520P2) with resolution of 3 A shows that the three arms bind non-overlapping epitopes spanning the CRD1 , CRD2, and CRD3 domains (Fig. 13). The 30027P2 Fab mostly binds the CRD1 domain and a small portion of CRD2, the 21520P2 Fab binds across CRD1 and CDR2 domains, and the 21519P2 Fab binds across CRD2 and CRD3 domains.

[0289] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims. The disclosures of all patents and non-patent literature cited herein are expressly incorporated in their entirety by reference.

Claims

What is claimed is:1 . A bispecific antigen-binding molecule comprising:(a) a first antigen-binding domain (D1 ) that binds a first epitope of human CD40; and(b) a second antigen-binding domain (D2) that binds a second epitope of human CD40.

2. The bispecific antigen-binding molecule of claim 1 , wherein the bispecific antigen-binding molecule:(i) binds human CD40 with a KD of less than 25 nM as measured by surface plasmon resonance at 25°C;(ii) binds human CD40 with a KD of less than 70 nM as measured by surface plasmon resonance at 37°C;(iii) binds human CD40 with a dissociative half-life (ti / 2) of greater than 75 minutes as measured by surface plasmon resonance at 25°C;(iv) binds a human CD40-expressing cell with an EC50 value of about 10 nM or less;(v) inhibits binding of human CD40 monomer to CD40L;(vi) inhibits CD40 ligand (CD40L)-induced activation; and / or(vii) does not significantly agonize CD40 in the absence of CD40L.

3. The bispecific antigen-binding molecule of 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) an HCDR1 comprising the amino acid sequence of SEQ ID NO:4, an HCDR2 comprising the amino acid sequence of SEQ ID NO:6, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:8;(b) an HCDR1 comprising the amino acid sequence of SEQ ID NO:24, an HCDR2 comprising the amino acid sequence of SEQ ID NO:26, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:28; and(c) an HCDR1 comprising the amino acid sequence of SEQ ID NO:34, an HCDR2 comprising the amino acid sequence of SEQ ID NO:36, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:38.

4. The bispecific antigen-binding molecule of 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 the LCDR1 comprises the amino acid sequence of SEQ ID NO:12, the LCDR2 comprises the amino acid sequence AAS, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:16.

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

6. The bispecific antigen-binding molecule of claim 5, wherein the D1 domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:4, an HCDR2 comprising the amino acid sequence of SEQ ID NO:6, an HCDR3 comprising the amino acid sequence of SEQ ID NO:8, an LCDR1 comprising the amino acid sequence of SEQ ID NO:12, an LCDR2 comprising the amino acid sequence AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:16.

7. The bispecific antigen-binding molecule of claim 6, wherein the D1 domain comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO:2.

8. The bispecific antigen-binding molecule of claim 5, wherein the D1 domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:24, an HCDR2 comprising the amino acid sequence of SEQ ID NO:26, an HCDR3 comprising the amino acid sequence of SEQ ID NO:28, an LCDR1 comprising the amino acid sequence of SEQ ID NO:12, an LCDR2 comprising the amino acid sequence AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:16.

9. The bispecific antigen-binding molecule of claim 8, wherein the D1 domain comprises an HCVR comprising the amino acid sequence of SEQ ID NO:22.

10. The bispecific antigen-binding molecule of any one of claims 1 to 9, wherein the D1 domain comprises a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:10.11 . The bispecific antigen-binding molecule of any one of claims 1 to 10, wherein the D2 domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:34, an HCDR2 comprising the amino acid sequence of SEQ ID NO:36, an HCDR3 comprising the amino acid sequence of SEQ ID NO:38, an LCDR1 comprising the amino acid sequence of SEQ ID NO:12, an LCDR2 comprising the amino acid sequence AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:16.

12. The bispecific antigen-binding molecule of claim 11 , wherein the D2 domain comprises an HCVR comprising the amino acid sequence of SEQ ID NO:32.

13. The bispecific antigen-binding molecule of claim 11 or 12, wherein the D2 domain comprises an LCVR comprising the amino acid sequence of SEQ ID NQ:10.

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

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

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

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

18. The bispecific antigen-binding molecule of any one of claims 1 to 4, wherein the D1 domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:34, an HCDR2 comprising the amino acid sequence of SEQ ID NO:36, an HCDR3 comprising the amino acid sequence of SEQ ID NO:38, an LCDR1 comprising the amino acid sequence of SEQ ID NO:12, an LCDR2 comprising the amino acid sequence AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:16.

19. The bispecific antigen-binding molecule of claim 18, wherein the D1 domain comprises an HCVR comprising the amino acid sequence of SEQ ID NO:32.

20. The bispecific antigen-binding molecule of claim 18 or 19, wherein the D1 domain comprises an LCVR comprising the amino acid sequence of SEQ ID NQ:10.21 . The bispecific antigen-binding molecule of any one of claims 1 -4 and 18-20, wherein the D2 domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:4, an HCDR2 comprising the amino acid sequence of SEQ ID NO:6, an HCDR3 comprising the amino acid sequence of SEQ ID NO:8, an LCDR1 comprising the amino acid sequence of SEQ ID NO:12, an LCDR2 comprising the amino acid sequence AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:16.

22. The bispecific antigen-binding molecule of claim 21 , wherein the D2 domain comprises an HCVR comprising the amino acid sequence of SEQ ID NO:2.

23. The bispecific antigen-binding molecule of claim 21 , wherein the D2 domain comprises an LCVR comprising the amino acid sequence of SEQ ID NO:10.

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

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

26. The bispecific antigen-binding molecule of any one of claims 1 to 25 that is a bispecific antibody.

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

28. The bispecific antigen-binding molecule of claim 27, wherein the human IgG heavy chain constant region is isotype lgG4 or IgG 1 .

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

30. The bispecific antigen-binding molecule of any one of claims 1-17 and 26-29, wherein the D1 comprises: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; or 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 NQ:20; or 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 NQ:20; or 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 NQ:20.31 . The bispecific antigen-binding molecule of any one of claims 1-17 and 26-30, wherein the D2 comprises: 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 NQ:20; or a heavy chain comprising the amino acid sequence of SEQ ID NQ:50 and a light chain comprising the amino acid sequence of SEQ ID NQ:20.

32. The bispecific antigen-binding molecule of any one of claims 1-17 and 26-31 which is a bispecific antibody comprising (i) a 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 NQ:20; and (ii) a 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 NQ:20.

33. The bispecific antigen-binding molecule of any one of claims 1-17 and 26-31 which is a bispecific antibody comprising (i) a 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 NQ:20; and (ii) a 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 NQ:20.

34. The bispecific antigen-binding molecule of any one of claims 1-17 and 26-31 which is a bispecific antibody comprising (i) a 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 NQ:20; and (ii) a D2 comprising a heavy chain comprising the amino acid sequence of SEQ ID NQ:50 and a light chain comprising the amino acid sequence of SEQ ID NQ:20.

35. The bispecific antigen-binding molecule of any one of claims 1-17 and 26-31 which is a bispecific antibody comprising (i) a 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) a D2 comprising a heavy chain comprising the amino acid sequence of SEQ ID NQ:50 and a light chain comprising the amino acid sequence of SEQ ID NQ:20.

36. The bispecific antigen-binding molecule of any one of claims 1 -4 and 18-29, wherein the D1 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:58 and a light chain comprising the amino acid sequence of SEQ ID NQ:20.

37. The bispecific antigen-binding molecule of any one of claims 1-4, 18-29, and 36, wherein the D2 comprises a heavy chain comprising the amino acid sequence of SEQ ID NQ:60 and a light chain comprising the amino acid sequence of SEQ ID NQ:20.

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

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

40. A nucleic acid molecule comprising a nucleotide sequence encoding the bispecific antigen-binding molecule of any one of claims 1 to 38.41 . A nucleic acid molecule comprising one or more nucleotide sequences set forth in Table 36.

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

43. A host cell comprising the expression vector of claim 42.

44. A method of producing a bispecific antigen-binding molecule, the method comprising culturing the host cell of claim 43 under conditions permitting production of the bispecific antigen-binding molecule, and recovering the bispecific antigen-binding molecule so produced.

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

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

47. The method of 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 organ transplant.

48. The method of claim 46 or 47, wherein the CD40-mediated disease or condition is an 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 (C I DP)) , 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, Sjogren's syndrome, skin graft, systemic lupus erythematosus, systemic sclerosis, transplant rejection, vasculitis, ANCA-associated vasculitis, ulcerative colitis, or Wegener granulomatosis.

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

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