CD40X 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. Also provided are methods for treating CD40 mediated diseases using the antigen binding molecules.

Description

Cross-references to related applications

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

[0002] References to sequence lists This application contains a sequence list that has been electronically submitted in XML format, and that sequence list is hereby incorporated herein by reference in its entirety. The XML copy created on June 13, 2024, is named 118003-50020.XML and has a size of 99,202 bytes. Technical Field

[0003] This disclosure relates to antigen-binding molecules that bind to CD40 and inhibit CD40L-induced activation, and methods of using them, for example, for the treatment of autoimmune diseases. Background Technology

[0004] CD40 is a cell surface receptor and 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 between CD40 and its ligand CD40L provides a co-stimulatory signal that is crucial for the survival of many cell types and is essential for immune response functions such as germinal center formation, antibody responses to T-dependent antigens, and “permitting” dendritic cell maturation and becoming effective to trigger T cell activation and differentiation (see, for example, Kawabe et al.). Immunity 1994, 1:167-178; Elgueta et al., Immunol.Rev. 2009, 229:152-172).

[0005] CD40-CD40L signaling is involved in autoimmune diseases primarily driven by autoantibodies, such as systemic rheumatic diseases in which autoantibodies play a significant role in disease progression (e.g., multiple sclerosis, autoimmune nephritis, rheumatoid arthritis, Sjögren's syndrome, and systemic lupus erythematosus), and non-rheumatic conditions with autoantibody components (e.g., myasthenia gravis, Graves' disease, and neuromyelitis optica) (see Karnell et al., Adv Drug Delivery Rev.2019, 141:92-103). Furthermore, because CD40-CD40L signaling is crucial for the activation of antigen-presenting cells, blocking antigen presentation by dendritic cells or B cells may affect CD8+ T cell responses in some diseases, such as multiple sclerosis (see, for example, Denic et al., 2019, 141:92-103). Expert Opinion Ther Targets 2013, 17:1053-1066). Altered CD40-CD40L signaling is also involved in other diseases and conditions, such as cardiovascular disease and transplantation (see, for example, Dakal et al., 2013, 17:1053-1066). 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 monoclonal antibodies targeting CD40L provided biological validation of the inhibition of the CD40-CD40L signaling pathway, but also revealed unexpected safety concerns due to thromboembolic complications (see Karnell et al.). Adv Drug Delivery Review. 2019, 141:92-103). Several monoclonal antibodies targeting CD40 are in clinical development; although no significant safety events have been reported for these antibodies, CD40 antibodies failed to meet the primary endpoint in several phase 2a trials (Kumar et al., 2019, 141:92-103). Biopharm. Drug Disposal. 2018, 39:245-255; Visvanathan et al., Ann Rheum Dis. 2019, 78:754-760). Summary of the Invention

[0007] In one aspect, this 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) The first antigen-binding domain (D1), which binds to the first epitope of human CD40; and (b) The second antigen-binding domain (D2) binds to the second epitope of human CD40.

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

[0009] In some embodiments, the bispecific antigen-binding molecule exhibits one or more of the following characteristics: (i) Combining human CD40, K D Less than 25 nM, as measured by surface plasmon resonance at 25°C; (ii) Combined with human CD40, K D Less than 70 nM, as measured by surface plasmon resonance at 37°C; (iii) Binding to human CD40, dissociation half-life (t 1 / 2 (More than 75 minutes, such as when measured by surface plasmon resonance at 25°C;) (iv) In combination with cells expressing human CD40, EC 50 The value is approximately 10 nM or less; (v) Inhibits the binding of human CD40 monomers to CD40L; (vi) Inhibit CD40 ligand (CD40L)-induced activation; and / or (vii) In the absence of CD40L, CD40 is not significantly stimulated.

[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 stimulate CD40 in the absence of CD40L.

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

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

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

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

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

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

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

[0018] In some embodiments, 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 NO:10, and 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 NO:10.

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

[0020] In some embodiments, 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 NO:10, and 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 NO:10.

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

[0022] In some embodiments, the bispecific antigen-binding molecule comprises a D2 domain, and the D1 domain comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO:4, HCDR2 comprising the amino acid sequence of SEQ ID NO:6, HCDR3 comprising the amino acid sequence of SEQ ID NO:8, LCDR1 comprising the amino acid sequence of SEQ ID NO:12, LCDR2 comprising the amino acid sequence AAS, and LCDR3 comprising the amino acid sequence of SEQ ID NO:16. In some embodiments, the D2 domain comprises: HCVR comprising the amino acid sequence of SEQ ID NO:2. In some embodiments, the D2 domain comprises: LCVR comprising the amino acid sequence of SEQ ID NO:10.

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

[0024] In some embodiments, 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 NO:10, and 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 NO:10.

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

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

[0027] In some embodiments, the bispecific antigen-binding molecule comprises D2, and D1 comprises: A heavy chain containing the amino acid sequence of SEQ ID NO:44 and a light chain containing the amino acid sequence of SEQ ID NO:20; or A heavy chain containing the amino acid sequence of SEQ ID NO:50 and a light chain containing the amino acid sequence of SEQ ID NO:20; or The heavy chain containing the amino acid sequence of SEQ ID NO:60 and the light chain containing the amino acid sequence of SEQ ID NO:20.

[0028] In some embodiments, the bispecific antigen-binding molecule is a bispecific antibody comprising: (i) D1, which 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; and (ii) D2, which 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 NO:20.

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

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

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

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

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

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

[0035] In another aspect, this disclosure provides nucleic acid molecules comprising a nucleotide sequence encoding an antigen-binding molecule that binds to CD40 (e.g., a single-specific 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 listed in Table 36.

[0036] In another aspect, this disclosure provides expression vectors and host cells comprising one or more nucleic acid molecules containing nucleotide sequences encoding antigen-binding molecules that bind to CD40 (e.g., single-specific or bispecific antigen-binding molecules as disclosed herein). In some embodiments, the expression vector and / or host cell comprises nucleic acid molecules containing one or more nucleotide sequences listed in Table 36.

[0037] On the other hand, this disclosure provides a method for generating an antigen-binding molecule that binds to CD40 (e.g., a single-specific or bispecific antigen-binding molecule as disclosed herein). In some embodiments, the method includes culturing host cells as disclosed herein under conditions that allow for the generation of the antigen-binding molecule, and recovering the antigen-binding molecule thus generated.

[0038] In another aspect, this disclosure provides a method for inhibiting CD40L-induced signaling. In some embodiments, the method includes contacting cells expressing CD40 with an antigen-binding molecule or pharmaceutical composition disclosed herein (e.g., a bispecific antigen-binding molecule comprising a sequence listed in any one of Tables 1 to 4 or 36, or a pharmaceutical composition comprising a bispecific antigen-binding molecule comprising a sequence listed in any one of Tables 1 to 4 or 36).

[0039] In another aspect, this disclosure provides methods for treating, improving, or preventing CD40-mediated diseases or conditions in a subject. In some embodiments, the method includes administering to 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 a sequence listed in any of Tables 1 to 4 or 36, or a pharmaceutical composition comprising a bispecific antigen-binding molecule comprising a sequence listed in any of Tables 1 to 4 or 36). In some embodiments, the CD40-mediated disease or condition is an autoimmune disease or condition, an inflammatory disease or condition, a cardiovascular disease or condition, or an organ transplant. In some embodiments, the CD40-mediated disease or condition is 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 thrombocytopenic purpura, 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, Sjögren's syndrome, skin grafts, systemic lupus erythematosus, systemic sclerosis, transplant rejection, vasculitis, ANCA-associated vasculitis, ulcerative colitis, or Wegener's granulomatosis. In some embodiments, the method further includes administering a second therapeutic agent to the subject.

[0040] On the other hand, this disclosure provides antigen-binding molecules (e.g., bispecific antigen-binding molecules) as disclosed herein, or pharmaceutical compositions comprising said antigen-binding molecules, for inhibiting CD40L-induced signaling. In some embodiments, the antigen-binding molecule is a bispecific antigen-binding molecule (e.g., a bispecific antibody) comprising a sequence listed in any one of Tables 1 to 4 or 36.

[0041] On the other hand, this disclosure provides antigen-binding molecules (e.g., bispecific antigen-binding molecules) or pharmaceutical compositions comprising said antigen-binding molecules as disclosed herein for treating, improving, or preventing CD40-mediated diseases or conditions in a subject. In some embodiments, the antigen-binding molecule is a bispecific antigen-binding molecule (e.g., a bispecific antibody) comprising a sequence listed in any one of Tables 1 to 4 or 36. In some embodiments, the CD40-mediated disease or condition is an autoimmune disease or condition, an inflammatory disease or condition, a cardiovascular disease or condition, or an organ transplant. In some embodiments, the CD40-mediated disease or condition is 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 thrombocytopenic purpura, 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, Sjögren's syndrome, skin grafts, systemic lupus erythematosus, systemic sclerosis, transplant rejection, vasculitis, ANCA-associated vasculitis, ulcerative colitis, or Wegener's granulomatosis.

[0042] On the other hand, this disclosure provides antigen-binding molecules (e.g., bispecific antigen-binding molecules) as disclosed herein for use in the manufacture of medicaments for treating, improving, or preventing CD40-mediated diseases or conditions in subjects. In some embodiments, the antigen-binding molecule is a bispecific antigen-binding molecule (e.g., a bispecific antibody) comprising a sequence listed in any of Tables 1 to 4 or 36. In some embodiments, the CD40-mediated disease or condition is an autoimmune disease or condition, an inflammatory disease or condition, a cardiovascular disease or condition, or an organ transplant. In some embodiments, the CD40-mediated disease or condition is 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 thrombocytopenic purpura, 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, Sjögren's syndrome, skin grafts, systemic lupus erythematosus, systemic sclerosis, transplant rejection, vasculitis, ANCA-associated vasculitis, ulcerative colitis, or Wegener's granulomatosis.

[0043] Other embodiments will become apparent from the following detailed description. Attached Figure Description

[0044] Figures 1A to 1C The effect of the CD40xCD40 bispecific antibody on IL6 production in human B cells from three different donors, in the presence of constant CD40L, is shown.

[0045] Figures 1D to 1E The effect of the CD40xCD40 bispecific antibody on IL6 production in human B cells from two different donors, in the presence of constant CD40L, is shown.

[0046] Figures 2A to 2C The effect of the CD40xCD40 bispecific antibody on IL10 production was shown in human B cells from three different donors in the presence of constant CD40L.

[0047] Figures 2D to 2E The effect of the CD40xCD40 bispecific antibody on IL10 production in human B cells from two different donors, in the presence of constant CD40L, is shown.

[0048] Figures 3A to 3C The effect of the CD40xCD40 bispecific antibody on TNFα production was shown in human B cells from three different donors in the presence of constant CD40L.

[0049] Figures 3D to 3E The effect of the CD40xCD40 bispecific antibody on TNFα production in human B cells from two different donors, in the presence of constant CD40L, is shown.

[0050] Figures 3F to 3G Analysis of the agonist activity of a CD40xCD40 bispecific antibody, measured by IL6 production from human B cells from two different donors, is shown.

[0051] Figures 4A to 4B The effect of the CD40xCD40 bispecific antibody on IL-12 / IL-23p40 production was shown in dendritic cells derived from human monocytes from two different donors in the presence of constant CD40L.

[0052] Figures 5A to 5B Analysis of the agonist activity of a CD40xCD40 bispecific antibody, measured by IL6 production from human B cells from two different donors, is shown.

[0053] Figures 6A to 6B Analysis of the agonist activity of a CD40xCD40 bispecific antibody, measured by IL10 from human B cells from two different donors, is shown.

[0054] Figure 7 An experimental timeline using the NP-KLH immune model, as disclosed in Example 9, is shown.

[0055] Figure 8A The effect of CD40xCD40 bispecific antibody on the frequency of B cells in NP-positive germinal centers was shown. p<0.05.

[0056] Figure 8B The effect of CD40xCD40 bispecific antibody on the frequency of NP IgG1 titer in mouse serum was shown. p<0.05; p<0.005.

[0057] Figure 9A The effect of CD40xCD40 bispecific antibody on the frequency of B cells in NP-positive germinal centers was shown.

[0058] Figure 9B The effect of CD40xCD40 bispecific antibody on the frequency of NP IgG1 titer in mouse serum was shown.

[0059] Figure 10 The experimental timeline using the mouse EAE model, as disclosed in Example 10, is shown.

[0060] Figure 11A The mean EAE symptom scores of mice treated with REGN16431 or REGN16432 are shown.

[0061] Figure 11B The mean EAE symptom scores of mice treated with REGN16334 or REGN16335 are shown.

[0062] Figure 11C The initial body weight percentage of mice treated with REGN16431 or REGN16432 is shown.

[0063] Figure 11D The initial body weight percentage of mice treated with REGN16334 or REGN16335 is shown.

[0064] Figure 12A The mean EAE symptom scores of mice treated with CD40xCD40 bispecific antibody are shown.

[0065] Figure 12B The percentage of initial body weight in mice treated with the CD40xCD40 bispecific antibody is shown.

[0066] Figure 13 The cryoEM reconstruction of CD40 composited with Fab arms 30027P2, 21519P2 and 21520P2 is shown. Detailed Implementation

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

[0068] Unless otherwise defined, 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 pertains.

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

[0070] As used herein, the term "CD40" refers to differentiation cluster 40 (CD40 / TNFRSF5), a costimulatory cell surface receptor that is part of the tumor necrosis factor (TNF) receptor superfamily. In some embodiments, CD40 is human CD40. In some embodiments, the CD40 protein comprises the amino acid sequence of human CD40 listed in UniProt accession number Q09LL4.

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

[0072] As used herein, the term "antibody" 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). As used herein, the term "antibody" includes immunoglobulin molecules and their multimers comprising four polypeptide chains, two heavy (H) chains, and two light (L) chains interconnected by disulfide bonds. For example (IgM). In typical antibodies, each heavy chain contains a heavy chain variable region (abbreviated as HCVR or V in this article). H The heavy-chain constant region contains three structural domains C. H 1. C H 2 and C H 3. Each light chain contains a light chain variable region (abbreviated as LCVR or V in this article). L ) and the light chain constant region. The light chain constant region contains a structural domain (C L 1). V H and V L The region can be further subdivided into highly variable regions (called complementary determinant regions (CDRs)), which are interspersed with more conservative regions (called frame regions (FRs)). Each V H and V L It consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments, the FRs of the antibody (or its antigen-binding portion) may be identical to the human germline sequence, or may be natural or artificially modified. The common amino acid sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0073] Methods and techniques for identifying CDRs within amino acid sequences of HCVR and LCVR 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 for identifying CDR boundaries 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). Generally, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia methods. See, for example, Kabat et al., "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Chothia et al., J Mol Biol (1987), 4:901–17; Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989); see also Dondelinger et al., Front. Immunol. (2018), 9:2278, doi:10.3389 / fimmu.2018.02278. Public databases can also be used to identify CDR sequences within antibodies.

[0074] As used herein, the term "antibody" also includes the antigen-binding fragment of a complete antibody molecule. As used herein, the terms "antigen-binding moiety," "antigen-binding fragment," "antigen-binding domain," etc., of an antibody include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The antigen-binding fragment of an antibody can be derived from, for example, a complete antibody molecule using any suitable standard technique such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding variable and optionally constant domains of the antibody. Such DNA is known and / or can be readily obtained from… For example DNA can be obtained from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or it can be synthesized. DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into suitable conformations, or to introduce codons, generate 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 composed of amino acid residues mimicking the hypervariable region of an antibody (e.g., a separated complementarity-determining region (CDR), such as a CDR3 peptide), or restricted FR3-CDR3-FR4 peptides. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deficient antibodies, chimeric antibodies, CDR-transplanted antibodies, binary antibodies, ternary antibodies, quaternary antibodies, microantibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed in the term "antigen-binding fragments" as used herein.

[0076] Antibody antigen-binding fragments typically contain at least one variable domain. Variable domains can have any size or amino acid composition and generally contain at least one CDR adjacent to or within one or more frame sequences. L V associated with the structural domain H In the antigen-binding fragment of the domain, V H and V L Domains can be arranged in any suitable arrangement relative to each other. For example, variable regions can be dimers and contain V. H -V H V H -V L or V L -V L Dimer. Alternatively, the antigen-binding fragment of the antibody may contain monomer V. H or V L Structural domain.

[0077] In some embodiments, the antigen-binding fragment of the 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 the antigen-binding fragment of the antibody include: (i) V H -C H 1; (ii) V H -C H 2; (iii) V H -C H 3; (iv) V H -C H 1-C H 2; (v) V H -C H 1-C H 2-CH 3; (vi) V H -C H 2-C H 3; (vii) V H -C L (viii) V L -C H 1; (ix) V L -C H 2; (x) V L -C H 3;(xi) V L -C H 1-C H 2;(xii) V L -C H 1-C H 2-C H 3; (xiii) V L -C H 2-C H 3; and (xiv) V L -C L In any configuration of the variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly connected to each other or connected via complete or partial hinge or linker regions. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids, creating a flexible or semi-flexible connection between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragment of the antibody may include the domains listed above and / or one or more monomers V H or V L Homodimers or heterodimers (or other polymers) of any variable and constant domain configurations of non-covalently associated domains (e.g., via disulfide bonds).

[0078] As used herein, the term "antibody" also includes multispecific (e.g., bispecific) antibodies. A multispecific antibody, or an antigen-binding fragment of an antibody, will typically include at least two distinct variable domains, each capable of specifically binding to a single antigen or a different epitope on the same antigen. In some embodiments, a multispecific antibody (e.g., a bispecific antibody) has an arm that binds to a first epitope of the antigen and an arm that binds to a second epitope of the same antigen.

[0079] Using conventional techniques available in the art, any form of multispecific antibody may be suitable for use in the context of the antibodies or antigen-binding fragments of antibodies disclosed herein. For example, this disclosure includes bispecific antibodies in which 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 forms that may be used in the context of this disclosure include, but are not limited to, scFv-based or binary antibody bispecific forms, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadromas, knots-intoholes, common light chains (e.g., common light chains with knots-intoholes, etc.), CrossMab, CrossFab, (SEED) bodies, leucine zippers, Duobody, IgG1 / IgG2, dual-action Fab (DAF)-IgG, and Mab. 2 Bispecific form ( See, for example Klein et al. 2012, mAbs 4:6, 1-11, and the references cited therein, for the purposes of the foregoing review. Bispecific antibodies can also be constructed using peptide / nucleic acid conjugations, for example, where non-natural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugations, which are then self-assembled into multimeric complexes with defined composition, valence, and geometry. See, for example Kazane et al. , J. Am. Chem.Soc [Epub: December 4, 2012]

[0080] As used herein, the term "human antibody" is intended to comprise antibodies having variable and constant regions derived from human immunoglobulin sequences. Nevertheless, human antibodies of this disclosure may comprise amino acid residues not encoded by human immunoglobulin sequences (e.g., via...). in vitro Random or site-specific mutagenesis formed or through in vivo Mutations introduced by somatic mutations), such as in CDRs, and particularly in CDR3. However, as used herein, the term “human antibody” is not intended to include antibodies in which a CDR sequence derived from a lineage of another mammalian species, such as a mouse, has been grafted onto a human frame sequence.

[0081] As used herein, the term "recombinant antibody" is intended to include all antibodies prepared, expressed, produced, or isolated by recombinant means. This term includes, but is not limited to, antibodies expressed using recombinant expression vectors transfected into host cells (e.g., Chinese hamster ovary (CHO) cells) or cell expression systems, antibodies isolated from recombinant human antibody libraries, and antibodies isolated from non-human animals (e.g., mice, such as mice transgenic to target human immunoglobulin genes) (see [link to relevant documentation]). example like Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295). In some embodiments, the recombinant antibody is a recombinant human antibody. In some embodiments, the recombinant human antibody has variable and constant regions derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies undergo... in vitro Mutagenesis (or, when used in animals with transgenic human Ig sequences, undergoing...) in vivo Somatic cell mutagenesis), and therefore the V of recombinant antibodies H District and V L The amino acid sequence of the region is as follows: Although it is derived from human lineage V H Sequence and V L The sequence is related to it, but may not be naturally present. in vivo It exists in human antibody germline libraries.

[0082] "Isolated antibodies" refer to antibodies that have been identified, isolated, and / or recovered from at least one component of their natural environment. For example, antibodies that have been isolated or removed from at least one component of an organism or from tissues or cells where antibodies are naturally present or produced are considered "isolated antibodies." Isolated antibodies also include recombinant intracellular antibodies. In situ Antibody. The isolated antibody is an antibody that has undergone at least one purification or isolation step. According to some embodiments, the isolated antibody may be substantially free of other cellular material and / or chemicals.

[0083] The term "specific binding" refers to the formation of a complex between an antibody or its antigen-binding fragment and an antigen that is relatively stable under physiological conditions. Specific binding can occur through at least approximately 1 x 10-1 -6 M or smaller, such as 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 The equilibrium dissociation constant of M is used to characterize (smaller K) D(Indicating 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™), biolayer interference assays (e.g., Octet® HTX biosensor), solution affinity ELISA, etc. In some embodiments, specific binding is measured in a surface plasmon resonance assay, for example at 25°C or 37°C. Antibodies or antigen-binding fragments that specifically bind to antigens from one species may or may not have cross-reactivity with other antigens, such as orthologous antigens from another species.

[0084] As used in this article, the term "K" D "" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction.

[0085] As used herein, the term “surface plasmon resonance” refers to an optical phenomenon that allows for the analysis of real-time biomolecular interactions by detecting changes in protein concentration within a biosensor matrix, for example, using a BIACORE™ system (Cytiva, Marlborough, MA).

[0086] As used herein, the term "epitope" refers to an antigenic determinant cluster that interacts with a specific antigen-binding site called a complementary site in a variable region of an antibody molecule. A single antigen may have more than one epitope. Thus, different antibodies can bind to different regions on an antigen and may have different biological effects. The term "epitope" can also refer to a site on an antigen to which B and / or T cells respond. It also refers to a region of an antigen bound by an antibody. Epitopes can be linear or discontinuous (e.g., conformational). Linear epitopes are epitopes generated from adjacent amino acid residues in a polypeptide chain. Conformational epitopes are generated from spatially juxtaposed amino acids from different segments of a linear polypeptide chain. In some embodiments, epitopes may comprise determinants clustered as chemically active surfaces of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in some embodiments may have specific three-dimensional structural properties and / or specific charge properties. Epitopes can also be defined as structural or functional. Functional epitopes are typically a subset of structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes typically contain at least three, and more often at least five or at least eight to ten, amino acids in a unique spatial conformation.

[0087] Methods for identifying epitopes of antigen-binding proteins (e.g., antibodies or antigen-binding fragments) include alanine scanning mutation analysis, peptide blotting analysis (Reineke), and other methods. Methods Mol Biol2004, 248:443-463), peptide cleavage analysis, crystallographic studies, and NMR analysis. Additionally, methods such as epitope exclusion, epitope extraction, and chemical modification of antigens can be used (Tomer, Prot Sci 2000, 9:487-496). Another method that can be used to identify amino acids within a polypeptide that interacts with an antigen-binding protein (e.g., an antibody or antigen-binding fragment) is hydrogen / deuterium exchange detected by mass spectrometry (HDX). See, for example, Ehring, Analytical Biochemistry 1999, 267:252-259; Engen and Smith, Anal Chem 2001, 73:256A-265A.

[0088] As used with respect to competitive binding, the term "competition" refers to an antigen-binding protein (e.g., an antibody or antigen-binding fragment) that binds to an antigen and inhibits or blocks the binding of another antigen-binding protein (e.g., an antibody or antigen-binding fragment) to the antigen. Unless otherwise stated, the term also includes competition between two antigen-binding proteins (e.g., antibodies) in two orientations, i.e., a first antigen binds to the antigen and blocks the binding of 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., an antibody) and the second antigen-binding protein (e.g., an antibody) may bind to the same epitope. Alternatively, the first and second antigen-binding proteins (e.g., antibodies) may bind to different epitopes, which may be overlapping or non-overlapping, wherein the 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 can be measured by methods known in the art (e.g., by real-time label-free biolayer interferometry).

[0089] As used with respect to nucleic acids or fragments thereof, the terms "substantially identical" and "substantially the same" mean that, when optimally aligned with another nucleic acid (or its complementary strand) by appropriate nucleotide insertions or deletions, at least about 90%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleotide bases exhibit nucleotide sequence identity, as measured by any well-known sequence identity algorithm (such as FASTA, BLAST, or GAP), as discussed below. In some cases, nucleic acid molecules having substantial identity with a reference nucleic acid molecule may encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0090] When applied to peptides, the terms "substantially identical" and "substantially the same" mean that two peptide sequences share at least about 90% sequence identity at optimal alignment, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, dissimilar residue positions differ due to conserved amino acid substitutions. A "conserved amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced by another amino acid residue on a side chain (R group) having similar chemical properties (e.g., charge or hydrophobicity). Typically, conserved amino acid substitutions will not substantially alter the functional properties of the protein.

[0091] Sequence analysis software is typically used to measure the sequence similarity of peptides. Protein analysis software uses similarity measures assigned to various substitutions, deletions, and other modifications (including conserved amino acid substitutions) to match similar sequences. For example, 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 peptides (such as homologous peptides from different biological species) or between wild-type proteins and their mutants. See, for example, GCG version 6.1. Peptide sequences can also be compared using FASTA with default or recommended parameters; the program in GCG version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignment of the best overlapping region between the query sequence and the search sequence and the percentage of sequence identity (Pearson, 2000, ibid.). Another preferred algorithm when comparing sequences of this disclosure with databases containing large numbers of sequences from different organisms is the computer program BLAST using default parameters, especially BLASTP or TBLASTN. (See, for example, Altschul et al., 1990, ...) J. Mol.Biol. 215: 403-410 and 1997 Nucleic Acids Res. 25:3389-3402).

[0092] When compared using the BLAST algorithm, a “variant” of a polypeptide containing the amino acid sequence specifically listed herein (such as an immunoglobulin, VH, VL, heavy chain, light chain, or CDR) refers to a polypeptide containing 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%) the same amino acid sequence as a reference polypeptide sequence (e.g., as listed in the sequence listing below), wherein the parameters of the algorithm are selected to give the maximum match between the corresponding sequences over the full length of the corresponding reference sequence. In some embodiments, a variant of the polypeptide includes a polypeptide having the amino acid sequence of a reference polypeptide sequence (e.g., as shown in the sequence listing below) but with one or more (e.g., 1 to 10, or less than 20, or less than 10) missense mutations (e.g., conserved substitutions), nonsense mutations, deletions, or insertions.

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

[0094] Monospecific CD40 antibody In some embodiments, this disclosure provides a monospecific CD40 antibody or an antigen-binding fragment thereof that specifically binds to CD40 (e.g., human CD40). In some embodiments, the CD40 antibody or its antigen-binding fragment comprises the amino acid sequence set HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3, which is contained within HCVR / LCVR amino acid sequence pairs selected from groups consisting of 2 / 10, 22 / 10, and 32 / 10. In some embodiments, CDRs within the HCVR and / or LCVR are identified according to the Kabat definition. In some embodiments, CDRs within the HCVR and / or LCVR are identified according to the Chothia definition. In some embodiments, CDRs within the HCVR and / or LCVR are identified according to the AbM definition. In some embodiments, CDRs within the HCVR and / or LCVR are identified according to the IMGT definition.

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

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

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

[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 with 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 with SEQ ID NO:10. In some embodiments, the CD40 antibody comprises: an HCVR comprising the amino acid sequence of SEQ ID NO:2 or composed thereof. In some embodiments, the CD40 antibody comprises: an LCVR comprising the amino acid sequence of SEQ ID NO:10 or composed thereof.

[0099] In some embodiments, the CD40 antibody comprises: a heavy chain comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:18; and / or a light chain comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with 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 NO:18. In some embodiments, the CD40 antibody comprises: a light chain comprising or consisting of the amino acid sequence of SEQ ID NO:20.

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

[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 with 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 with SEQ ID NO:10. In some embodiments, the CD40 antibody comprises: an HCVR comprising the amino acid sequence of SEQ ID NO:22 or composed thereof. In some embodiments, the CD40 antibody comprises: an LCVR comprising the amino acid sequence of SEQ ID NO:10 or composed thereof.

[0102] In some embodiments, the CD40 antibody comprises: a heavy chain comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:28; and / or a light chain comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with 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 NO:28. In some embodiments, the CD40 antibody comprises: a light chain comprising or consisting of the amino acid sequence of SEQ ID NO:20.

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

[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 with 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 with SEQ ID NO:10. In some embodiments, the CD40 antibody comprises: an HCVR comprising the amino acid sequence of SEQ ID NO:32 or composed thereof. In some embodiments, the CD40 antibody comprises: an LCVR comprising the amino acid sequence of SEQ ID NO:10 or composed thereof.

[0105] In some embodiments, the CD40 antibody comprises: a heavy chain comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:40; and / or a light chain comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with 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 NO:40. In some embodiments, the CD40 antibody comprises: a light chain comprising or consisting of the amino acid sequence of SEQ ID NO:20.

[0106] In some embodiments, the CD40 antibody has at least 85% sequence identity with 30027P2, such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In some embodiments, the CD40 antibody has the amino acid sequence of 30027P2.

[0107] In some embodiments, the CD40 antibody has at least 85% sequence identity with 21519P2, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In some embodiments, the CD40 antibody has the amino acid sequence of 21519P2.

[0108] In some embodiments, the CD40 antibody has at least 85% sequence identity with 21520P2, such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In some embodiments, the CD40 antibody comprises or is composed of the amino acid sequence of 21520P2.

[0109] CD40xCD40 bispecific antigen-binding molecule This disclosure also provides a multispecific antigen-binding molecule that specifically binds 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 first antigen-binding domain (D1) of a first epitope binding to CD40 (e.g., human CD40) and a second antigen-binding domain (D2) of a second epitope binding to CD40 (e.g., human CD40). In some embodiments, D1 and D2 do not compete with each other for binding to CD40 (e.g., human CD40). In some embodiments, D1 and D2 compete with each other for binding to CD40 (e.g., human CD40).

[0110] In some embodiments, the bispecific antigen-binding molecule comprises two distinct heavy chain immunoglobulin variable regions, wherein at least one heavy chain immunoglobulin variable region comprises an HCDR1-HCDR2-HCDR3 amino acid sequence set contained within the HCVR amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 22, and 32. In some embodiments, each heavy chain immunoglobulin variable region comprises an HCDR1-HCDR2-HCDR3 amino acid sequence set contained within the HCVR amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 22, and 32. In some embodiments, CDRs within the HCVR are identified according to the Kabat definition. In some embodiments, CDRs are identified according to the Chothia definition. In some embodiments, CDRs are identified according to the AbM definition. In some embodiments, CDRs are identified according to the IMGT definition.

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

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

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

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

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

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

[0117] In some embodiments, the D1 domain comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO:4, HCDR2 comprising the amino acid sequence of SEQ ID NO:6, and HCDR3 comprising the amino acid sequence of SEQ ID NO:8. In some embodiments, the D1 domain comprises: HCDR1 composed of the amino acid sequence of SEQ ID NO:4, HCDR2 composed of the amino acid sequence of SEQ ID NO:6, and HCDR3 composed of the amino acid sequence of SEQ ID NO:8. In some embodiments, the D1 domain comprises: HCVR comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:2. In some embodiments, the D1 domain comprises: HCVR comprising or composed of the amino acid sequence of SEQ ID NO:2.

[0118] In some embodiments, the D1 domain comprises a heavy chain containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:46. In some embodiments, the D1 domain comprises a heavy chain containing or consisting of the amino acid sequence of SEQ ID NO:46.

[0119] In some embodiments, the D1 domain comprises a heavy chain containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:52. In some embodiments, the D1 domain comprises a heavy chain containing 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: LCDR1 comprising the amino acid sequence of SEQ ID NO:12, LCDR2 comprising the amino acid sequence AAS, and LCDR3 comprising the amino acid sequence of SEQ ID NO:16.

[0121] In some embodiments, the D1 domain comprises an LCVR containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:10. In some embodiments, the D1 domain comprises an LCVR containing the amino acid sequence of SEQ ID NO:10.

[0122] In some embodiments, the D1 domain comprises a light chain comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:20. In some embodiments, the D1 domain comprises a light chain comprising the amino acid sequence of SEQ ID NO:20.

[0123] In some embodiments, the D1 domain comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO:24, HCDR2 comprising the amino acid sequence of SEQ ID NO:26, and HCDR3 comprising the amino acid sequence of SEQ ID NO:28. In some embodiments, the D1 domain comprises: HCDR1 composed of the amino acid sequence of SEQ ID NO:24, HCDR2 composed of the amino acid sequence of SEQ ID NO:26, and HCDR3 composed of the amino acid sequence of SEQ ID NO:28. In some embodiments, the D1 domain comprises: HCVR comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:22. In some embodiments, the D1 domain comprises: HCVR comprising or composed of the amino acid sequence of SEQ ID NO:22.

[0124] In some embodiments, the D1 domain comprises a heavy chain containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:42. In some embodiments, the D1 domain comprises a heavy chain containing or consisting of the amino acid sequence of SEQ ID NO:42.

[0125] In some embodiments, the D1 domain comprises a heavy chain containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:46. In some embodiments, the D1 domain comprises a heavy chain containing 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: LCDR1 comprising the amino acid sequence of SEQ ID NO:12, LCDR2 comprising the amino acid sequence AAS, and LCDR3 comprising the amino acid sequence of SEQ ID NO:16.

[0127] In some embodiments, the D1 domain comprises an LCVR containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:10. In some embodiments, the D1 domain comprises an LCVR containing the amino acid sequence of SEQ ID NO:10.

[0128] In some embodiments, the D1 domain comprises a light chain comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:20. In some embodiments, the D1 domain comprises a light chain comprising the amino acid sequence of SEQ ID NO:20.

[0129] In some embodiments, the bispecific antigen-binding molecule comprises D2, which binds to a second epitope of human CD40, wherein the D2 domain comprises a heavy chain immunoglobulin chain comprising: HCDR1 comprising the amino acid sequence of SEQ ID NO:34, HCDR2 comprising the amino acid sequence of SEQ ID NO:36, and HCDR3 comprising the amino acid sequence of SEQ ID NO:38. In some embodiments, the D2 domain comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO:34, HCDR2 comprising the amino acid sequence of SEQ ID NO:36, and HCDR3 comprising the amino acid sequence of SEQ ID NO:38. In some embodiments, the D2 domain comprises: HCVR comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:32. In some embodiments, the D2 domain comprises: HCVR comprising or comprising the amino acid sequence of SEQ ID NO:32.

[0130] In some embodiments, the D2 domain comprises a heavy chain containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:44. In some embodiments, the D2 domain comprises a heavy chain containing or consisting of the amino acid sequence of SEQ ID NO:44.

[0131] In some embodiments, the D2 domain comprises a heavy chain containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:50. In some embodiments, the D2 domain comprises a heavy chain containing 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: LCDR1 comprising the amino acid sequence of SEQ ID NO:12, LCDR2 comprising the amino acid sequence AAS, and LCDR3 comprising the amino acid sequence of SEQ ID NO:16.

[0133] In some embodiments, the D2 domain comprises an LCVR containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:10. In some embodiments, the D2 domain comprises an LCVR containing the amino acid sequence of SEQ ID NO:10.

[0134] In some embodiments, the D2 domain comprises a light chain comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:20. In some embodiments, the D2 domain comprises a light chain comprising the amino acid sequence of SEQ ID NO:20.

[0135] In some embodiments, the bispecific antigen-binding molecule comprises a D1 domain binding to a first epitope of human CD40 and a D2 domain binding to a second epitope of human CD40, wherein the D1 domain comprises a heavy chain immunoglobulin chain comprising: HCDR1 comprising the amino acid sequence of SEQ ID NO:34, HCDR2 comprising the amino acid sequence of SEQ ID NO:36, and HCDR3 comprising the amino acid sequence of SEQ ID NO:38. In some embodiments, the D1 domain comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO:34, HCDR2 comprising the amino acid sequence of SEQ ID NO:36, and HCDR3 comprising the amino acid sequence of SEQ ID NO:38. In some embodiments, the D1 domain comprises: HCVR comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:32. In some embodiments, the D1 domain comprises: an amino acid sequence comprising or consisting of HCVR containing SEQ ID NO:32.

[0136] In some embodiments, the D1 domain comprises a heavy chain containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:58. In some embodiments, the D1 domain comprises a heavy chain containing 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: HCDR1 comprising the amino acid sequence of SEQ ID NO:4, HCDR2 comprising the amino acid sequence of SEQ ID NO:6, and HCDR3 comprising the amino acid sequence of SEQ ID NO:8. In some embodiments, the D2 domain comprises: HCDR1 consisting of the amino acid sequence of SEQ ID NO:4, HCDR2 consisting of the amino acid sequence of SEQ ID NO:6, and HCDR3 consisting of the amino acid sequence of SEQ ID NO:8. In some embodiments, the D2 domain comprises: HCVR comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:2. In some embodiments, the D2 domain comprises: 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 containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:60. In some embodiments, the D2 domain comprises a heavy chain containing or consisting of the amino acid sequence of SEQ ID NO:60.

[0139] In some embodiments, the D1 and / or D2 domains further comprise 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 domains comprise an LCVR comprising an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:10. In some embodiments, the D1 and / or D2 domains comprise an LCVR comprising the amino acid sequence of SEQ ID NO:10. In some embodiments, the D1 and / or D2 domains comprise a light chain containing an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:20. In some embodiments, the D1 and / or D2 domains comprise a light chain containing the amino acid sequence of SEQ ID NO:20.

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

[0141] In some embodiments, the bispecific antigen-binding molecule comprises: The D1 domain comprises: an HCVR containing an amino acid sequence having 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 with SEQ ID NO:2; and an LCVR containing an amino acid sequence having 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 with SEQ ID NO:10; and The D2 domain comprises: an HCVR containing an amino acid sequence having 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 with SEQ ID NO:32, and an LCVR containing an amino acid sequence having 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 with SEQ ID NO:10.

[0142] In some embodiments, the bispecific antigen-binding molecule comprises: a D1 domain comprising: 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 NO:10; and a D2 domain comprising: 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 NO:10.

[0143] In some embodiments, the bispecific antigen-binding molecule comprises: a D1 domain comprising: 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 NO:20; and a D2 domain comprising: 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 NO:20.

[0144] In some embodiments, the bispecific antigen-binding molecule comprises: a D1 domain comprising: 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 NO: 20; and a D2 domain comprising: a heavy chain comprising or consisting of the 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: The D1 domain, which binds to the first epitope of human CD40, comprises: HCDR1 containing the amino acid sequence of SEQ ID NO:24, HCDR2 containing or consisting of the amino acid sequence of SEQ ID NO:26, HCDR3 containing or consisting of the amino acid sequence of SEQ ID NO:28, LCDR1 containing or consisting of the amino acid sequence of SEQ ID NO:12, LCDR2 containing or consisting of the amino acid sequence AAS, and LCDR3 containing or consisting of the amino acid sequence of SEQ ID NO:16; and The D2 domain, which binds to the second epitope of human CD40, comprises: HCDR1 containing or consisting of the amino acid sequence of SEQ ID NO:34, HCDR2 containing or consisting of the amino acid sequence of SEQ ID NO:36, HCDR3 containing or consisting of the amino acid sequence of SEQ ID NO:38, LCDR1 containing or consisting of the amino acid sequence of SEQ ID NO:12, LCDR2 containing or consisting of the amino acid sequence AAS, and LCDR3 containing or consisting of the amino acid sequence of SEQ ID NO:16.

[0146] In some embodiments, the bispecific antigen-binding molecule comprises: The D1 domain comprises: an HCVR containing an amino acid sequence having 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 with SEQ ID NO:2; and an LCVR containing an amino acid sequence having 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 with SEQ ID NO:10; and The D2 domain comprises: an HCVR containing an amino acid sequence having 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 with SEQ ID NO:32, and an LCVR containing an amino acid sequence having 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 with SEQ ID NO:10.

[0147] In some embodiments, the bispecific antigen-binding molecule comprises: a D1 domain comprising: 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 NO:10; and a D2 domain comprising: 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 NO:10.

[0148] In some embodiments, the bispecific antigen-binding molecule comprises: a D1 domain comprising: 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 comprising: 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 NO:20.

[0149] In some embodiments, the bispecific antigen-binding molecule comprises: a D1 domain comprising: 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 NO:20; and a D2 domain comprising: a heavy chain comprising or consisting of the 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.

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

[0151] In some embodiments, the bispecific antigen-binding molecule comprises: The D1 domain comprises: an HCVR containing an amino acid sequence having 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 with SEQ ID NO:32; and an LCVR containing an amino acid sequence having 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 with SEQ ID NO:10; and The D2 domain comprises: an HCVR containing an amino acid sequence having 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 with SEQ ID NO:2, and an LCVR containing an amino acid sequence having 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 with SEQ ID NO:10.

[0152] In some embodiments, the bispecific antigen-binding molecule comprises: a D1 domain comprising: 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 NO:10; and a D2 domain comprising: 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 NO:10.

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

[0154] The bispecific antigen-binding molecules disclosed herein can be bispecific antibodies. In some cases, bispecific antibodies contain the constant region of the human IgG heavy chain. In some cases, the constant region of the human IgG heavy chain is isotype IgG1. In some cases, the constant region of the human IgG heavy chain is isotype IgG4.

[0155] The first antigen-binding domain and the second antigen-binding domain may be directly or indirectly linked to each other to form the bispecific antigen-binding molecule of the present invention. Alternatively, the first antigen-binding domain and the second antigen-binding domain may each be linked to a separate polymerizing domain. Association of one polymerizing domain with the other promotes association between the two antigen-binding domains, thereby forming a bispecific antigen-binding molecule. As used herein, "polymerizing domain" means any macromolecule, protein, polypeptide, peptide, or amino acid capable of associating with a second polymerizing domain having the same or similar structure or composition. For example, the polymerizing domain may be a polypeptide containing the immunoglobulin CH3 domain. Non-limiting examples of polymerizing components are the Fc portion of immunoglobulins (containing the CH2-CH3 domain), for example, the Fc domain selected from isotypes IgG1, IgG2, IgG3, and IgG4, and any allotype of IgG within each isotype group.

[0156] In some embodiments, the bispecific antigen-binding molecule of this disclosure comprises two polymerizing domains, such as two Fc domains, each of which is individually part of a separate antibody heavy chain. The first and second polymerizing domains may be the same IgG isoform, such as, for example, IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4. Alternatively, the first and second polymerizing domains may be different IgG isoforms, such as, for example, IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0157] In some embodiments, the polymerizing domain is an Fc fragment or amino acid sequence of 1 to 200 amino acids in length, containing at least one cysteine ​​residue. In other embodiments, the polymerizing domain is a cysteine ​​residue, or a short peptide containing cysteine. Other polymerizing domains include peptides or polypeptides comprising leucine zippers, helical-cyclic motifs, or coiled-helical motifs, or combinations thereof.

[0158] In some embodiments, the bispecific antigen-binding molecule is a CD40xCD40 bispecific antibody having at least 85% sequence identity with REGN16334, such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In some embodiments, the bispecific antigen-binding molecule is a CD40xCD40 bispecific antibody having the amino acid sequence of REGN16334.

[0159] In some embodiments, the bispecific antigen-binding molecule is a CD40xCD40 bispecific antibody having at least 85% sequence identity with REGN16335, such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In some embodiments, the bispecific antigen-binding molecule is a bispecific antibody having the amino acid sequence of REGN16335.

[0160] In some embodiments, the bispecific antigen-binding molecule is a CD40xCD40 bispecific antibody having at least 85% sequence identity with REGN16431, such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In some embodiments, the bispecific antigen-binding molecule is a CD40xCD40 bispecific antibody having the amino acid sequence of REGN16431.

[0161] In some embodiments, the bispecific antigen-binding molecule is a CD40xCD40 bispecific antibody having at least 85% sequence identity with REGN16432, such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In some embodiments, the bispecific antigen-binding molecule is a CD40xCD40 bispecific antibody having the amino acid sequence of REGN16432.

[0162] In some embodiments, the bispecific antigen-binding molecule is a CD40xCD40 bispecific antibody having at least 85% sequence identity with REGN20484, such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In some embodiments, the bispecific antigen-binding molecule is a CD40xCD40 bispecific antibody having the amino acid sequence of REGN20484.

[0163] Sequence variants Compared to the corresponding germline sequences derived from individual antigen-binding domains, the antigen-binding molecules of this disclosure may contain one or more amino acid substitutions, insertions, and / or deletions in the frame and / or CDR regions of the heavy chain and / or light chain variable domains. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available from, for example, public antibody sequence databases. The antigen-binding molecules of this disclosure may contain antigen-binding fragments derived from any of the exemplary amino acid sequences disclosed herein, wherein one or more amino acids in one or more frame and / or CDR regions are mutated to corresponding residues in the germline sequence of the derived antibody, or mutated to corresponding residues in another human germline sequence, or mutated to conserved amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy chain and light chain variable region sequences disclosed herein, those skilled in the art can readily generate numerous antibody and antigen-binding fragments containing one or more single germline mutations or combinations thereof. In some embodiments, V H and / or V L All frame and / or CDR residues within the domain are mutated back to residues found in the original germline sequence of the originally derived antigen-binding domain. In other embodiments, only certain residues are mutated back to the original germline sequence, for example, mutated residues found only in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or mutated residues found only in CDR1, CDR2, or CDR3. In other embodiments, one or more frame and / or CDR residues are mutated to different germline sequences. (Right nowThe antigen-binding domain may contain corresponding residues of a germline sequence (different from the germline sequence of the originally derived antigen-binding domain). Furthermore, the antigen-binding domain may contain any combination of two or more germline mutations within the framework and / or CDR region, for example, where certain individual residues are mutated to corresponding residues of a specific germline sequence, while certain other residues different from the original germline sequence are maintained or mutated to corresponding residues of a different germline sequence. Once obtained, one or more desired properties of the antigen-binding domain containing one or more germline mutations can be readily tested, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties, reduced immunogenicity, etc. Bispecific antigen-binding molecules containing one or more antigen-binding domains obtained in this general manner are covered within this disclosure.

[0164] This disclosure also includes antigen-binding molecules in which one or both antigen-binding domains comprise variants of any of the disclosed HCVR, LCVR, and / or CDR amino acid sequences having one or more conserved substitutions. For example, this disclosure includes antigen-binding molecules comprising an antigen-binding domain having an HCVR, LCVR, and / or CDR amino acid sequence having, for example, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 conserved amino acid substitution relative to any of the disclosed HCVR, LCVR, and / or CDR amino acid sequences. A “conserved amino acid substitution” is an amino acid substitution in which an amino acid residue is replaced by another amino acid residue of a side chain (R group) having similar chemical properties (e.g., charge or hydrophobicity). Generally, conserved amino acid substitutions will not substantially alter the functional properties of the protein. Examples of amino acid groups with side chains having similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains are cysteine ​​and methionine. Preferred conservative amino acid substituents are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, conservative substitutions are found in Gonnet... et al. Any variation with positive values ​​in the PAM250 log-likelihood matrix disclosed in (1992) Science 256: 1443-1445. “Moderately conservative” is replaced by any variation with non-negative values ​​in the PAM250 log-likelihood matrix.

[0165] This disclosure also includes antigen-binding molecules comprising an antigen-binding domain, the HCVR, LCVR, and / or CDR amino acid sequences of which are substantially identical to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. In some embodiments, the antigen-binding molecule comprises HCVR, LCVR, and / or CDR amino acid sequences having at least 85% sequence identity with the sequences disclosed in Table 36, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequences disclosed in Table 36. In some embodiments, the antigen-binding molecule comprises HCVR, LCVR, and / or CDR amino acid sequences having at least 85% sequence identity with the sequences disclosed in Table 36, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequences disclosed in Table 36, wherein the differences in amino acid residues relative to the sequences disclosed in Table 36 are conserved substitutions or moderately conserved substitutions.

[0166] Antigen-binding proteins containing Fc modification In some embodiments, the CD40 antigen-binding molecules disclosed herein (e.g., CD40xCD40 bispecific antigen-binding molecules, such as those disclosed in any of Tables 1 to 4) include an Fc domain containing one or more modifications or mutations that enhance or weaken antibody binding to the FcRn receptor. For example, this disclosure includes antigen-binding molecules containing one or more mutations in the CH2 and / or CH3 regions of the Fc domain, wherein the mutations increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., in endosomes with a pH range of about 5.5 to about 6.0). When administered to animals, such mutations can result in an increased serum half-life of the antibody.

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

[0168] In some embodiments, the CD40xCD40 bispecific antigen-binding molecule comprises an Fc domain containing one or more mutation pairs or groups selected from the group consisting of: 250Q and 248L ( For example, T250Q and M248L); 252Y, 254T and 256E ( For example, M252Y, S254T and T256E; 428L and 434S ( For example, M428L and N434S); and 433K and 434F ( For example, H433K and N434F).

[0169] In some embodiments, the CD40xCD40 bispecific antigen-binding molecule of this disclosure comprises a modified Fc domain having reduced effector function. As used herein, “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 containing the modified Fc exhibits a reduced severity or degree 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 comparative molecule containing a wild-type naturally occurring Fc portion. In some embodiments, “modified Fc domain having reduced effector function” is an Fc domain with reduced or weakened binding to an Fc receptor (e.g., FcγR).

[0170] In some embodiments, the modified Fc domain with reduced binding to an Fc receptor (e.g., an Fcγ receptor, such as FcγRI, FcγRIIA, FcγRIIB, or FcγRIIIA) is a variant IgG1 Fc or variant IgG4 Fc containing one or more substitutions or modifications in the hinge region and / or CH region (e.g., CH2). For example, the modified Fc domain may contain a variant IgG1 Fc, wherein at least one amino acid in the IgG1 Fc hinge region and / or CH region is replaced by a corresponding amino acid from the IgG2 Fc hinge region and / or CH region. Alternatively, the modified Fc domain may contain a variant IgG4 Fc, wherein at least one amino acid in the IgG4 Fc hinge region and / or CH region is replaced by a corresponding amino acid from the IgG2 Fc hinge region and / or CH region. In some embodiments, the modified Fc domain contains a modification in which each of positions 233-236 by EU numbering is G-occupied or unoccupied. Non-limiting exemplary modified Fc regions that may be used in the context of this disclosure are set forth in U.S. Patent No. 11,518,807, the disclosure of which is hereby incorporated by reference in its entirety, as well as any functionally equivalent variations of the modified Fc regions set forth therein. Other modified Fc domains and Fc modifications that may be used in the context of this disclosure include any modifications 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 disclosure of each of which is incorporated herein by reference.

[0171] In some embodiments, the bispecific antigen-binding molecules disclosed herein comprise immunoglobulin heavy chains that are heterodimers (i.e., differing from each other by at least one amino acid) and have different affinities for affinity reagents such as protein A. In some embodiments, one of the heavy chains includes one or more modifications in its Fc domain that reduce or eliminate the binding of the Fc domain to protein A. In some embodiments, one of the heavy chains includes H435R / Y436F (via the EU numbering system) substitution in its CH3 region. Non-limiting exemplary modified Fc regions that may be used in the context of this disclosure are set forth in U.S. Patent No. 8,586,713, the entire disclosure of which is hereby incorporated by reference.

[0172] All possible combinations of the aforementioned Fc domain mutations and other mutations within the antibody variable domains disclosed herein are envisioned within the scope of this disclosure.

[0173] Polynucleotides, vectors and host cells On the other hand, this disclosure provides nucleic acid molecules comprising one or more polynucleotide sequences encoding antigen-binding molecules disclosed herein, as well as vectors (e.g., expression vectors) encoding such polynucleotide sequences and host cells in which such vectors have been introduced.

[0174] The polynucleotides disclosed herein may encode all or part of the antigen-binding molecules, antibodies, or antigen-binding fragments disclosed herein. In some cases, a single polynucleotide may encode both the HCVR and LCVR of the antibody or antigen-binding fragment (e.g., referring to the CDR definitions contained within the respective amino acid sequence definitions of HCVR and LCVR, referring to the amino acid sequence definitions of the CDRs of HCVR and LCVR respectively, or referring to the amino acid sequence definitions of HCVR and LCVR respectively), or the HCVR and LCVR may be encoded by a single polynucleotide (i.e., a pair of polynucleotides). In the latter case, where the HCVR and LCVR are encoded by a single polynucleotide, the polynucleotides may be combined in a single vector or may be contained in a single vector (i.e., a pair of vectors). In any case, the host cell used to express the polynucleotide or vector may contain all the components for generating the component portion of the antibody or its antigen-binding fragment. For example, the host cell may contain separate vectors, each encoding the HCVR and LCVR of the antibody or its antigen-binding fragment as discussed above or herein. Similarly, one or more polynucleotides and one or more vectors can be used to express the full-length heavy chain and full-length light chain of antibodies as discussed above or herein. For example, a host cell may contain a single vector having polynucleotides encoding both the heavy chain and light chain of an antibody, or a host cell may contain a separate vector having polynucleotides encoding the heavy chain and 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 to 4. In some embodiments, the nucleic acid molecule comprises one or more polynucleotide sequences listed in Table 36.

[0176] In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence encoding CD40 HCVR, wherein the CD40 HCVR comprises HCDR1, HCDR2, and HCDR3 as specified in SEQ ID NOs: 4, 6, and 8, SEQ ID NOs: 24, 26, and 28, or SEQ ID NOs: 34, 36, and 38, respectively. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence encoding CD40 HCVR, wherein the CD40 HCVR comprises or is composed 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 encoding an LCVR, the LCVR comprising: LCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO:12, LCDR2 comprising the amino acid sequence AAS, and LCDR3 comprising the amino acid sequence of SEQ ID NO:16. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence encoding an LCVR, the LCVR comprising the sequence of SEQ ID NO:10.

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

[0179] On the other hand, this disclosure also provides recombinant expression vectors carrying one or more nucleic acid molecules as disclosed herein, and host cells in 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., Chinese hamster ovary (CHO) cell). A method for generating the antigen-binding molecules of this disclosure is also provided by culturing the host cell under conditions that allow for the generation of antigen-binding molecules and recovering the antigen-binding molecules thus generated.

[0180] Characterization of CD40xCD40 bispecific antigen-binding molecule This disclosure includes antibodies and antigen-binding fragments thereof that bind to human CD40 with high affinity, such as bispecific antigen-binding molecules that bind to two different epitopes of CD40. In some embodiments, the antibody and its antigen-binding fragment (e.g., a bispecific antigen-binding molecule) bind to CD40 and inhibit CD40L-induced activation, but do not have agonist activity and / or cytotoxic effector function.

[0181] In some embodiments, this disclosure includes bispecific antigen-binding molecules (e.g., bispecific antibodies as disclosed herein, such as bispecific antibodies having the amino acid sequence REGN16334, REGN16335, REGN16431, REGN16432, or REGN20484) that bind to human CD40. （ For example, at 25°C or 37°C), K D Less than about 75 nM, such as through surface plasmon resonance ( For example, Measured using the assay method described in Example 2 of this document. In some embodiments, the antigen-binding molecule of this disclosure binds to human CD40,K D Less than approximately 75 nM, less than approximately 70 nM, less than approximately 60 nM, less than approximately 50 nM, less than approximately 40 nM, less than approximately 30 nM, less than approximately 25 nM, less than approximately 20 nM, less than approximately 15 nM, less than approximately 10 nM, less than approximately 5 nM, less than approximately 1 nM, less than approximately 500 pM, less than approximately 400 pM, less than approximately 300 pM, less than approximately 200 pM, less than approximately 100 pM, less than approximately 90 pM, less than approximately 80 pM, less than approximately 70 pM, less than approximately 60 pM, less than approximately 50 pM, less than approximately 40 pM, less than approximately 30 pM, less than approximately 20 pM, less than approximately 10 pM, less than approximately 5 pM, less than approximately 4 pM, less than approximately 2 pM, less than approximately 1 pM, less than approximately 0.5 pM, less than approximately 0.2 pM, less than approximately 0.1 pM, or less than approximately 0.05 pM. pM, such as by surface plasmon resonance (e.g., using a measurement form as defined in Example 2 of this document or a substantially similar measurement).

[0182] In some embodiments, this disclosure includes bispecific antigen-binding molecules (e.g., bispecific antibodies disclosed herein, such as those having the amino acid sequence REGN16334, REGN16335, REGN16431, REGN16432, or REGN20484) that have an improved half-life compared to monospecific antibodies (e.g., parental CD40 antibodies). In some embodiments, this disclosure includes bispecific antigen-binding molecules that bind to human CD40 (e.g., bispecific antibodies disclosed herein), with a dissociation half-life (t... ½ The time is greater than approximately 70 minutes, as measured at 25°C by surface plasmon resonance (e.g., using an assay format as defined in Example 2 herein or a substantially similar assay). In some embodiments, the antibody or antigen-binding fragment of this disclosure binds to human CD40, t ½ Greater than about 75 minutes, greater than about 80 minutes, greater than about 85 minutes, or greater than about 90 minutes, as measured at 25°C by surface plasmon resonance (e.g., using a measurement format as defined in Example 2 of this document or a substantially similar measurement).

[0183] In some embodiments, this disclosure includes a bispecific antigen-binding molecule (e.g., a bispecific antibody as disclosed herein, such as a bispecific antibody having the amino acid sequence REGN16334, REGN16335, REGN16431, REGN16432, or REGN20484) that inhibits the binding of human CD40 (e.g., a monomeric form of hCD40) to human CD40L. In some embodiments, the inhibition of CD40-CD40L binding is measured using an ELISA-based blocking assay as described in Example 4 herein. In some embodiments, the bispecific antigen-binding molecule inhibits the binding of human CD40 (e.g., a monomeric form of hCD40) to human CD40L by at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, or more, for example, using an assay format as defined in Example 4 herein or a substantially similar assay.

[0184] In some embodiments, this disclosure includes bispecific antigen-binding molecules (e.g., bispecific antibodies disclosed herein, such as those having the amino acid sequence REGN16334, REGN16335, REGN16431, REGN16432, or REGN20484) that specifically interact (e.g., bind) with cells expressing CD40. The extent to which the antigen-binding molecule binds to cells expressing CD40 can be assessed by flow cytometry, as shown in Example 5 below. For example, in some embodiments, this disclosure provides a CD40xCD40 bispecific antibody that specifically binds to cells expressing CD40 on their cell surface (e.g., primary human B cells or human B cell lines, such as Ramos 2G6.4C10). In some embodiments, this disclosure provides a CD40xCD40 bispecific antibody, EC, that binds to cells or cell lines expressing CD40. 50 Values ​​of approximately 10 nM or less, for example, approximately 0.5 nM to approximately 10 nM, for example, EC 50 The concentrations were approximately 1 nM, approximately 1.5 nM, approximately 2 nM, approximately 2.5 nM, approximately 3 nM, approximately 3.5 nM, approximately 4 nM, approximately 4.5 nM, approximately 5 nM, approximately 5.5 nM, approximately 6 nM, approximately 6.5 nM, approximately 7 nM, approximately 7.5 nM, approximately 8 nM, approximately 8.5 nM, approximately 9 nM, approximately 9.5 nM, or approximately 10 nM, as determined by flow cytometry as described in Example 5 or a substantially similar assay.

[0185] In some embodiments, this disclosure includes bispecific antigen-binding molecules (e.g., bispecific antibodies as disclosed herein, such as bispecific antibodies having the amino acid sequence REGN16334, REGN16335, REGN16431, REGN16432, or REGN20484) that inhibit CD40L-induced activation. In some embodiments, CD40L-induced activation is measured using a reporter gene assay (such as a luciferase-based reporter gene assay) that quantitatively assesses receptor activation in CD40-expressing cells by measuring downstream gene expression. In some embodiments, the reporter gene assay is the assay described in Example 6 herein. In some embodiments, CD40L-induced activation is measured by a cytokine secretion assay (e.g., secretion of IL-6, IL-10, IL-23, or TNFα) in the presence of CD40L. In some embodiments, the cytokine secretion assay is performed in primary cells expressing CD40 (e.g., human B cells). In some embodiments, the cytokine secretion assay is performed in stable cell lines (e.g., cell lines expressing both CD40 and a reporter gene, such as luciferase). In some embodiments, the cytokine secretion assay is the assay described in Example 7 of this document. 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 reference value.

[0186] In some embodiments, this disclosure includes bispecific antigen-binding molecules that do not significantly activate CD40 in the absence of CD40L (e.g., bispecific antibodies disclosed herein, such as bispecific antibodies having the amino acid sequence REGN16334, REGN16335, REGN16431, REGN16432, or REGN20484). As used herein, “does not significantly activate CD40 in the absence of CD40L” means that in the presence of a bispecific antigen-binding molecule and in the absence of CD40L, the activation level of CD40 is less than 15%, for example less than 13%, less than 10%, less than 8%, or less than 6%, for example, as measured by downstream gene expression or cytokine secretion. In some embodiments, the agonistic effect of CD40 is measured by a reporter gene assay (such as a luciferase-based reporter gene assay) that quantitatively assesses receptor activation in cells expressing CD40 by measuring downstream gene expression. In some embodiments, the agonistic effect of CD40 is measured by an assay of cytokine secretion (e.g., secretion of IL-6, IL-10, IL-23, or TNFα) in the absence of CD40L. In some embodiments, the assay is the one described in Example 6 or Example 8 herein.

[0187] Epitope plotting and related techniques In some embodiments, the epitopes on CD40 bound by the antigen-binding molecule of this disclosure (e.g., the first epitope of human CD40 bound by the first antigen-binding domain (D1) or the second epitope of human CD40 bound by the second antigen-binding domain (D2)) may be composed of three or more epitopes of the CD40 protein. (For example, Epitopes consist of a single, continuous sequence of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids. Alternatively, an epitope may consist of multiple non-continuous amino acids (or amino acid sequences) of CD40. As used herein, the term “epitope” refers to an antigenic determinant that interacts with a specific antigen-binding site called a complementary site in the variable region of an antibody molecule. A single antigen may have more than one epitope. Thus, different antibodies can bind to different regions on the antigen and can have different biological effects. Epitopes can be conformational or linear. Conformational epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are epitopes generated from adjacent amino acid residues in a polypeptide chain. In some cases, an epitope may contain a sugar, phosphoryl, or sulfonyl moiety on the antigen.

[0188] Various techniques known to those skilled in the art can be used to determine whether an antibody's antigen-binding domain "interacts with one or more amino acids" within a peptide or protein. Exemplary techniques that can be used to determine epitopes or binding domains of a particular antibody or antigen-binding domain include, for example, conventional cross-blocking assays, such as those used in... Antibodies Those point mutagenesis described in Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY) (e.g., alanine scanning mutagenesis, arginine scanning mutagenesis) wait Peptide blot analysis (Reineke, 2004) Methods Mol Biol 248:443-463), protease protection, and peptide cleavage analysis. Additionally, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify amino acids within peptides that interact with antibodies is hydrogen / deuterium exchange detected by mass spectrometry. Generally, the hydrogen / deuterium exchange method involves deuterating the protein of interest, followed by binding an antibody to the deuterated protein. Next, the protein / antibody complex is transferred to water to allow hydrogen-deuterium exchange to occur at all residues except those protected by the antibody (which remain deuterated). After dissociation of the antibody, the target protein is cleaved by a protease and analyzed by mass spectrometry, thereby revealing the deuterated residues corresponding to the specific amino acids that interact with the antibody. See, for example, Ehring (1999). Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal.Chem.73 :256A-265A. X-ray crystal structure analysis can also be used to identify amino acids within peptides that interact with antibodies.

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

[0190] In some embodiments, this disclosure provides a CD40xCD40 bispecific antibody comprising a first antigen-binding domain (D1) that binds to a first epitope of human CD40 and a second antigen-binding domain (D1) that binds to a second epitope of human CD40, wherein the first 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, this disclosure provides a CD40xCD40 bispecific antibody comprising a first antigen-binding domain (D1) that binds to a first epitope of human CD40 and a second antigen-binding domain (D2) that binds to a second epitope of human CD40, wherein D1 and D2 do not compete with each other for binding to human CD40.

[0192] Those skilled in the art can determine specific antigen-binding molecules using conventional methods known in the art. （ For example, whether the antibody or its antigen-binding domain binds to the same epitope as or competes with the reference antigen-binding molecule of this disclosure. For example, to determine whether a test antibody binds to the same epitope on CD40 as the reference bispecific antigen-binding molecule of this disclosure, the reference bispecific molecule is first bound to the CD40 protein. Next, the ability of the test antibody to bind to the CD40 molecule is evaluated. If, after saturation binding with the reference bispecific antigen-binding molecule, the test antibody is able to bind to CD40, it can be concluded that the test antibody binds to a different epitope on CD40 from the reference bispecific antigen-binding molecule. On the other hand, if, after saturation binding with the reference bispecific antigen-binding molecule, the test antibody cannot bind to the CD40 molecule, the test antibody may bind to the same CD40 epitope as the epitope bound by the reference bispecific antigen-binding molecule of this disclosure. Further routine experiments can then be performed. （ For example, peptide mutation and binding assays can be used to confirm whether the observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference bispecific antigen-binding molecule, or whether steric hindrance (or another phenomenon) is the cause of the observed lack of binding. Such experiments can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. According to certain embodiments of this disclosure, if, for example, an excess of 1, 2, 5, 10, 20, or 100 times of one antigen-binding protein inhibits the binding of another antigen-binding protein by at least 50%, but preferably 75%, 90%, or even 99%, as measured in a competitive binding assay, then both antigen-binding proteins bind to the same (or overlapping) epitope (see, for example, Junghans et al.). Cancer Res(1990:50:1495-1502). Alternatively, if substantially all amino acid mutations in an antigen that reduce or eliminate the binding of one antigen-binding protein reduce or eliminate the binding of another antigen-binding protein, then the two antigen-binding proteins are considered to bind to the same epitope. If only a subset of amino acid mutations that reduce or eliminate the binding of one antigen-binding protein reduces or eliminates the binding of another antigen-binding protein, then the two antigen-binding proteins are considered to have “overlapping epitopes.”

[0193] To determine whether an antibody or its antigen-binding domain competes with a reference antigen-binding molecule for binding, the above binding method is performed in two orientations: In the first orientation, the reference antigen-binding molecule is bound to the CD40 protein under saturation conditions, and then the binding of the test antibody to the CD40 molecule is evaluated. In the second orientation, the test antibody is bound to the CD40 molecule under saturation conditions, and then the binding of the reference antigen-binding molecule to the CD40 molecule is evaluated. If, in both orientations, only the first (saturated) antigen-binding molecule can bind to the CD40 molecule, it is concluded that the test antibody and the reference antigen-binding molecule compete for binding to CD40. As will be understood by those skilled in the art, an antibody competing for binding with a reference antigen-binding molecule may not necessarily bind to the same epitope as the reference antibody, but the binding of the reference antibody can be spatially blocked by binding to overlapping or adjacent epitopes.

[0194] Preparation of antigen-binding domains and construction of multispecific antigen-binding molecules Antigen-binding domains specific to a particular antigen can be prepared using any antibody-generating technique known in the art. Once obtained, two distinct antigen-binding domains can be suitably arranged relative to each other using conventional methods to produce the bispecific antigen-binding molecule of this disclosure. (A discussion of exemplary bispecific antibody forms that can be used to construct the bispecific antigen-binding molecule of this disclosure is provided elsewhere herein.) In some embodiments, individual components of a multispecific antigen-binding molecule （ For example, one or more of the heavy and light chains are derived from chimeric antibodies, humanized antibodies, or fully human antibodies. Methods for preparing such antibodies are well known in the art. For example, VELOCIMMUNE can be used. TM The technique involves preparing one or more of the heavy and / or light chains of the bispecific antigen-binding molecule disclosed herein. VELOCIMMUNE is used. TM The technology (or any other human antibody generation technology) initially isolates a specific antigen with a human variable region and a mouse constant region. （For example, high-affinity chimeric antibodies (CD40). Antibodies are characterized and selected for desired properties (including affinity, selectivity, epitopes, etc.). Mouse constant regions are replaced with desired human constant regions to generate fully human heavy and / or light chains that can be incorporated into bispecific antigen-binding molecules.

[0195] In some embodiments, genetically engineered animals can be used to prepare human bispecific antigen-binding molecules. For example, genetically modified mice that cannot rearrange and express endogenous mouse immunoglobulin light chain variable sequences can be used, wherein the mice express only one or two human light chain variable domains encoded by human immunoglobulin sequences operatively linked to the mouse κ constant gene at the endogenous mouse κ locus. Such genetically modified mice can be used to generate fully human bispecific antigen-binding molecules comprising two distinct heavy chains associated with the same light chain, the same light chain containing variable domains derived from one of two distinct human light chain variable region gene segments. （ See, for example, US 2011 / 0195454, the entire contents of which are incorporated herein by reference for a detailed discussion of such engineered mice and their use in generating bispecific antigen-binding molecules. As used herein, “fully human” means an antigen-binding molecule, such as an antibody or its antigen-binding fragment or immunoglobulin domain, comprising an amino acid sequence encoded by DNA derived from a human sequence along the full length of each polypeptide of the antigen-binding molecule, antibody, antigen-binding fragment, or its immunoglobulin domain. In some cases, the fully human sequence is derived from a human endogenous protein. In other cases, the fully human protein or protein sequence comprises a chimeric sequence in which each component sequence is derived from a human sequence. While not bound by any one theory, chimeric proteins or chimeric sequences are generally designed to minimize the generation of immunogenic epitopes at the component sequence junctions, for example, compared to any wild-type human immunoglobulin region or domain.

[0196] bioequivalent This disclosure covers antigen-binding molecules having an amino acid sequence that differs from the amino acid sequence of the antibody but retains the ability to bind CD40. Such variant molecules contain one or more additions, deletions, or substitutions of amino acids when compared to the parental sequence, but exhibit biological activity substantially equivalent to that of the antigen-binding molecule described herein. Similarly, nucleic acid sequences encoding antigen-binding molecules of this disclosure cover sequences containing one or more additions, deletions, or substitutions of nucleotides when compared to the disclosed sequences, but encoding antigen-binding molecules substantially bioequivalent to those disclosed herein.

[0197] This disclosure includes antigen-binding molecules that are bioequivalent to any of the exemplary antigen-binding molecules listed herein. If, for example, two antigen-binding proteins (e.g., bispecific antibodies) are pharmaceutical equivalents or substitutes that do not show significant differences in absorption rate and extent when administered at the same molar dose (single or multiple doses) under similar experimental conditions, they are considered bioequivalent. If some antibodies are equivalent in extent of absorption but not in rate of absorption, the antibody is considered an equivalent or substitute, and can still be considered bioequivalent because such differences in absorption rate are intentional and reflected in the label, are not necessary for achieving effective bodily drug concentrations with prolonged use, and are considered medically irrelevant to the specific pharmaceutical product under investigation.

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

[0199] In one embodiment, if a patient can switch between a first antigen-binding protein (e.g., a reference product) and a second antigen-binding protein (e.g., a biologic) once or more without an expected increased risk of side effects (including clinically significant changes in immunogenicity or reduced efficacy) compared to continuous therapy without such switching, then the two antigen-binding proteins are bioequivalent.

[0200] In one embodiment, two antigen-binding proteins are bioequivalent if they both function through one or more co-operating mechanisms targeting one or more conditions of use (provided those mechanisms are known).

[0201] Bioequivalence can be achieved through in vivo and in vitro Methods for proof. Non-limiting examples of bioequivalence measurements include, for example, (a) in humans or other mammals. in vivo (a) Testing in which the concentration of an antibody or its metabolites changes over time in blood, plasma, serum or other biological fluids; (b) Correlation with and reasonable prediction of bioavailability data in the human body. in vitro (c) In humans or other mammals in vivo The study includes (d) the measurement of the appropriate acute pharmacological effects of an antibody (or its target) over time; and (d) in a well-controlled clinical trial establishing the safety, efficacy, bioavailability, or bioequivalence of the antibody.

[0202] Bioequivalent variants of the exemplary bispecific antigen-binding molecules listed herein can be constructed, for example, by various substitutions of residues or sequences, or by deleting terminal or internal residues or sequences required for non-biological activity. For example, cysteine ​​residues required for non-biological activity can be deleted or substituted with other amino acids to prevent the formation of unwanted or incorrect intramolecular disulfide bridges during renaturation. In other embodiments, bioequivalent antibodies may comprise the exemplary bispecific antigen-binding molecules listed herein, comprising amino acid changes that modify the glycosylation characteristics of the antibody, such as mutations that eliminate or remove glycosylation.

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

[0204] In some embodiments, the pharmaceutical composition comprises an injectable formulation, such as a dosage form for intravenous, subcutaneous, intradermal, and intramuscular injection, infusion, etc. These injectable formulations can be prepared by known methods. For example, injectable formulations can be prepared, for instance, by dissolving, suspending, or emulsifying the aforementioned antibody or its salt in a sterile aqueous or oily medium conventionally used for injection. As an aqueous medium for injection, such as physiological saline, isotonic solutions containing glucose and other adjuvants, they can be used in combination with suitable solubilizers (such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (a polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As an oily medium, such as sesame oil, soybean oil, etc., they can be used in combination with solubilizers (such as benzyl benzoate, benzyl alcohol, etc.). The resulting injectable formulation can be filled into a suitable ampoule.

[0205] The dosage of the antigen-binding molecule administered to a patient according to this disclosure can vary depending on the patient's age and body size, symptoms, condition, route of administration, etc. Dosage is typically calculated based on weight or body surface area. The frequency and duration of treatment can be adjusted depending on the severity of the condition. Effective dosages and dosing regimens for administering the pharmaceutical compositions disclosed herein can be determined empirically; for example, by periodically assessing and monitoring patient progress and adjusting the dosage accordingly. Furthermore, interspecies scaling of dosages can be performed using methods well known 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 drug compositions. For example Encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, for example, Wu et al., 1987). J. Biol.Chem. 262:4429-4432). Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered via any convenient route, such as by infusion or bolus, or by absorption through the epithelial or mucosal lining of the skin. For example (Oral mucosa, rectal mucosa, and intestinal mucosa, etc.), and may be administered in combination with other biologically active agents. In some embodiments, the pharmaceutical compositions disclosed herein are administered intravenously. In some embodiments, the pharmaceutical compositions disclosed herein are administered subcutaneously.

[0207] In some embodiments, antigen-binding molecules, such as those disclosed herein, or pharmaceutical compositions comprising antigen-binding molecules are contained within a container. Therefore, in another aspect, containers comprising antigen-binding molecules or pharmaceutical compositions disclosed herein are provided. For example, in some embodiments, antibody or pharmaceutical compositions are contained within a container selected from the group consisting of glass vials, syringes, pen delivery devices, and autoinjectors.

[0208] In some embodiments, the antigen-binding molecules or pharmaceutical compositions of this disclosure are delivered subcutaneously or intravenously using standard needles and syringes. In some embodiments, the syringe is a pre-filled syringe. In some embodiments, a pen delivery device or an autoinjector is used to deliver the pharmaceutical compositions of this disclosure (e.g., for subcutaneous delivery). The pen delivery device can be reusable or disposable. Reusable pen delivery devices typically utilize a replaceable cartridge containing the pharmaceutical composition. Once all the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Instead, the disposable pen delivery device is pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

[0209] Examples of suitable pen-based and auto-injector 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), NOVOPENJUNIOR™ (Novo Nordisk, Copenhagen, Denmark), and BD™ pen (Becton Dickinson, Franklin Lakes, New Jersey). OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTIPEN STARLET™ (Sanofi-Aventis, Frankfurt, Germany). Examples of disposable pen delivery devices for subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, the SOLOSTAR™ pen (Sanofi-Aventis), FLEXPEN™ (Novo Nordisk), KWIKPEN™ (Eli Lilly), and SURECLICK.TM Automatic injectors (Amgen, Thousand Oaks, CA), PENLET TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA) TM Pen (Abbott Labs, Abbott Park IL, Illinois).

[0210] In some embodiments, a controlled-release system is used to deliver the drug composition. In one embodiment, a pump can be used (see Langer, ibid.; Sefton, 1987). CRC Crit.Ref. Biomed.Eng. 14:201). In another embodiment, polymeric materials may be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, the controlled-release system may be placed near the target of the composition, thus requiring only a portion of the systemic dose (see...). For example Other controlled release systems are discussed in Goodson's 1984 review in Medical Applications of Controlled Release, ibid., Vol. 2, pp. 115-138. Langer, 1990, Science 249: 1527-1533.

[0211] In some embodiments, the pharmaceutical composition used as described herein is prepared into a dosage form suitable for containing a unit dose of the active ingredient. Such unit dose dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. In some embodiments, the dosage form contains an amount of antigen-binding molecule of about 5 to about 1000 mg, for example, about 5 to about 500 mg, about 5 to about 100 mg, or about 10 to about 250 mg.

[0212] Therapeutic uses In another aspect, this disclosure provides methods for using antigen-binding molecules of this disclosure (e.g., the CD40xCD40 bispecific antigen-binding molecule disclosed herein). In some embodiments, this disclosure provides methods for inhibiting CD40L-induced CD40 activation, for example, in or on cells expressing CD40 (e.g., B cells, dendritic cells, monocytes, platelets, or macrophages). In some embodiments, the method includes contacting CD40-expressing cells with the CD40xCD40 bispecific antigen-binding molecule disclosed herein.

[0213] In some embodiments, this disclosure provides a method for treating, improving, or preventing CD40-mediated diseases or conditions 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. As used herein, a CD40-mediated disease or condition is any disease or condition caused or aggravated by the activity of CD40 (e.g., activation of downstream signaling due to binding of CD40 to its ligand CD40L). In some embodiments, a CD40-mediated disease or condition is due to a mutation in a gene in the CD40 or CD40L or CD40 signaling pathway.

[0214] In some embodiments, CD40-mediated diseases or conditions are autoimmune diseases or conditions, inflammatory diseases or conditions, cardiovascular diseases or conditions, or organ transplantation. In some embodiments, CD40-mediated diseases or conditions are autoimmune diseases or conditions. In some embodiments, CD40-mediated diseases or conditions are Addison's disease, autoimmune hemolytic anemia, autoimmune thyroid diseases (e.g., thyroiditis, Graves' disease, or Hashimoto's thyroiditis), Crohn's disease, diabetes (e.g., type 1 diabetes), experimental autoimmune encephalomyelitis (EAE), focal segmental glomerulosclerosis (FSGS), glomerulonephritis, Guillain-Barré syndrome, graft-versus-host disease (GVHD) (e.g., acute GVHD or chronic GVHD), hemolytic anemia, hidradenitis suppurativa (HS), immune thrombocytopenic purpura, inflammatory bowel disease, inflammatory neuropathy (e.g., acute inflammatory demyelinating polyneuropathy). Arthritis of the skin (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 cholangitis, psoriasis, psoriatic arthritis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Sjögren's syndrome, skin grafts, spondyloarthritis, systemic lupus erythematosus, systemic sclerosis, transplant rejection, vasculitis, ANCA-associated vasculitis (e.g., granulomatous polyangiitis (GPA), microscopic polyangiitis (MPA) and / or eosinophilic GPA (EGPA)), ulcerative colitis, or Wegener's 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 to 4)) prevents or delays the exacerbation of disease symptoms or the progression of disease in subjects with CD40-mediated diseases or conditions.

[0216] Dosage and administration regimen In some embodiments, the amount of CD40 antigen-binding molecule (e.g., CD40xCD40 bispecific antigen-binding molecule (e.g., bispecific antibody)) administered to a subject according to the methods disclosed herein is a therapeutically effective amount. As used herein, the phrase "therapeutically effective amount" means the amount that produces the desired effect of its administration.

[0217] In some embodiments, a CD40 antigen-binding molecule (e.g., a CD40xCD40 bispecific antibody) is administered to the subject at a weight-based dose. "Weight-based dose" (For example, The dose (in mg / kg) is the dose of the antigen-binding molecule that will be altered according to the subject's weight.

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

[0219] Typically, appropriate doses of antigen-binding molecules can range from about 0.001 to about 200.0 mg per kilogram of recipient body weight, and generally from about 1 to 50 mg per kilogram of body weight. For example, antigen-binding molecules can be administered at individual doses of about 0.1 mg / kg, about 0.2 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 3 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 40 mg / kg, and about 50 mg / kg. These values ​​and ranges between them are also intended to form part of this disclosure.

[0220] In some embodiments, the antigen-binding molecule is administered at a fixed dose between about 5 mg and about 2500 mg. In some embodiments, the antigen-binding molecule is in the form 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, about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about 750 mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg, about 875 mg, about 875 mg. Administered at a fixed dose of approximately 1000 mg, about 900 mg, about 925 mg, about 950 mg, about 975 mg, about 1000 mg, about 1500 mg, about 2000 mg, or about 2500 mg. Values ​​and ranges between these 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 the subject at a frequency of approximately four times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, once every twelve weeks, or less, as long as a therapeutic response is achieved.

[0222] In some embodiments, multiple doses of a CD40 antigen-binding molecule (e.g., a CD40xCD40 bispecific antibody) are administered to a subject over a defined time period. In some embodiments, the method of this disclosure includes sequentially administering multiple doses of the antigen-binding molecule to a subject. As used herein, “sequentially administering” means administering each dose of the antigen-binding molecule to the subject at different time points, for example, on different dates spaced apart by predetermined intervals (e.g., hours, days, weeks, or months). In some embodiments, the method of this disclosure includes sequentially administering a single initial dose of the antigen-binding molecule to a patient, followed by one or more second doses of the antigen-binding molecule, and optionally subsequently administering one or more third doses of the antigen-binding molecule.

[0223] The terms “initial dose,” “second dose,” and “third dose” refer to the temporal sequence of the administration of antigen-binding molecules. Thus, the “initial dose” is the dose administered at the start of a treatment regimen (also known as a “loading dose”); the “second dose” is the dose administered after the initial dose; and the “third dose” is the dose administered after the second dose. In some embodiments, the initial dose, second dose, and third dose may all contain the same amount of antigen-binding molecules, but may differ in their frequency of administration. In some embodiments, the amount of antigen-binding molecules contained in the initial dose, second dose, and / or third dose may differ from one another during the treatment process (e.g., directional up or down adjustments, depending on the situation). In some embodiments, one or more doses (e.g., 1, 2, 3, 4, or 5) are administered at the start of a treatment regimen as a “loading dose,” followed by subsequent doses (e.g., “maintenance doses”) administered on a less frequent basis. In some embodiments, the initial dose and one or more second doses each contain the same amount of antigen-binding molecules. In other embodiments, the initial dose contains a first amount of antigen-binding molecules, and one or more second doses each contain a second amount of antigen-binding molecules. For example, the first amount of antigen-binding molecules can be 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 5 times or more of the second amount of antigen-binding molecules.

[0224] In some embodiments, each second and / or third dose is administered 1 to 14 weeks after the immediately preceding dose (e.g., 1, 1½, 2, 2½, 3, 3½, 4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½, 12, 12½, 13, 13½, 14, 14½ or more). As used herein, the phrase “immediately preceding dose” means, in a sequence of multiple administrations, the dose of the antigen-binding molecule administered to the patient before the administration of the immediately following next dose in the sequence, without intermediate doses.

[0225] The methods disclosed herein may include administering any number of second and / or third doses of the antigen-binding molecule to a patient. For example, in some embodiments, only a single second dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) second doses are administered to the patient. Similarly, in some embodiments, only a single third dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) third doses are administered to the patient.

[0226] In some embodiments involving multiple second doses, each second dose is administered at the same frequency as the other second doses. For example, each second dose may be administered to the patient 1, 2, 3, or 4 weeks immediately following the preceding dose. Similarly, in some embodiments involving multiple third doses, each third dose is administered at the same frequency as the other third doses. Alternatively, the frequency of administration of the second and / or third doses to the patient may vary during the treatment regimen. The physician may also adjust the administration frequency during the treatment process based on the individual patient's needs following a clinical examination.

[0227] Combination therapy In another aspect, this disclosure includes compositions and therapeutic formulations comprising any of the exemplary antibodies and bispecific antigen-binding molecules described herein, combined with one or more additional therapeutic agents, and treatment methods including administering such combinations to a subject in need. In some embodiments, the additional therapeutic agent is an immunomodulator or an anti-inflammatory agent. In some embodiments, the additional therapeutic agent is an immunosuppressive therapy. In some embodiments, the additional therapeutic agent is surgery.

[0228] Exemplary additional therapeutic agents that can be administered in combination with or in combination with the antigen-binding molecules of this disclosure include, for example, another CD40-CD40L inhibitor (e.g., CD40 antibodies, such as icali [CFZ-533], bulrucizumab [ASKP1240], BI). 655064, cd5D12 or FFP104; or CD40L antibodies or antigen-binding proteins, such as dapirozumab (PEG), dazolibep [VIB4920], frasalimab [INX-021], letolizumab [BMS-986004], MR-1, rupulizumab [BG9588], tergoprurubab [AT-1501] or tolaolizumab [IDEC-131]; B-cell depletion agents (e.g., direct agents, such as CD19 antibody, CD20 antibody, CD22 antibody), or indirect agents, such as BLyS inhibitors or APRIL inhibitors); plasma cell depletion agents (e.g. Examples include B-cell maturation antigen (BCMA) targets, BCMAxCD3 bispecific antibodies, CD38 antibodies (e.g., daratumumab), proteasome inhibitors, histone deacetylase inhibitors, B-cell activating factor (BAFF) inhibitors, and APRIL inhibitors; steroids (e.g., corticosteroids, such as topical, systemic, oral, or inhaled corticosteroids, including but not limited to betamethasone, clobetasol, dexamethasone, fluocinolone acetonide, fluocinolone acetate, halometasol, hydrocortisone, methylprednisolone, prednisone, prednisolone, or triamcinolone); and nonsteroidal external medicines, such as, but not limited to, PDE4 inhibitors or calcineurin inhibitors. Nonsteroidal anti-inflammatory drugs (NSAIDs), such as but not limited to celecoxib, diclofenac, etodoxacin, fenprofen, flurbiprofen, ibuprofen, ketoprofen, meclofenamic acid, meloxicam, nabumetone, naproxen, oxapazine, piroxicam, rofecoxib, salicylates, sulfasalazine, sulindac, or tometidine; anti-inflammatory antibodies or biologics (e.g., TNFα antibodies or biologics, such as but not limited to adalimumab, sertozumab, etanercept, golimumab, or infliximab; IL-1 antibodies or biologics, such as but not limited to LY2189102, anakinin, canatumab, glycosylmab, or linacip; IL-6 / IL -6R antibodies or biologics, such as, but not limited to, thalidomide, cetuximab, or tocilizumab; IL-17A / IL-17R antibodies or biologics, such as, but not limited to, bimecrolimus, brodamarumab, ixekizumab, or secukinumab; or IL-12 / IL-23 antibodies or biologics, such as, but not limited to, AMG139, BI655066, brazkumab, brenzumab, gusejinumab, millizumab, resalizumab, tilatizumab, or ustekinumab); JAK inhibitors, such as, but not limited to, abuxitinib, baricitinib, fentatinib, filgrastim, ruxolitinib, tofacitinib, or utpatinib;Immunosuppressants (e.g., systemic immunosuppressants such as, but not limited to, methotrexate, cyclophosphamide, imidazolidinedione, chlorambucil, cyclosporine, mycophenolate mofetil, or azathioprine); disease-modifying antirheumatic drugs (DMARDs) such as, but not limited to, apromid, azathioprine, baricitinib, cyclophosphamide, cyclosporine, hydroxychloroquine, leflunomide, methotrexate, mycophenolate mofetil, sulfasalazine, or tofacitinib; exogenously administered therapeutic proteins such as enzymes (e.g., enzyme replacement therapy); radiotherapy; chemotherapy; intravenous immunoglobulin therapy; or surgery or surgical procedures (such as, but not limited to, splenectomy, lymph node dissection, thyroidectomy, plasma exchange, leukocyte ablation, or cell, tissue, or organ transplantation).

[0229] In some embodiments, the CD40 antigen-binding molecules disclosed herein (e.g., CD40xCD40 bispecific antigen-binding molecules, such as those disclosed in any of Tables 1 to 4) may be administered in combination with or in combination with B cell depletion agents and / or plasma cell depletion agents. In some embodiments, the depletion agent is an agent that directly targets B cells, such as an agent that binds to molecules on the surface of B cells. In some embodiments, the B cell depletion agent is a CD19 antibody (e.g., MEDI-551), a CD20 antibody (e.g., rituximab, oligrizumab, or oligrizumab), or a CD22 antibody (e.g., epazolizumab). In some embodiments, the depletion agent is an agent that indirectly targets B cells and / or plasma cells, such as by targeting B cells or plasma cell survival factors. In some embodiments, the B cell depletion agent is a BLyS / BAFF inhibitor (e.g., belimumab, BR3-Fc, or AMG-623), an APRIL inhibitor (e.g., attacipex), or a BLyS receptor 3 / BAFF receptor inhibitor (e.g., anti-BR3). In some embodiments, the depleting agent is a BCMA target, such as a BCMAxCD3 bispecific antibody, a chimeric antigen receptor against BCMA, or a BCMA antibody conjugated with a cytotoxic drug (e.g., linvosartuzumab, REGN5459, pacanotuzumab, or teritumumab).

[0230] Additional therapeutically active components may be administered precisely before, simultaneously with, or shortly after the administration of the antigen-binding molecule of this disclosure. For the purposes of this disclosure, such administration regimens are considered as a “combination” of administering the antigen-binding molecule with additional therapeutically active components.

[0231] This disclosure includes pharmaceutical compositions wherein the antigen-binding molecule of the invention is co-formulated with one or more of other therapeutically active components as described elsewhere herein.

[0232] Example The following examples are provided to provide a complete disclosure and description of how to prepare and use the methods and compositions of this disclosure to those skilled in the art, and are not intended to limit the scope of what the inventors consider to be their invention. Efforts have been made to ensure the accuracy of the figures used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise stated, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at atmospheres or close to atmospheres.

[0233] Example 1: Construction of CD40xCD40 bispecific antibody Generation of parental CD40 antibodies Antibodies against CD40 are obtained by immunizing VELOCIMMUNE® mice (i.e., engineered mice containing DNA encoding the variable regions of the heavy and κ chains of human immunoglobulins) with human CD40 antigen (human CD40 extracellular domain with a C-terminal MMH tag; SEQ ID NO:53).

[0234] Following immunization, antibodies are isolated directly from antigen-positive mouse B cells, for example, as described in U.S. Patent No. 7,582,298, which is incorporated herein by reference. Using this method, fully human CD40 antibodies (i.e., antibodies having both human variable and human constant domains) are obtained. The antibodies generated using this method are characterized and selected for desired properties, including affinity, selectivity, etc.

[0235] CD40 antibodies generated using this method include antibodies numbered 30027P2, 21519P2, and 21520P2. Certain biological characteristics of the exemplary CD40 antibodies generated according to the method of this example are described in detail in the examples set forth below.

[0236] Table 1: Amino acid sequence identifiers of parental CD40 monoclonal antibodies Table 2: Nucleic acid sequence identifiers of parental CD40 monoclonal antibodies Generation of CD40xCD40 bispecific antibodies A bispecific antibody comprising a first CD40 binding arm (“first antigen-binding domain”) and a second CD40 binding arm (“second antigen-binding domain”) is constructed using standard methods, wherein the two CD40 binding arms contain different HCVRs that pair with a common light chain. Two distinct heavy chain constant regions (e.g., as described in U.S. Patent No. 11,518,807) are used for each CD40 binding arm. An exemplary CD40xCD40 bispecific antibody is generated according to this example and comprises the sequences shown in Tables 3 to 4 below.

[0237] As shown in Table 3, for REGN16334 and REGN16431, the first CD40 binding arm (“D1”) contains the HCVR sequence of the parental antibody 21519P2, and the second CD40 binding arm (“D2”) contains the HCVR sequence of the parental antibody 21520P2; REGN16334 and REGN16431 have different constant region modifications to reduce Fc receptor binding and effector function.

[0238] For REGN16335 and REGN16432, the first CD40 binding arm (“D1”) contains the HCVR sequence of the parental antibody 30027P2, and the second CD40 binding arm (“D2”) contains the HCVR sequence of the parental antibody 21520P2; REGN16335 and REGN16432 have different constant region modifications to reduce Fc receptor binding and effector function.

[0239] For REGN20484, the first CD40 binding arm (“D1”) contains the HCVR sequence of the parent antibody 21520P2, and the second CD40 binding arm (“D2”) contains the HCVR sequence of the parent antibody 30027P2.

[0240] REGN16334, REGN16335, REGN16431, REGN16432, and REGN20484 all contain a common light chain sequence.

[0241] Example 2: Biacore binding kinetics of CD40 bivalent parental antibody and CD40xCD40 bispecific antibody The equilibrium dissociation constants (K0) of CD40 bivalent and bispecific monoclonal antibodies (mAbs) were determined using a Biacore 4000 biosensor based on real-time surface plasmon resonance (SPR). DAll binding studies were performed at 25°C and 37°C in a running buffer of 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v / v surfactant Tween-20, pH 7.4 (HBS-ET). The Biacore CM5 sensor surface was first derivatized with a monoclonal mouse anti-human Fc antibody (REGN2567) via amine coupling to capture both the CD40 bivalent parental antibody and the CD40xCD40 bispecific antibody. 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)) were injected at a flow rate of 30 µL / min for 4 minutes or at a flow rate of 50 µL / min for 5 minutes in a series of 3-fold dilutions prepared in HBS-ET running buffer. The dissociation of different CD40 reagents binding to the CD40 bivalent parental antibody and the CD40xCD40 bispecific antibody was monitored in HBS-ET run buffer for 10 minutes. At the end of each cycle, the regenerated CD40 bivalent parental antibody and the CD40xCD40 bispecific antibody were injected with 20 mM H3PO4 for 12 seconds to capture the surface.

[0242] The association rate (ka) and dissociation rate (kd) were determined by fitting the real-time binding sensor map to a 1:1 binding model with mass transfer constraints using Scrubber 2.0c curve fitting software. The binding dissociation equilibrium constant (K) was also determined. D The dissociation half-life (t½) and the kinetic rate are calculated as: K D (M) = k d / k a , and t ½ (min) = [ln(2) / (60 k d )).

[0243] The binding kinetic parameters of different CD40 reagents with CD40 bivalent parental antibody and CD40xCD40 bispecific antibody at 25℃ and 37℃ are shown in Tables 5 to 10.

[0244] As shown in Table 5, at 25℃, the CD40 bivalent parental antibody and the CD40xCD40 bispecific antibody bind to hCD40-MMH,K D The values ​​ranged from 101 pM to 25.1 nM. At 25°C, the CD40 bivalent parental antibody bound mfCD40-MMH, K... D The values ​​ranged from 901 pM to 205 nM (Table 6). The CD40 bivalent parental antibody did not bind to mCD40-MMH at 25°C (Table 7).

[0245] As shown in Table 8, at 37°C, the KD values ​​of CD40 bivalent parental antibody and CD40xCD40 bispecific antibody binding to hCD40-MMH range from 101 pM to 69.9 nM. At 37°C, the CD40 bivalent parental antibody binding to mfCD40-MMH has a KD value of 3.16 nM (Table 9). The CD40 bivalent parental antibody does not bind to mCD40-MMH at 37°C (Table 10).

[0246] Table 5: Interactions of hCD40-MMH with CD40 bivalent parental antibody and CD40xCD40 bispecific antibody at 25℃ Used dynamic combined parameters Table 6: Interactions of mfCD40-MMH with CD40 bivalent parental antibody and CD40xCD40 bispecific antibody at 25℃ Used dynamic combined parameters NT = Untested Table 7: Interactions of mCD40-MMH with CD40 bivalent parental antibody and CD40xCD40 bispecific antibody at 25℃ Used dynamic combined parameters NB = No binding was observed under experimental conditions; NT = Not tested. Table 8: Interactions of hCD40-MMH with CD40 bivalent parental antibody and CD40xCD40 bispecific antibody at 37℃ Used dynamic combined parameters Table 9: Interactions of mfCD40-MMH with CD40 bivalent parental antibody and CD40xCD40 bispecific antibody at 37℃ Used dynamic combined parameters IC = Uncertain; NT = Untested Table 10: Interactions of mCD40-MMH with CD40 bivalent parental antibody and CD40xCD40 bispecific antibody at 37℃ Used dynamic combined parameters NB = No binding was observed under experimental conditions; NT = Not tested. Example 3: Cross-competition between different CD40 monoclonal antibodies Binding competition between different CD40 monoclonal antibodies (mAbs) was determined using a real-time label-free biolayer interferometry (BLI) assay on the Octet HTX biosensor platform (Pall ForteBio Corp.). In addition to the parental antibodies 21519P2, 21520P2, and 30027P2, a comparative CD40 antibody (REGN11209) with the heavy and light chain sequences of iscalimab was also tested (see US 8,828,396). All experiments were performed at 25°C in 10 mM HEPES buffer at pH 7.4 (HBS-EP) containing 150 mM NaCl, 3 mM EDTA, 1 mg / mL BSA, 0.02% NaN3, and 0.05% v / v surfactant Tween-20, with plates shaken at 1000 rpm.

[0247] To evaluate the ability of one antibody to compete with another for binding to CD40, approximately 0.47 nM to 0.54 nM of the recombinant human CD40 extracellular domain (hCD40-MMH; SEQ ID NO: 53) expressed with C-terminal myc-myc-hexahistidine was first captured onto the Octet biosensor tip (Fortebio Inc, #18-5122) coated with an anti-pentahistidine antibody by immersing the biosensor tip in a well containing 10 µg / mL hCD40-MMH solution for 90 seconds. The antigen-captured biosensor tip was then permeated with a first CD40 monoclonal antibody (hereinafter referred to as “mAb-1”) by immersing it in a well containing 50 µg / mL mAb-1 solution for 4 minutes. The biosensor tip was then immersed in a well containing 50 µg / mL a second CD40 monoclonal antibody (hereinafter referred to as “mAb-2”) solution for 3 minutes. The biosensor tip was washed in HBS-EBT buffer between each step of the experiment. The binding reaction was monitored in real time, and the binding reaction at the end of each step was recorded. The binding reaction of mAb-2 with hCD40-MMH pre-conjugated with mAb-1 was compared with the binding reaction of hCD40-MMH alone (isotype control sample), and mAb-1 was considered a competitor of mAb-2 if the pre-conjugated mAb-1 reduced the binding of mAb-2 by more than 50%.

[0248] The competitors for 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.

[0249] Table 11: Cross-competition for binding to hCD40-MMH among different CD40 monoclonal antibodies Example 4: ELISA assay for evaluating the blocking activity of CD40xCD40 bispecific antibody An ELISA-based blocking assay was developed to determine the ability of the CD40xCD40 bispecific antibody to block the binding of hCD40 monomers to plate-coated hCD40L. The recombinant human CD40-mmH protein (hCD40-MMH; REGN3094; SEQ ID NO:53) used in the experiment contained a portion of the human CD40 extracellular domain (amino acid P20-R193) fused to a 2xMyc peptide and a 6xhistidine residue at the C-terminus of human CD40, and human CD40L (accession number NM_000074.2) with a 9xHis-2x(SGGG)-IGER (9His-hCD40L) at the N-terminus and an extracellular domain containing amino acids E108-L261, was commercially available from Biolegend.

[0250] In the blocking assay, 9His-hCD40L was passively absorbed overnight at 4°C in PBS at a concentration of 5 μg / mL on a 96-well microtiter plate. Non-specific binding sites were then blocked using a solution of 0.5% (w / v) BSA in PBS. In separate 96-well microtiter plates, a fixed amount of 40 nM hCD40-mmH was premixed with one of the following antibodies in PBS + 0.5% BSA at concentrations ranging from 977 pM to 1 µM: (1) CD40xCD40 bispecific antibodies (REGN16431, REGN16432, REGN16634, and REGN16335); (2) parental CD40 mAb (a bivalent antibody containing two fragment antigen-binding [Fab] arms identical to one of the anti-CD40Fab of the CD40xCD40 antibody); (3) CD40x irrelevant antibodies (a bivalent antibody incorporating one Fab arm identical to one of the anti-CD40 Fab of the CD40xCD40 antibody and another Fab arm specific to irrelevant antigens); and (4) human IgG4 isotype control antibodies with Fc mutations (REGN7540 and REGN4513). The fixed concentration of hCD40-mmH was selected to be close to 50% of the concentration that generates the maximum binding of 9His-hCD40L to the plate (EC). 50After incubation for one hour, the antibody-antigen complex was transferred to a microtiter plate coated with 9His-hCD40L. After incubation at room temperature for one hour, the plate was washed, and the hCD40-mmH protein bound to the plate was detected using a horseradish peroxidase (HRP)-conjugated goat anti-c-Myc antibody. The plate was then developed using TMB substrate solution (BDBiosciences) according to the manufacturer's recommended procedure, and the absorbance (OD) at 450 nm was measured on a SpectraMax i3x microplate reader. 450 ).

[0251] The binding data were analyzed using a sigmoid (four-parameter logic) dose-response model with GraphPad Prism™ software to determine the IC. 50 The value, defined as the antibody concentration required to block 50% binding of 40 nM hCD40-mmH to the plate-coated 9His-hCD40L, indicates blocking efficacy. The percentage of blocking effect at the highest test concentration (1 µM) is calculated using the formula shown below: The largest signal was the interpolated binding signal of 40 nM hCD40-mmH from the hCD40-mmH concentration-response curve. Antibodies blocking greater than 50% of hCD40 binding were classified as blocking agents. Antibodies blocking equal to or less than 50% of binding were classified as non-blocking agents. The IC50 of non-blocking agents was not determined. 50 value.

[0252] result The ability of CD40xCD40 bispecific antibodies to block the binding of human CD40 monomers to plate-coated human CD40L was evaluated 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 showed concentration-dependent blocking of hCD40 to hCD40L binding, with blocking rates ranging from 93% to 98% at the highest antibody concentration tested (1 µM). The IC50 values ​​of these bispecific antibodies... 50 The values ​​were similar, approximately 32 nM. Four parental anti-CD40 antibodies (H4sH21519P2, H4sH21520P2, REGN17288, REGN17544) showed maximum blockade ranging from 91% to 94% and IC50 values ​​ranging from 52 nM to 71 nM. 50Values. Four CD40x-independent antibodies (REGN17551, REGN17552, REGN17548, and RENG17549) also inhibited the binding of hCD40 to hCD40L, with a blocking range of 83% to 87%. Two parental CD40 antibodies (H4sH30027P2 and REGN17289) and two CD40x-independent antibodies (REGN17553 and REGN17550) showed minimal blocking activity of approximately 10% and were classified as non-blocking agents. In this experiment, two human IgG4 isotype control antibodies with Fc mutations (REGN7540 and REGN4513) did not show blocking activity as expected.

[0253] Table 12: Summary of CD40 antibodies that block the binding of human CD40 monomers to human CD40L NBL: No blocking (blocking percentage of 50% or less) ND: Not measured (no observations were observed for calculating IC) 50 (S-shaped curve fitting of values) Example 5: Binding of CD40xCD40 bispecific antibody to cell surface CD40 as measured by flow cytometry The binding of the CD40xCD40 bispecific antibody to human CD40 (hCD40) or [other specific antibodies] was evaluated using flow cytometry. Crab-eating macaques The ability of cells to express CD40 (mfCD40) was assessed. Cells were engineered to express hCD40 (accession number P25942-1) or mfCD40 (accession number XP_005569274.1) by transfecting them with either a neomycin-resistant pRG984 plasmid encoding full-length hCD40 (amino acid M1-Q277, HEK293 / D9 / hCD40) or a neomycin-resistant pRG984 plasmid encoding full-length mfCD40 (amino acid M1-Q282, HEK293 / D9 / mfCD40). Human embryonic kidney 293 (HEK293) cells stably expressing a luciferase reporter gene under the control of activated B cells' nuclear factor κ-light chain enhancer (NF-κB) and enhanced green fluorescent protein (HEK293 / D9). The binding of anti-CD40 antibodies to endogenously expressed hCD40 on the cell surface was evaluated using Ramos2G6.4C10 cells. This included using flow cytometry with a commercially available CD40 antibody to show CD40-negative HEK293 / D9 cells with no detectable CD40 expression as a background binding control.

[0254] The experiment was conducted according to the following procedure: HEK293 / D9 / hCD40, HEK293 / D9 / mfCD40, or HEK293 / D9 cells were placed in a Ca-free environment.2+ / Mg 2+ Rinse once with 1xPBS buffer and incubate at 37°C for 10 minutes with enzyme-free cell dissociation solution to detach cells from the culture flask. Wash the dissociated cells or Ramos 2G6.4G10 suspension cells with 1xPBS and use a Cellometer. TM Cell counting was performed using an Auto T4 cell counter (Nexcelom Bioscience, Lawrence, MA). Cells were then resuspended in 1xPBS to a final volume of 1x10⁻⁶. 7 Each mL, and at room temperature (RT), was treated with 2.5 μM CellTrace. TM The reagent (Invitrogen, Carlsbad, CA) was used to stain for 20 minutes to generate a unique fluorescent signature for each cell line (Ramos2G6.4C10 cells were stained with CellTrace). TM CFSE, HEK293 / D9 / mfCD40 cells for CellTrace TM Purple, HEK293 / D9 cells are CellTrace TM Yellow, and HEK293 / D9 / hCD40 cells were unstained). CellTrace was terminated by adding FBS to 1xPBS to a final concentration of 25%, followed by incubation at room temperature for 5 minutes to quench unbound dye in solution. TM Labeling reaction. Wash cells with 1xPBS and mix equal amounts of each of the four stained and unstained cell lines in a 1:1:1:1 ratio for multiplex detection. Pack approximately 2 x 10⁻⁶ cells per well. 5 Cells were seeded into 96-well Corning plates and separated into live and dead cells according to the manufacturer's recommended procedure using LIVE / DEAD. TMCells were stained with Fixable Near-IR (Thermo Fisher Scientific, Waltham, MA) in 1xPBS at 4°C for 20 minutes. Cells were washed with 1xPBS (flow cytometry staining buffer) containing 2% FBS (w / v) by centrifugation using a benchtop centrifuge 5810R (Eppendorf®, Hamburg, Germany). Cells were incubated in flow cytometry staining buffer with serial dilutions of the following antibodies ranging from 1.7 pM to 100 nM at 4°C for 30 minutes: (1) a CD40xCD40 bispecific antibody, or (2) a parental CD40 antibody (a bivalent antibody containing two fragment antigen-binding [Fab] arms identical to one of the two anti-CD40 Fabs of the bispecific CD40xCD40 antibody), or (3) a CD40x irrelevant antibody (a bivalent antibody incorporating one Fab arm identical to one of the anti-CD40 Fabs of the CD40xCD40 antibody and another Fab arm specific to irrelevant antigens (e.g., birch pollen major allergen Bet v1), or (4) a human IgG4 isotype control antibody with an Fc mutation. After washing, cells were detected for antibody binding at 4°C for 30 minutes using a 2.5 μg / ml goat anti-human IgG antibody specifically conjugated to the Fcγ fragment (APC) (Jackson Immunoresearch, West Grove, PA). Cells were washed and then fixed at room temperature for 20 minutes in a solution of 50% Cytofix™ fixation buffer (BD, Franklin Lakes, NJ) in flow cytometry staining buffer. Cells were washed and resuspended in flow cytometry staining buffer and stored at 4°C for downstream flow cytometry analysis.

[0255] Fluorescence signals were acquired and recorded on a ZE5 cell analyzer (Bio-Rad, Hercules, CA) according to the manufacturer's recommended procedures. Flow cytometry data analysis was performed using the open-source R packages flowCore, flowStats, flowDensity, ggCyto, and flowAI. Multiplexed samples were deconvolved and analyzed based on its unique CellTrace feature. TMFluorescence signatures were used to identify individual cell populations. Median fluorescence intensity (MFI) of APCs was recorded to indicate the binding strength of each antibody within a specific concentration range. Antibodies with an MFI greater than 1500 at the highest tested concentration (100 nM) were classified as specifically binding antibodies. Furthermore, the direct binding signal (APC MFI) was analyzed as a function of antibody concentration, and the data were fitted using a sigmoid (four-parameter logic) dose-response model with GraphPad Prism™ software. EC was measured. 50 The value, defined as the antibody concentration at which 50% of the maximum binding occurs, is used as an indicator of antibody binding potency. Only EC values ​​of specifically binding antibodies are reported. 50 value.

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

[0257] Four CD40xCD40 bispecific antibodies (REGN16431, REGN16432, REGN16334, and REGN16335) showed concentration-dependent specific binding to hCD40 expressed on HEK293 / D9 / hCD40 or Ramos 2G6.4C10 cells. On HEK293 / D9 / hCD40 cells, the MFI values ​​at the highest tested concentration (100 nM) ranged from 28,932 to 30,315, and EC50 was also observed. 50 The MFI values ​​ranged from 1.5 nM to 2.0 nM, or 3,748 to 3,852 on Ramos 2G6.4C10 cells, with EC50 values ​​ranging from 1.0 nM to 2.0 nM. These four bispecific antibodies similarly showed specific binding to mfCD40 expressed on HEK293 / D9 / mfCD40 cells, with MFI values ​​ranging from 41,714 to 48,653, and EC50 values ​​ranging from 1.0 nM to 2.0 nM. 50 The values ​​range from 2.1 nM to 9.1 nM.

[0258] Six parental CD40 antibodies (H4sH21519P2, H4sH21520P2, H4sH30027P2, REGN17288, REGN17544, and REGN17289) showed specific binding to HEK293 / D9 / hCD40 or Ramos 2G6.4C10 cells, with MFI values ​​ranging from 20,213 to 20,911 on HEK293 / D9 / hCD40 cells, and EC50 values... 50The values ​​ranged from 0.72 nM to 1.3 nM, or in Ramos 2G6.4C10 cells, the MFI values ​​ranged from 2,408 to 2,664, and the EC values ​​were... 50 The values ​​ranged from 0.45 nM to 1.5 nM. All parental CD40 antibodies showed specific binding to HEK293 / D9 / mfCD40 cells, with MFI values ​​ranging from 11,539 to 33,930, and EC50 values. 50 The value ranges from 1.0 nM to 10 nM.

[0259] Six CD40x-independent antibodies (REGN17551, REGN17552, REGN17553, REGN17548, REGN17549, and REGN17550) showed specific binding to HEK293 / D9 / hCD40 or Ramos 2G6.4C10 cells, with MFI values ​​ranging from 21,394 to 24,591 on HEK293 / D9 / hCD40 cells, and EC50 values... 50 The MFI values ​​ranged from 1.1 nM to 8.2 nM, or from 1,833 to 3,140 on Ramos 2G6.4C10 cells. Four CD40x-independent antibodies (REGN17552, REGN17553, REGN17549, and REGN17550) showed specific binding to HEK293 / D9 / mfCD40 cells, with MFI values ​​ranging from 4,719 to 39,448, but two CD40x-independent antibodies (REGN17551 and REGN17548) did not specifically bind to mfCD40 on HEK293 / D9 / mfCD40 cells.

[0260] All tested CD40 antibodies did not bind to negative control HEK293 / D9 cells: binding signal below 950 MFI. As expected, no detectable cell surface binding was observed for the two Fc-mutated hIgG4 isotype control antibodies (REGN7540 and REGN4513) up to the highest antibody concentration tested (100 nM).

[0261] Example 6: Bioassay to evaluate the regulation of CD40 signaling by CD40xCD40 bispecific antibody CD40 is a member of the tumor necrosis factor receptor superfamily (TNFRSF), which activates the immune system in response to the binding of its ligand CD40L. To assess the regulation of CD40 signaling, a bioassay was developed to quantitatively evaluate receptor activation by measuring gene expression downstream of NFκB (nuclear factor κB) nuclear translocation. A luciferase-based reporter gene assay was designed in three different human cell lines endogenously expressing CD40 (Ramos.2G6.4C10, Raji, and THP-1). Cells were transduced with NFκB-luciferase reporter lentivirus (QIAGEN CLS-013L-8), and stable reporter cell lines were selected and maintained in medium containing 1 μg / ml puromycin.

[0262] For the bioassay, cells were seeded at 20,000 cells / well in 96-well plates in assay medium (RPMI-1640 containing 10% FBS, penicillin / streptomycin / glutamine). The antibody was then serially diluted 1:3 in assay medium to a final concentration ranging from 100 nM to 1.7 pM (under conditions without additional test molecules) and added to the cells, with or without a constant concentration of human CD40L (500 pM, 900 pM, or 10 nM for Ramos, Raji, or THP1 reporter cells, respectively). To obtain a range of activation, hCD40L was serially diluted 1:3 to a final concentration ranging from 100 nM to 1.7 pM (under conditions without additional ligands) and added to the cells. After incubation at 37°C / 5% CO2 for 5 hours, luciferase activity was measured on an Envision multi-plate reader (PerkinElmer) after the addition of ONE-Glo™ (Promega) reagent. All conditions are tested in duplicate.

[0263] EC was determined using nonlinear regression (4-parameter logic) with GraphPad Prism™ software. 50 or IC 50 Value. Based on the relative luminous unit (RLU) value, the suppression percentage is calculated using the equation: RLU 抗体最大值 "and "RLU 抗体最小值 "These represent the maximum and minimum luminescence values ​​achieved using antibodies." RLU 配体最大值 "and "RLU 配体恒定值 "Values ​​achieved through the maximum and constant concentrations of CD40L." RLU 背景值 "RLU" indicates the absence of any CD40L value. For antibodies that show enhancement in the presence of CD40L, use "RLU".抗体最大值 "To calculate the inhibition that leads to a negative inhibition value."

[0264] result As shown in Table 14, the CD40xCD40 bispecific antibodies REGN16431, REGN16432, REGN16334, and REGN16335 exhibited minimal activation ranging from 4% to 6% in Ramos.2G6.4C10 / NFκB-luc cells in the absence of CD40L, and showed inhibition ranging from 90% to 94% in the presence of 500 pM CD40L. IC50 50 The effective concentrations ranged from 38.6 to 85.4 pM. The CD40 bivalent antibody and the CD40 x Irrel (irrelevant, non-CD40 target) antibody showed activation ranging from 4% to 49% in the absence of CD40L and inhibition ranging from 28% to 95% in the presence of 500 pM CD40L. In the Ramos bioassay, all four antibodies showed signal transduction enhancement in the presence of CD40L, with inhibition ranging from -110% to -142%.

[0265] As shown in Table 15, the CD40xCD40 bispecific antibodies REGN16431, REGN16432, REGN16334, and REGN16335 exhibited minimal activation ranging from 5% to 13% in Raji / NFκB-luc cells in the absence of CD40L, and showed inhibition ranging from 68% to 113% in the presence of 900 pM CD40L. 50 The values ​​ranged from 49.4 to 107 pM. In the Raji bioassay, the CD40 bivalent antibody and the CD40 x Irrel antibody showed activation ranging from 7% to 25% in the absence of CD40L and inhibition ranging from 33% to 92% with 900 pM CD40L.

[0266] As shown in Table 16, the CD40xCD40 bispecific antibodies REGN16431, REGN16432, REGN16334, and REGN16335 exhibited minimal activation ranging from 1% to 6% in THP-1 / NFκB-luc cells in the absence of CD40L, and showed inhibition ranging from 97% to 98% in the presence of 10 nM CD40L. 50The concentrations ranged from 148 to 553 pM. The CD40 bivalent antibody and CD40 x Irrel antibody showed activation ranging from 0% to 5% in the absence of CD40L and inhibition ranging from 54% to 101% in the presence of 10 nM CD40L. In the THP-1 bioassay, all four antibodies showed signal transduction enhancement in the presence of CD40L, with inhibition ranging from -45% to -92%.

[0267] Control mAb1, control mAb2, and control mAb3, along with irrelevant human IgG antibodies, showed little or no activation (0%–13%) in all cells without CD40L and showed inhibition (3%–23%) in the presence of CD40L. In Ramos.2G6.4C10 / NFκB-luc, Raji / / NFκB-luc, and THP-1 / NFκB-luc cells, CD40L showed signaling activation, EC 50 The values ​​were 436 pM, 691 pM, and 1.47 nM, respectively.

[0268] Table 14: CD40 levels in Ramos.2G6.4C10 / NFκB-luc cells with or without human CD40L Anti-CD40 x CD40 bispecific regulation of signal transduction The value represents the EC value in the presence of CD40L. 50 Calculate. All other values ​​are IC. 50 .

[0269] #Due to the hook effect, IC 50 The value was obtained by excluding RLU values ​​from conditions containing the three highest concentrations of antibodies.

[0270] Table 15: CD40 signaling in the presence or absence of human CD40L using Raji / NFκB-luc cells Anti-CD40 x CD40 bispecific regulation Table 16: CD40 signaling in the presence or absence of human CD40L using THP-1 / NFκB-luc cells Anti-CD40 x CD40 bispecific regulation The value represents the EC value in the presence of CD40L. 50 Calculations were performed. EC was not determined when no best-fit value was found. 50 Value. All other values ​​are IC. 50 .

[0271] Example 7: CD40xCD40 bispecific antibody in vitro CD40L blocking activity B-cell assay #1 To determine the efficacy of the CD40xCD40 bispecific antibody in blocking CD40L stimulation, the production of IL-6, IL-10, and TNFα cytokines was quantified in antibody-treated primary human B cell cultures in the presence of soluble CD40L. B cells were cultured at 2 x 10⁻⁶ cells / year. 5 Cells were seeded per well in RPMI 1640 medium containing 15% FBS and 1x penicillin-streptomycin in 96 U-shaped plates. CD40xCD40 bispecific antibody was simultaneously added to the cells in the presence of a constant dose of IL-4 (10 pM) and CD40L (500 nM). A dose-response model of CD40L with a constant dose of IL-4 (10 pM) was included as a control. Cells were cultured at 37°C for 3 days, and the cell culture supernatant was collected for analysis.

[0272] Cytokine levels in the supernatant were analyzed using the MSD V-PLEX pro-inflammatory group 1. MSD V-PELX assays were performed using cell culture supernatant diluted 1:2 according to the manufacturer's instructions. MSD plates were read using a MESO QuickPlex Sq 120MM instrument and MSD Discovery Workbench software. Data analysis was performed using Graphpad Prism software. The obtained data points were transformed using the equation X=Log(X), and linear regression analysis was performed on the transformed data and fitted to an sigmoid dose-response curve. The IC50 values ​​were derived from this analysis. 50 .

[0273] result All four antibodies tested (REGN16334, REGN16335, REGN16431, and REGN16432) blocked the production of IL-6 by primary human B cells from three donors in response to CD40L stimulation. Figure 1A , 1B and 1C), IL-10 ( Figure 2A , 2B and 2C) and TNFα ( Figure 3A , 3B (and 3C). IC50 of antibody and COMP11209 (compared to CD40 antibody). 50Values ​​are shown in Tables 17 (IL-6), 18 (IL-10), and 19 (TNFα). Maximum percentage of blockade was calculated using the highest doses of REGN16334, REGN16335, REGN16431, and REGN16432 relative to the mean cytokine levels in untreated cells.

[0274] Table 17: Response of CD40xCD40 bispecific antibody to IL-6 production in human B cells in the presence of constant CD40L. Influence Table 18: Response of CD40xCD40 bispecific antibody to IL-10 production in human B cells in the presence of constant CD40L. Influence Table 19: Effects of CD40xCD40 bispecific antibody on TNFα production in human B cells in the presence of constant CD40L. Influence B-cell assay #2 To determine the efficacy of the CD40xCD40 bispecific antibody in blocking CD40L stimulation, the production of IL-6, IL-10, and TNFα cytokines was quantified in antibody-treated primary human B cell cultures in the presence of soluble CD40L. B cells were cultured at 2 x 10⁻⁶ cells / year. 5 B cells were seeded per well in RPMI 1640 medium containing 15% FBS and 1x penicillin-streptomycin in a 96°C U-shaped plate and incubated with CD40xCD40 bispecific antibody at 37°C for 30 minutes. After antibody incubation, constant doses of IL-4 (10 pM) and CD40L (500 nM) were added to the B cell culture in the presence of CD40xCD40 bispecific antibody. A dose-response relationship between CD40L and a constant dose of IL-4 (10 pM) was included as a control. Simultaneously, to quantify the agonistic activity of the CD40xCD40 bispecific antibody, B cells were incubated with the antibody in the presence of a constant dose of IL4 (10 pM) and in the absence of CD40L. Cells were cultured at 37°C for 3 days, and the cell culture supernatant was collected for analysis.

[0275] Cytokine levels in the supernatant were analyzed using the MSD V-PLEX pro-inflammatory group 1. MSD V-PELX assays were performed using cell culture supernatant diluted 1:2 according to the manufacturer's instructions. MSD plates were read using a MESO QuickPlex Sq 120MM instrument and MSD Discovery Workbench software. Data analysis was performed using Graphpad Prism software. The obtained data points were transformed using the equation X=Log(X), and linear regression analysis was performed on the transformed data and fitted to an sigmoid dose-response curve. The IC50 values ​​were derived from this analysis. 50 .

[0276] result All three CD40xCD40 bispecific antibodies tested (REGN16334, REGN16335, and REGN20484) blocked IL-6 production from primary human B cells from two donors in response to CD40L stimulation. Figure 1D and 1E ), IL10 ( Figure 2D and 2E ) and TNFα ( Figure 3D and 3E Cytokines. IC50 of antibodies and COMP11209. 50 The values ​​are shown in Tables 20 to 22. The maximum percentage of blockade was calculated using the highest doses of REGN16334, REGN16335, and REGN20484 relative to the mean cytokine levels in untreated cells. Agonistative activity analysis of antibodies REGN16334, REGN16335, and REGN20484 showed that, in both donors, the induction of cytokine IL6 was comparable to that in cells treated with only IL4 (without antibody). Figure 3F and 3G ).

[0277] Table 20: Response of CD40xCD40 bispecific antibody to IL-6 production in human B cells in the presence of constant CD40L. Influence Table 21: Response of CD40xCD40 bispecific antibody to IL-10 production in human B cells in the presence of constant CD40L. Influence Table 22: Effects of CD40xCD40 bispecific antibody on TNFα production in human B cells in the presence of constant CD40L. Influence Dendritic cell assay To determine the efficacy of the CD40xCD40 bispecific antibody in blocking CD40L stimulation, IL-12 / IL-23p40 cytokine production was quantified in antibody-treated primary human monocyte-derived dendritic cell (MDDC) cultures in the presence of soluble CD40L. To generate MDDCs, peripheral blood mononuclear cells (PBMCs) from healthy human donors were isolated from leukocyte packs obtained from the New York Blood Center by Ficoll-Paque density gradient centrifugation. CD14+ cells were purified from PBMCs using CD14 human microbeads via positive selection. The purified CD14+ cells were then cultured at 3 x 102 times. 6 MDDCs were seeded per well in RPMI 1640 medium containing 10% FBS, 1x penicillin-streptomycin, 800 U / mL GM-CSF, and 500 U / mL IL-4 in 6-well plates. On days 3 and 5, the medium was replenished, and the full amount of GM-CSF and IL-4 was added back to the cells. On day 6, MDDCs were collected and seeded at 1x10⁻⁶ cells per well. 6 Cells were plated in 96-U plates. CD40xCD40 bispecific antibody and a constant dose of CD40L (20 nM) were simultaneously added to the cells. A dose-response including CD40L served as a control. Cells were cultured at 37°C for 4 days, and the cell culture supernatant was collected for analysis.

[0278] Cytokine levels in the supernatant were analyzed using MSD V-PLEX cytokine assay 1. MSD V-PLEX assays were performed using cell culture supernatant diluted 1:2 or 1:20 according to the manufacturer's instructions. MSD plates were read using a MESO QuickPlexSq 120MM instrument and MSD Discovery Workbench software. Data analysis was performed using Graphpad Prism software. The obtained data points were transformed using the equation X=Log(X), and linear regression analysis was performed on the transformed data and fitted to an sigmoid dose-response curve. The IC50 values ​​were derived from this analysis. 50 .

[0279] result All four antibodies tested (REGN16334, REGN16335, REGN16431, and REGN16432) blocked the production of IL-12 / IL-23p40 by primary human monocyte-derived dendritic cells from two donors in response to CD40L stimulation. Figures 4A to 4B IC50 of antibody and COMP11209 50 The values ​​are shown in Table 23.

[0280] Table 23: Effects of CD40xCD40 bispecific antibody on IL-12 / L in human dendritic cells in the presence of constant CD40L. The effects of IL-23p40 Example 8: CD40xCD40 bispecific antibody in human B cells in vitro agonist activity Simultaneously, to quantify the agonistic activity of the CD40xCD40 bispecific antibody, B cells were incubated with the antibody in the presence of a constant dose of IL-4 (10 pm) and in the absence of CD40L. Cells were cultured at 37°C for 3 days, and the cell culture supernatant was collected for analysis. Cytokine levels in the supernatant were analyzed using MSD V-PLEX pro-inflammatory group 1. MSD V-PELX assays were performed using cell culture supernatant diluted 1:2 according to the manufacturer's instructions. MSD plates were read using a MESO QuickPlexSq 120MM instrument and MSD Discovery Workbench software. Data analysis was performed using Graphpad Prism software. The obtained data points were transformed using the equation X=Log(X), and linear regression analysis was performed on the transformed data and fitted to an sigmoid dose-response curve. The IC50 values ​​were then derived from this analysis. 50 .

[0281] result Activational activity assays of antibodies REGN16334, REGN16335, REGN16431, and REGN16432 showed that, in both donors, the cytokine IL-6 (… Figures 5A to 5B ) and IL-10 ( Figures 6A to 6B The induction of IL-4 was within the range comparable to that of cells not treated with antibodies (IL-4 only).

[0282] Example 9: Effects of CD40xCD40 bispecific antibody in a mouse NP-KLH immunization model To determine the effect of CD40 blockade on antigen-specific antibody responses, the widely used T-dependent immunization model, the NP-KLH immunization model, was used in human CD40 homozygous mice (replacing mouse CD40). Mice were immunized with 25 µg of NP-KLH by subcutaneous injection of 100 µL of a 1:1 mixture of NP-KLH and alum (emulsified by shaking for 30 minutes) in the flank. For the group receiving alum alone, a 1:1 mixture of 100 µL of PBS and alum was administered. Figure 7As shown, mice were administered CD40xCD40 bispecific antibodies (REGN16334, REGN16335, REGN16431, or REGN16432) or isotype controls (REGN4439 or REGN4460) 3 days prior to immunization, and twice weekly for two weeks after immunization, at a dose of 1 mg / kg. Following the immunization and antibody treatment regimens, mice were sacrificed, and blood and inguinal lymph nodes were collected. Blood was collected from mice in all groups via cardiac puncture and transferred to BD microtainer tubes (catalog number 365967) for serum separation.

[0283] For tissue processing, lymph nodes were homogenized using the posterior end of a 3 mL syringe on a 74-micron cell filter in 2 mL RPMI medium + 10% FBS, and the single-cell suspension was filtered through a Millipore plate filter (100 µm) into 2 mL deep-well plates. Cells were centrifuged at 400 g for 4 min and resuspended in 200 µL PBS. Cells were transferred to 96-well U-shaped plates, centrifuged at 400 g for 4 min, and stained with live / dead cell markers for 15 min at room temperature. Cells were washed and incubated with Fc blocking agent at 4 °C for 15 min, followed by antibody staining at 4 °C for 30 min with the antibody mixture shown in Table 24. After staining, cells were washed twice with MACS buffer, fixed with BD Cytofix (catalog number 554655) diluted 1:4 in PBS for 15 min, resuspended in MACS buffer, and stored at 4 °C. On the day of acquisition, cells were washed with BDPerm / wash, incubated in BD Perm / wash buffer (catalog number 554723) for 20 minutes, and stained with intracellular antibodies [Table 24] for 30 minutes. Cells were washed twice, fixed with BD Cytofix, and then resuspended in MACS buffer. Cells were then acquired in a FACSymphony A5 instrument and analyzed using OMIQ software. NP+ germinal center B cells were identified as: viable cells → exclusion group- (exclusion group included TCRb, CD200R3, Ly6G, CD49b, and CD11b) → non-marginal zone B cells (CD1d-) → CD19+B220+ → CD38-IgD- → GC positive (GL7+CD95+) → NP+. Statistical significance was assessed for normality using the Shaprio-Wilk test and determined in GraphPad Prism using the Kruskal-Wallis test and Dunn post-hoc multiple comparison test.

[0284] Table 24: Flow Cytometry Group Experiment #1 - Methods and Results In one experiment, the level of NP-specific IgG1 in the final serum was detected by ELISA. NP-2-BSA was diluted to 4 µg / mL in PBS and coated into 384-well plates overnight at 4°C with 25 µL / well solution. The plates were washed four times with wash buffer and blocked for 1 hour at room temperature with 50 µL / well of 0.5% BSA in PBS. The plates were washed four times with wash buffer, and group A mouse serum was diluted 1:100, and group BG mouse serum was diluted 1:10,000. The serum was further serially diluted 3-fold (8 times) with 0.5% BSA in PBS and added to the plates at 12.5 µL / well. The diluted serum was incubated on the plates at room temperature for 1 hour, followed by four washes. For detection, 25 µL of rat anti-mouse IgG1 HRP-conjugated antibody diluted 1:1000 in 0.5% BSA in PBS was added to the plates and incubated for 1 hour at room temperature. The plate was washed seven times and developed by adding 25 µL of OptEIA™ TMB substrate solution. After 20 minutes, the reaction was terminated by adding 25 µL of 2N sulfuric acid. The absorbance (OD450) at 450 nm was measured on a Molecular Devices SpectraMax M5 plate reader. The relative levels of circulating NP-specific IgG1 in serum were expressed in titer units, defined as the dilution factor required to achieve an OD450 reading equal to twice the background OD450. Graph analysis was performed using GraphPad Prism software (version 7.0). Statistical significance was assessed for normality using the Shaprio-Wilk test and determined in GraphPad Prism by the Kruskal-Wallis test and Dunn's post-hoc multiple comparison test.

[0285] In this NP-KLH immunization model, prophylactic treatment with CD40xCD40 bispecific antibodies blocked the formation of antigen-specific germinal center B cells in draining lymph nodes, as quantified by flow cytometry. All CD40xCD40 bispecific antibodies, compared to their associated isotype controls, reduced the frequency of NP-specific germinal center B cells at 1 mg / kg. Figure 8A (See Table 25). Prophylactic treatment with 1 mg / kg of CD40xCD40 bispecific antibody also blocked the generation of a high-affinity NP-IgG1 antibody response, as measured by NP-specific IgG1 titers in serum, with most samples below the limit of quantitation. Figure 8B (and Table 26).

[0286] Table 25: Effect of CD40xCD40 bispecific antibody on the frequency of B cells in NP+ germinal centers SD = standard deviation; N / A = not applicable; ns = not statistically significant. p<0.05.

[0287] Table 26: Effect of CD40xCD40 bispecific antibody on the frequency of NP IgG1 titer in mouse serum SD = standard deviation; N / A = not applicable; ns = not statistically significant. p<0.05; p<0.005.

[0288] Experiment #2 - Methods and Results In another experiment, the level of NP-specific IgG1 in the final serum was detected by ELISA. NP-2-BSA was diluted to 4 µg / mL in PBS and coated into 384-well plates overnight at 4°C with 25 µL / well solution. The plates were washed five times with wash buffer and blocked for 1 hour at room temperature with 50 µL / well of 0.5% BSA in PBS. The plates were washed five times with wash buffer, and serum from groups AB mice was diluted 1:100, and serum from group CL mice was diluted 1:10,000. The serum was further serially diluted 3-fold (8 times) with 0.5% BSA in PBS and added to the plates at 12.5 µL / well. The diluted serum was incubated on the plates at room temperature for 1 hour, followed by four washes. For detection, 25 µL of rat anti-mouse IgG1 HRP-conjugated antibody diluted 1:1,000 in 0.5% BSA in PBS was added to the plates and incubated for 1 hour at room temperature. The plate was washed seven times and developed by adding 25 µL of OptEIA™ TMB substrate solution. After 20 minutes, the reaction was terminated by adding 25 µL of 2N sulfuric acid. The absorbance (OD450) at 450 nm was measured on a Molecular Devices SpectraMax M5 plate reader. The relative levels of circulating NP-specific IgG1 in serum were expressed in titer units, defined as the dilution factor required to achieve an OD450 reading equal to twice the background OD450. Data analysis was performed using GraphPad Prism. Statistical significance was assessed for normality using the Shaprio-Wilk test and determined in GraphPad Prism by the Kruskal-Wallis test and Dunn's post-hoc multiple comparison test.

[0289] In this experiment, prophylactic treatment with CD40xCD40 bispecific antibodies blocked the formation of antigen-specific germinal center B cells in draining lymph nodes, as quantified by flow cytometry. All tested CD40xCD40 bispecific antibodies, compared to isotype controls, reduced the frequency of NP-specific germinal center B cells at doses of 1 mg / kg or higher. Figure 9A (See Table 27). Prophylactic treatment with 1 mg / kg of CD40xCD40 bispecific antibody also blocked the generation of a high-affinity NP-IgG1 antibody response, as measured by NP-specific IgG1 titers in serum, with most samples below the limit of quantitation. Figure 9B (and Table 28).

[0290] Table 27: Effect of CD40xCD40 bispecific antibody on the frequency of B cells in NP+ germinal centers SD = standard deviation; N / A = not applicable; ns = not statistically significant. p<0.05.

[0291] Table 28: Effect of CD40xCD40 bispecific antibody on the frequency of NP IgG1 titer in mouse serum SD = standard deviation; N / A = not applicable; ns = not statistically significant. p<0.05.

[0292] Example 10: The effect of prophylactic administration of CD40xCD40 bispecific antibody in a mouse model of experimental autoimmune encephalomyelitis (EAE) To determine the impact of CD40 blockade on autoimmune diseases, an experimental autoimmune encephalomyelitis (EAE) model was used in mice, in which the entire mouse CD40 gene was blocked by full-length human CD40 (CD40) receptors. hu / hu (Mice) as an alternative. Mice were immunized by subcutaneous injection of 0.1 mL of an emulsion containing complete Freund's adjuvant in the upper and lower back (0.2 mL / mouse for total). Two hours post-immunization, mice were administered 150 ng of pertussis toxin (PTX) intraperitoneally at a dose of 0.1 mL / mice. PTX was repeated 24 hours later. Three days prior to immunization, mice were administered a CD40 x CD40 bispecific antibody or an isotype control at 25 mg / kg, and this was continued twice weekly for five weeks post-immunization. See also Figure 10Mice were monitored and weighed twice weekly for the first two weeks after immunization, and monitored and weighed daily from weeks 3 to 5. Mice were scored according to the scoring guidelines outlined in Table 29. Mice were euthanized after two consecutive days of a symptom score of 4, or when the score was 4.5 or 5, or when the mice had lost 30% of their initial body weight.

[0293] Table 29: Mouse EAE Scoring Guidelines Experiment #1 - Results In a study using an experimental autoimmune encephalomyelitis (EAE) model, prophylactic blockade of CD40 using the CD40xCD40 bispecific antibodies REGN16334, REGN16335, REGN16431, and REGN16432 reduced the severity of EAE disease symptom scores compared to antibody-free or allotype-controlled treatment groups. Figures 11A to 11B (See Table 30). In addition to reducing symptom severity, all CD40 blocking antibodies reduced the percentage of weight loss in mice compared to the antibody-free or isotype-controlled treatment groups. Figures 11C to 11D (See Table 31). In the group treated with CD40 blocking antibodies, the frequency of disease scores in mice was also reduced (Table 32).

[0294] Table 30: Average EAE Symptom Score Table 31: Average Weight Loss Table 32: Number of mice exhibiting EAE symptoms Experiment #2 - Results In another trial, prophylactic blockade of CD40 with the CD40xCD40 bispecific antibodies REGN16335 or REGN20484 reduced the severity of EAE disease symptom scores compared to the antibody-free or allotype control groups. Figure 12A (See Table 33). In addition to reducing symptom severity, all CD40 blocking antibodies reduced the percentage of weight loss in mice compared to the antibody-free or isotype-controlled treatment groups. Figure 12B (See Table 34). In the group treated with CD40 blocking antibodies, the frequency of disease scores in mice was also reduced (Table 35).

[0295] Table 33: Average EAE Symptom Scores Table 34: Average Weight Loss Table 35: Number of mice exhibiting EAE symptoms Example 11: CryoEM analysis of CD40xCD40 bispecific antibody To better understand the binding of REGN16335 and REGN16334 to CD40, structural analysis was performed using cryo-electron microscopy (cryo-EM). Fab fragments were prepared enzymatically. Fab fragment 30027P2 corresponds to arm 1 of REGN16335, Fab fragment 21519P2 corresponds to arm 1 of REGN16334, and Fab fragment 21520P2 corresponds to arm 2 of both REGN16335 and REGN16334.

[0296] A 3D reconstruction of the CD40 complex with three Fab arms (30027P2, 21519P2, and 21520P2) at 3 Å resolution shows that these three arms bind non-overlapping epitopes spanning the CRD1, CRD2, and CRD3 domains. Figure 13 ). 30027P2Fab mainly combines the CRD1 structural domain and a small portion of the CRD2, 21520P2 Fab combines across the CRD1 and CRD2 structural domains, and 21519P2 Fab combines across the CRD2 and CRD3 structural domains.

[0297] This invention is not limited to the specific embodiments described herein. In fact, various modifications to the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and drawings. Such modifications are intended to fall within the scope of the appended claims. All disclosures of patent and non-patent documents cited herein are expressly incorporated herein by reference in their entirety.

Claims

1. A bispecific antigen binding molecule comprising: (a) a first antigen binding domain (D1) that binds to a first epitope of human CD40; and (b) a second antigen binding domain (D2) that binds to 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, K D less than 25 nM as measured by surface plasmon resonance at 25 °C; (ii) binds human CD40, K D less than 70 nM, as measured by surface plasmon resonance at 37 °C; (iii) binds human CD40 with a dissociation half-time (t 1 / 2 ) greater than 75 minutes as measured by surface plasmon resonance at 25°C; (iv) binds to a cell expressing human CD40, EC 50 a 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) HCDR1 comprising the amino acid sequence of SEQ ID NO:4, HCDR2 comprising the amino acid sequence of SEQ ID NO:6, and HCDR3 comprising the amino acid sequence of SEQ ID NO:8; (b) HCDR1 comprising the amino acid sequence of SEQ ID NO:24, HCDR2 comprising the amino acid sequence of SEQ ID NO:26, and HCDR3 comprising the amino acid sequence of SEQ ID NO:28; and (c) HCDR1 comprising the amino acid sequence of SEQ ID NO:34, HCDR2 comprising the amino acid sequence of SEQ ID NO:36, and HCDR3 comprising the amino acid sequence of SEQ ID NO:

38.

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) HCDR1 comprising the amino acid sequence of SEQ ID NO:4, HCDR2 comprising the amino acid sequence of SEQ ID NO:6, HCDR3 comprising the amino acid sequence of SEQ ID NO:8, LCDR1 comprising the amino acid sequence of SEQ ID NO: 12, LCDR2 comprising the amino acid sequence AAS, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 16; or (b) HCDR1 comprising the amino acid sequence of SEQ ID NO:24, HCDR2 comprising the amino acid sequence of SEQ ID NO:26, HCDR3 comprising the amino acid sequence of SEQ ID NO:28, LCDR1 comprising the amino acid sequence of SEQ ID NO: 12, LCDR2 comprising the amino acid sequence of AAS, and 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: HCDR1 comprising the amino acid sequence of SEQ ID NO:4, HCDR2 comprising the amino acid sequence of SEQ ID NO:6, HCDR3 comprising the amino acid sequence of SEQ ID NO:8, LCDR1 comprising the amino acid sequence of SEQ ID NO: 12, LCDR2 comprising the amino acid sequence of AAS, and 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: HCDR1 comprising the amino acid sequence of SEQ ID NO:24, HCDR2 comprising the amino acid sequence of SEQ ID NO:26, HCDR3 comprising the amino acid sequence of SEQ ID NO:28, LCDR1 comprising the amino acid sequence of SEQ ID NO: 12, LCDR2 comprising the amino acid sequence of AAS, and 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: a 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: HCDR1 comprising the amino acid sequence of SEQ ID NO:34, HCDR2 comprising the amino acid sequence of SEQ ID NO:36, HCDR3 comprising the amino acid sequence of SEQ ID NO:38, LCDR1 comprising the amino acid sequence of SEQ ID NO: 12, LCDR2 comprising the amino acid sequence of AAS, and 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: a 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: a LCVR comprising the amino acid sequence of SEQ ID NO:

10.

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

16.

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

10.

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

16.

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

10.

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

10.

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

10.

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

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 NO: 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 NO:

10.

26. The bispecific antigen binding molecule of any one of claims 1 to 25, which 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 of the isotype IgG4 or IgG1.

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 reduce binding to an Fc receptor.

30. The bispecific antigen binding molecule of any one of claims 1 to 17 and 26 to 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 NO: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 NO: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 NO:

20.

31. The bispecific antigen binding molecule of any one of claims 1 to 17 and 26 to 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 NO:20; or a heavy chain comprising the amino acid sequence of SEQ ID NO:50 and a light chain comprising the amino acid sequence of SEQ ID NO:

20.

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

20.

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

20.

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

20.

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

20.

36. The bispecific antigen-binding molecule 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 NO:

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 NO:60 and a light chain comprising the amino acid sequence of SEQ ID NO:

20.

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

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 of the nucleotide sequences listed 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 that allow for 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 expressing 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 to 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 an 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 (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’s granulomatosis.

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

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