Anti-CD40 antibodies and uses thereof

Abstract

The present disclosure relates to anti-CD40 (TNF receptor superfamily member 5) antibodies, antigen binding fragments and uses thereof.

Description

[0001] Priority declaration

[0002] This application claims the benefit of PCT application number PCT / CN2022 / 087003, filed on April 15, 2022. The entire contents of the foregoing application are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to anti-CD40 (TNF receptor superfamily member 5) antibodies and uses thereof. Background Art

[0004] An autoimmune disease is a condition caused by an abnormal immune response to a normal body part. There are at least 80 autoimmune diseases. The causes of autoimmune diseases are often not very clear. Some autoimmune diseases, such as lupus, run in families, while others may be triggered by infections or other environmental factors. Some common autoimmune diseases include, for example, celiac disease, type 1 diabetes, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.

[0005] The recent clinical and commercial success of therapeutic antibodies has generated great interest in using antibodies to treat various immune-related diseases. There is a need to develop antibody-based therapies for the treatment of autoimmune diseases. Summary of the invention

[0006] The present disclosure relates to anti-CD40 antibodies, antigen-binding fragments thereof, and uses thereof.

[0007] In one aspect, the present disclosure relates to an antibody or antigen-binding fragment thereof that binds to CD40 (TNF receptor superfamily member 5), the antibody or antigen-binding fragment thereof comprising: a heavy chain variable region (VH), the VH comprising complementarity determining regions (CDRs) 1, 2, and 3, in some embodiments, the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL), the VL comprising CDR1, 2, and 3, in some embodiments, the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL The CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VLCDR3 amino acid sequence, and in some embodiments, the selected VHCDR1, 2, and 3 amino acid sequence and the selected VL CDR1, 2, and 3 amino acid sequence are one of:

[0008] (1) the selected VH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1, 2, 3, respectively, and the selected VLCDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 4, 5, 6, respectively;

[0009] (2) the selected VH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 7, 8, 9, respectively, and the selected VLCDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 10, 11, 12, respectively;

[0010] (3) the selected VH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 13, 14, 15, respectively, and the selected VL CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 16, 17, 18, respectively;

[0011] (4) the selected VH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 19, 20, 21, respectively, and the selected VL CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 22, 23, 24, respectively;

[0012] (5) the selected VH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 25, 26, 27, respectively, and the selected VL CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 28, 29, 30, respectively; and

[0013] (6) The selected VH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 31, 32, 33, respectively, and the selected VL CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 34, 35, 36, respectively.

[0014] In some embodiments, VH comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NOs: 1, 2, and 3, respectively, and VL comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NOs: 4, 5, and 6, respectively. In some embodiments, VH comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NOs: 7, 8, and 9, respectively, and VL comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NOs: 10, 11, and 12, respectively. In some embodiments, VH comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NOs: 13, 14, and 15, respectively, and VL comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NOs: 16, 17, and 18, respectively. In some embodiments, VH comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NOs: 19, 20 and 21, respectively, and VL comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NOs: 22, 23 and 24, respectively. In some embodiments, VH comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NOs: 25, 26 and 27, respectively, and VL comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NOs: 28, 29 and 30, respectively. In some embodiments, VH comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NOs: 31, 32 and 33, respectively, and VL comprises CDR1, 2, 3 having amino acid sequences as shown in SEQ ID NOs: 34, 35 and 36, respectively.

[0015] In some embodiments, the antibody or its antigen binding fragment specifically binds to human, monkey or dog CD40. In some embodiments, the antibody or its antigen binding fragment is a human antibody or its antigen binding fragment. In some embodiments, the antibody or its antigen binding fragment is a single chain variable fragment (scFV) or a multispecific antibody (e.g., a bispecific antibody). In some embodiments, the antibody or its antigen binding fragment is a human IgG1 antibody or its antigen binding fragment (optionally with YTE and / or LALA mutations) or a human IgG4 antibody or its antigen binding fragment (optionally with YTE).

[0016] In one aspect, the present disclosure relates to a nucleic acid comprising a polynucleotide encoding a polypeptide comprising:

[0017] (1) an immunoglobulin heavy chain or a fragment thereof, wherein the immunoglobulin heavy chain or the fragment thereof comprises a heavy chain variable region (VH), wherein the VH comprises complementary determining regions (CDRs) 1, 2, and 3, wherein CDRs 1, 2, and 3 comprise the residues of SEQ ID NOs: 1, 2, and 3, respectively.

[0018] and 3, and in some embodiments, when paired with a light chain variable region (VL) comprising the amino acid sequence as shown in SEQ ID NO:37, the VH binds CD40;

[0019] (2) an immunoglobulin light chain or a fragment thereof, comprising a VL, wherein the VL comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein CDRs 1, 2, and 3 comprise the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, respectively, and in some embodiments, when paired with a VH comprising the amino acid sequence shown in SEQ ID NO: 65, the VL binds to CD40;

[0020] (3) an immunoglobulin heavy chain or a fragment thereof, wherein the immunoglobulin heavy chain or the fragment thereof comprises a heavy chain variable region (VH), wherein the VH comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein CDRs 1, 2, and 3 comprise SEQ ID NOs: 7, 8, respectively.

[0021] and 9, and in some embodiments, when paired with a light chain variable region (VL) comprising the amino acid sequence as shown in SEQ ID NO:39, the VH binds CD40;

[0022] (4) an immunoglobulin light chain or a fragment thereof, comprising a VL, wherein the VL comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein CDRs 1, 2, and 3 comprise the amino acid sequences shown in SEQ ID NOs: 10, 11, and 12, respectively, and in some embodiments, when paired with a VH comprising the amino acid sequence shown in SEQ ID NO: 38, the VL binds to CD40;

[0023] (5) an immunoglobulin heavy chain or a fragment thereof, comprising a VH, wherein the VH comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein CDRs 1, 2, and 3 comprise the amino acid sequences shown in SEQ ID NOs: 13, 14, and 15, respectively, and in some embodiments, when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 41, the VH binds to CD40; or

[0024] (6) an immunoglobulin light chain or a fragment thereof, comprising a VL, wherein the VL comprises CDR1, 2, and 3, wherein CDR1, 2, and 3 comprise the amino acid sequences shown in SEQ ID NO:16, 17, and 18, respectively, and in some embodiments, when paired with a VH comprising the amino acid sequence shown in SEQ ID NO:40, the VL binds to CD40.

[0025] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, the immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, 3, and CDR1, 2, 3 comprising the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, respectively. In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof, the immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, 3, and CDR1, 2, 3 comprising the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, respectively. In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, the immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, 3, and CDR1, 2, 3 comprising the amino acid sequences shown in SEQ ID NOs: 7, 8, and 9, respectively. In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof, the immunoglobulin light chain or a fragment thereof comprising a VL, the VL comprising CDR1, 2, 3, and CDR1, 2, 3 comprising the amino acid sequences shown in SEQ ID NOs: 10, 11, and 12, respectively. In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, the immunoglobulin heavy chain or a fragment thereof comprising a VH, the VH comprising CDR1, 2, 3, and CDR1, 2, 3 comprising the amino acid sequences shown in SEQ ID NOs: 13, 14, and 15, respectively. In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof, the immunoglobulin light chain or a fragment thereof comprising a VL, the VL comprising CDR1, 2, 3, and CDR1, 2, 3 comprising the amino acid sequences shown in SEQ ID NOs: 16, 17, and 18, respectively.

[0026] In some embodiments, when paired with VL, VH specifically binds to human, monkey or dog CD40, or when paired with VH, VL specifically binds to human, monkey or dog CD40. In some embodiments, the immunoglobulin heavy chain or fragment thereof is a human immunoglobulin heavy chain or fragment thereof (e.g., a human IgG1 heavy chain or fragment thereof, optionally with a YTE and / or LALA mutation; or a human IgG4 heavy chain or fragment thereof, optionally with a YTE), and the immunoglobulin light chain or fragment thereof is a human immunoglobulin light chain or fragment thereof. In some embodiments, the nucleic acid encodes a single-chain variable fragment (scFv) or a multispecific antibody (e.g., a bispecific antibody). In some embodiments, the nucleic acid is a cDNA.

[0027] In one aspect, the disclosure relates to a vector comprising one or more of the nucleic acids as described herein. In one aspect, the disclosure relates to a vector comprising two of the nucleic acids as described herein, which in some embodiments encode a VL region and a VH region that jointly bind to CD40. In one aspect, the disclosure relates to a pair of vectors, each of which in some embodiments comprises one of the nucleic acids as described herein, which in some embodiments jointly encode a VL region and a VH region that jointly bind to CD40.

[0028] In one aspect, the disclosure relates to a cell comprising a vector or vector pair as described herein. In some embodiments, the cell is a CHO cell. In one aspect, the disclosure relates to a cell comprising one or more of the nucleic acids as described herein. In one aspect, the disclosure relates to a cell comprising two of the nucleic acids as described herein. In some embodiments, the two nucleic acids together encode a VL region and a VH region that together bind CD40.

[0029] In one aspect, the present disclosure relates to a method of producing an antibody or an antigen-binding fragment thereof, the method comprising: (a) culturing a cell as described herein under conditions sufficient for the cell to produce the antibody or antigen-binding fragment; and (b) collecting the antibody or antigen-binding fragment produced by the cell.

[0030] In one aspect, the present disclosure relates to an antibody or antigen-binding fragment thereof that binds CD40, the antibody or antigen-binding fragment thereof comprising: a heavy chain variable region (VH), the VH comprising an amino acid sequence that is at least 90% identical to a selected VH sequence, and a light chain variable region (VL), the VL comprising an amino acid sequence that is at least 90% identical to a selected VL sequence, in some embodiments, the selected VH sequence and the selected VL sequence are one of the following: (1) the selected VH sequence is SEQ ID NO:65, and the selected VL sequence is SEQ ID NO:37; (2) the selected VH sequence is SEQ ID NO:38, and the selected VL sequence is SEQ ID NO:39; and (3) the selected VH sequence is SEQ ID NO:40, and the selected VL sequence is SEQ ID NO:41.

[0031] In some embodiments, VH comprises the sequence of SEQ ID NO: 65, and VL comprises the sequence of SEQ ID NO: 37. In some embodiments, VH comprises the sequence of SEQ ID NO: 38, and VL comprises the sequence of SEQ ID NO: 39. In some embodiments, VH comprises the sequence of SEQ ID NO: 40, and VL comprises the sequence of SEQ ID NO: 41.

[0032] In some embodiments, the antibody or antigen binding fragment thereof specifically binds to human, monkey or dog CD40. In some embodiments, the antibody or antigen binding fragment thereof is a human antibody or antigen binding fragment thereof. In some embodiments, the antibody or antigen binding fragment is a single chain variable fragment (scFV) or a multispecific antibody (e.g., a bispecific antibody). In some embodiments, the antibody or antigen binding fragment is a human IgG1 antibody or antigen binding fragment thereof (optionally with YTE and / or LALA mutations) or a human IgG4 antibody or antigen binding fragment thereof (optionally with YTE).

[0033] In one aspect, the disclosure relates to an antibody or antigen-binding fragment thereof comprising the VH CDR1, 2, 3 and VL CDR1, 2, 3 of the antibody or antigen-binding fragment thereof as described herein.

[0034] In one aspect, the disclosure relates to an antibody or antigen-binding fragment thereof that cross-competes with an antibody or antigen-binding fragment thereof as described herein.

[0035] In one aspect, the disclosure relates to an antibody drug conjugate comprising an antibody or antigen-binding fragment thereof as described herein covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic agent or a cytostatic agent.

[0036] In one aspect, the present disclosure relates to a method of treating a subject suffering from cancer, the method comprising: administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described herein or an antibody drug conjugate as described herein. In some embodiments, the subject suffers from a solid tumor. In some embodiments, the cancer is melanoma, pancreatic cancer, mesothelioma, or a hematological malignancy. In some embodiments, the cancer is non-Hodgkin's lymphoma, lymphoma, or chronic lymphocytic leukemia.

[0037] In one aspect, the present disclosure relates to a method of reducing tumor growth rate, the method comprising contacting tumor cells with an effective amount of a composition comprising an antibody or antigen binding fragment thereof as described herein or an antibody drug conjugate as described herein. In one aspect, the present disclosure relates to a method of killing tumor cells, the method comprising contacting the tumor cells with an effective amount of a composition comprising an antibody or antigen binding fragment thereof as described herein or an antibody drug conjugate as described herein. In one aspect, the present disclosure relates to a method of suppressing an immune response in a subject, the method comprising: administering to the subject an effective amount of a composition comprising an antibody or antigen binding fragment thereof as described herein or an antibody drug conjugate as described herein. In some embodiments, the subject suffers from an autoimmune disease.

[0038] In one aspect, the present disclosure relates to a method for treating an autoimmune disease, the method comprising administering to a subject an effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described herein or an antibody drug conjugate as described herein. In some embodiments, the autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, atopic dermatitis, or multiple sclerosis.

[0039] In one aspect, the present disclosure relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described herein and a pharmaceutically acceptable carrier. In one aspect, the present disclosure relates to a pharmaceutical composition comprising an antibody drug conjugate as described herein and a pharmaceutically acceptable carrier.

[0040] In one aspect, the present disclosure relates to an antibody or antigen-binding fragment thereof that binds to CD40, the antibody or antigen-binding fragment thereof comprising an Fc region, which in some embodiments lacks ADCC activity or has reduced ADCC activity compared to a wild-type Fc region. In some embodiments, the KD between the antibody or antigen-binding fragment thereof and FcRn (e.g., human FcRn) is less than 1×10 -5 M, less than 5×10 -6 M, less than 1×10 -6 M, less than 5×10 -7 M, less than 1×10 -7 M or less than 5×10 -8 M. In some embodiments, the Fc region is of IgG1 or IgG4 subtype. In some embodiments, the Fc region comprises a YTE mutation. In some embodiments, the Fc region comprises a LALA mutation. In some embodiments, when administered to a subject, the half-life of an antibody or antigen-binding fragment thereof as described herein is at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, or at least 18 days. In some embodiments, the subject is a mouse. In some embodiments, the subject is genetically modified to express human or humanized CD40.

[0041] As used herein, the term "cancer" refers to a cell with autonomous growth ability. Examples of such cells include cells with abnormal states or conditions characterized by rapid proliferation of cell growth. The term is intended to include cancerous growth (e.g., tumors), carcinogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, regardless of their histopathological type or invasion stage. Malignant tumors of various organ systems are also included, such as the respiratory system, cardiovascular system, renal system, reproductive system, blood system, nervous system, liver system, gastrointestinal system, and endocrine system; and adenocarcinomas, which include malignant tumors, such as most colon cancers, renal cell carcinomas, prostate cancers, and / or testicular tumors, non-small cell lung cancers, and small intestinal cancers. "Naturally occurring" cancers include any cancer that is not experimentally induced by implanting cancer cells into a subject, and include, for example, spontaneously occurring cancers, cancers caused by exposure of patients to carcinogens, cancers caused by insertion of transgenic oncogenes or knockout of tumor suppressor genes, and cancers caused by infection (e.g., viral infection). The term "cancer" is recognized in the art and refers to a malignant tumor of epithelial or endocrine tissue. The term also includes carcinosarcoma, which includes malignant tumors composed of carcinoma and sarcoma tissue. "Adenocarcinoma" refers to a cancer derived from glandular tissue or in which the tumor cells form a recognizable glandular structure. The term "sarcoma" is recognized in the art and refers to a malignant tumor derived from mesenchyme. The term "hematopoietic neoplastic disease" includes diseases involving hyperplastic / tumor cells of hematopoietic origin. Hematopoietic neoplastic diseases can be caused by myeloid cells, lymphocytes or erythroid cells or their precursor cells.

[0042] As used herein, the term "antibody" refers to any antigen binding molecule that contains at least one (e.g., one, two, three, four, five or six) complementary determining regions (CDRs) (e.g., any one of the three CDRs from an immunoglobulin light chain or any one of the three CDRs from an immunoglobulin heavy chain), and is capable of specifically binding to an epitope. Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, the antibody may contain the Fc region of a human antibody. The term antibody also includes derivatives such as bispecific antibodies, single-chain antibodies, double antibodies, linear antibodies, and multispecific antibodies formed by antibody fragments.

[0043] As used herein, the term "antigen binding fragment" refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen. In some embodiments, the antigen binding fragment contains at least one variable domain (e.g., a heavy chain variable domain or a light chain variable domain). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments.

[0044] As used herein, the term "human antibody" refers to an antibody encoded by an endogenous nucleic acid present in a human (e.g., a rearranged human immunoglobulin heavy chain or light chain locus). In some embodiments, human antibodies are collected from humans or produced in human cell cultures (e.g., human hybridoma cells). In some embodiments, human antibodies are produced in non-human cells (e.g., mouse or hamster cell lines). In some embodiments, human antibodies are produced in bacteria or yeast cells. In some embodiments, human antibodies are produced in transgenic non-human animals (e.g., cattle) containing unrearranged or rearranged human immunoglobulin loci (e.g., heavy chain or light chain human immunoglobulin loci).

[0045] As used herein, the term "chimeric antibody" refers to an antibody containing sequences present in at least two different antibodies (e.g., antibodies from two different mammalian species, such as human and mouse antibodies). A non-limiting example of a chimeric antibody is an antibody containing a variable domain sequence (e.g., all or part of a light chain and / or heavy chain variable domain sequence) of a non-human (e.g., mouse) antibody and a constant domain of a human antibody. Other examples of chimeric antibodies are described herein and are known in the art.

[0046] As used herein, the term "humanized antibody" refers to a non-human antibody, which contains a minimum sequence derived from a non-human (e.g., mouse) immunoglobulin, and contains a sequence derived from a human immunoglobulin. In a non-limiting example, a humanized antibody is a human antibody (receptor antibody), wherein the hypervariable region (e.g., CDR) residues of the receptor antibody are replaced by hypervariable regions (e.g., CDR) residues from a non-human antibody (e.g., donor antibody) with desired specificity, affinity and ability, such as mouse, rat or rabbit antibody. In some embodiments, the Fv framework residues of human immunoglobulin are replaced by corresponding non-human (e.g., mouse) immunoglobulin residues. In some embodiments, a humanized antibody may contain residues not found in a receptor antibody or a donor antibody. These modifications may be performed to further improve antibody performance. In some embodiments, humanized antibodies include at least one and generally substantially all of two variable domains, wherein all or substantially all of the hypervariable loops (CDRs) correspond to the hypervariable loops of non-human (e.g., mouse) immunoglobulins, and all or substantially all of the framework regions are framework regions of human immunoglobulins. Humanized antibodies may also include at least a portion of an immunoglobulin constant region (Fc), generally a constant region of a human immunoglobulin. Humanized antibodies can be produced using molecular biology methods known in the art. Non-limiting examples of methods for producing humanized antibodies are described herein.

[0047] As used herein, the term "single-chain antibody" refers to a single polypeptide containing at least two immunoglobulin variable domains (e.g., variable domains of mammalian immunoglobulin heavy or light chains) that are capable of specific binding to an antigen. Non-limiting examples of single-chain antibodies are described herein.

[0048] As used herein, the term "multimeric antibody" refers to an antibody containing four or more (e.g., six, eight, or ten) immunoglobulin variable domains. In some embodiments, the multimeric antibody is capable of cross-linking a target molecule (e.g., CD40) on the surface of a mammalian cell (e.g., a human T cell) to at least one second target molecule (e.g., CTLA-4).

[0049] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification, and describe an animal (human or non-human) for which treatment is provided according to the method of the present invention. Veterinary and non-veterinary applications are contemplated by the present invention. Human patients can be adults or adolescents (e.g., people under 18 years of age). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. Including, for example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, pigs (e.g., pigs, miniature pigs), horses, dogs, cats, cattle, and other domestic animals, farm animals, and zoo animals.

[0050] As used herein, when referring to an antibody, the phrase "specifically binds" means that the antibody preferentially interacts with its target molecule (e.g., CD40) over other molecules because the interaction depends on the presence of a specific structure (i.e., antigenic determinant or epitope) on the target molecule; in other words, the agent recognizes and binds to molecules that include a specific structure, rather than all molecules as is generally the case. An antibody that specifically binds to a target molecule may be referred to as a target-specific antibody. For example, an antibody that specifically binds to a CD40 molecule may be referred to as a CD40-specific antibody or an anti-CD40 antibody.

[0051] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to amino acid polymers of any length of at least two amino acids.

[0052] As used herein, the terms "polynucleotide," "nucleic acid molecule," and "nucleic acid sequence" are used interchangeably herein to refer to nucleotide polymers of any length of at least two nucleotides, and include but are not limited to DNA, RNA, DNA / RNA hybrids, and modifications thereof.

[0053] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail.

[0054] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The experimental scheme for analyzing the effect of anti-CD40 antibodies on immune responses in hCD40 mice is shown. Anti-CD40 antibodies were administered on days 0 and 4. OVA and CFA were administered on day 1. Anti-OVA antibody levels were determined by ELISA on days 10 and 17.

[0056] Figure 2 Shown are anti-OVA antibody levels measured by ELISA at day 10. hCD40 mice were administered PBS (G1), blelumab analogs (G2), 12B5-IgG4-FLAA (G3), 16D5-IgG4-FLAA (G4), 2F8-IgG4-FLAA (G5), 6A4-IgG4-FLAA (G6), or 15B4-IgG4-FLAA (G7).

[0057] Figure 3 Shown are anti-OVA antibody levels at day 17 as determined by ELISA.

[0058] Figure 4 Shown are changes in the concentrations of blelumab analogs (G1), 12B5-IgG4-FLAA (G2), 6A4-IgG4-FLAA (G3) and 2F8-IgG4-FLAA (G4) in the serum of hCD40 mice over time, and changes in the concentrations of blelumab analogs (G5), 12B5-IgG4-FLAA (G6), 6A4-IgG4-FLAA (G7) and 2F8-IgG4-FLAA (G8) in the serum of C57BL / 6 mice over time.

[0059] Figure 5Shown are changes in the concentrations of 12B5-IgG1-LALA-YTE (G1), 12B5-IgG1-N297A-YTE (G2), 12B5-IgG4-YTE (G3), and 12B5-IgG4-FLAA (G4) in the serum of hFcRn mice over time.

[0060] Figure 6 Anti-OVA antibody levels at day 10 as determined by ELISA are shown. hCD40 / hFcRn mice were administered PBS (G1), breklumab analogs (G2-G4), 12B5-IgG1-LALA-YTE (G5-G7), or 2F8-IgG1-LALA-YTE (G8-G10). Anti-CD40 antibodies were administered on day 0. OVA and CFA were administered on day 1.

[0061] Figure 7 Shown are anti-OVA antibody levels at day 17 as determined by ELISA.

[0062] Figure 8 Shown are anti-OVA antibody levels at day 21 as determined by ELISA.

[0063] Fig. 9 The CDR sequences of anti-CD40 antibodies (2F8, 6A4, and 12B5) as well as the CDR sequences of their related anti-CD40 antibodies as defined by Kabat numbering are listed.

[0064] Fig.10 The CDR sequences of anti-CD40 antibodies (2F8, 6A4, and 12B5) and the CDR sequences of their related anti-CD40 antibodies defined by Chothia numbering are listed.

[0065] Fig.11 The amino acid sequences of the heavy chain variable regions and light chain variable regions of the anti-CD40 antibodies (2F8, 6A4, and 12B5) are listed.

[0066] Fig.12 Cytotoxicity data for 12B5-IgG1-LALA-YTE, 12B5-IgG1, rituximab analogs, and human IgG1 are shown.

[0067] Fig.13 Cytotoxicity data for 12B5-IgG1-LALA-YTE, rituximab analogs, and human IgG1 are shown.

[0068] Fig.14 The inhibitory effects of 12B5-IgG1-LALA-YTE and human IgG1 on PBMC proliferation are shown.

[0069] Fig.15A The inhibitory effect of 12B5-IgG1-LALA-YTE on B cell proliferation is shown.

[0070] Fig. 15B The inhibitory effect of hIgG1 on B cell proliferation is shown.

[0071] Fig.16 Shown are the body weights of hCD40 / hFcRn mice injected with MOG to induce the EAE model and then treated with hIgG1 (G2) or anti-hCD40 antibody 2F8-IgG1-LALA-YTE (G3). hCD40 / hFcRn mice injected with PBS were used as controls (G1).

[0072] Fig.17 Shown are clinical scores of hCD40 / hFcRn mice injected with MOG to induce the EAE model and then treated with hIgG1 (G2) or anti-hCD40 antibody 2F8-IgG1-LALA-YTE (G3). hCD40 / hFcRn mice injected with PBS were used as controls (G1).

[0073] Fig.18 Shown are H&E (hematoxylin and eosin) staining images of the spinal cord of hCD40 / hFcRn mice injected with MOG to induce the EAE model and then treated with hIgG1 (G2) or anti-hCD40 antibody 2F8-IgG1-LALA-YTE (G3). hCD40 / hFcRn mice injected with PBS were used as controls (G1). Inflammatory cell infiltration was examined in the images.

[0074] Fig.19 Shown are LFB (Luxol Fast Blue) stained images of spinal cords from hCD40 / hFcRn mice injected with MOG to induce the EAE model and then treated with hIgG1 (G2) or anti-hCD40 antibody 2F8-IgG1-LALA-YTE (G3). hCD40 / hFcRn mice injected with PBS were used as controls (G1). Spinal cord demyelination was examined in the images.

[0075] Fig. 20 Shown are the body weights of hCD40 mice injected with CII emulsion to induce CIA model and then treated with PBS (G2) or anti-hCD40 antibody 12B5-IgG1-LALA-YTE (G3). hCD40 mice injected with PBS only were used as controls (G1).

[0076] Fig.21Shown are clinical scores of hCD40 mice injected with CII emulsion to induce CIA model and then treated with PBS (G2) or anti-hCD40 antibody 12B5-IgG1-LALA-YTE (G3). hCD40 mice injected with PBS only were used as controls (G1).

[0077] Fig. 22 The morbidity of hCD40 mice injected with CII emulsion to induce CIA model and then treated with PBS (G2) or anti-hCD40 antibody 12B5-IgG1-LALA-YTE (G3) is shown. hCD40 mice injected with PBS only were used as control (G1).

[0078] Fig.23 H&E staining images of joint tissues collected from the limbs of hCD40 mice injected with CII emulsion to induce CIA model and then treated with PBS (G2) or anti-hCD40 antibody 12B5-IgG1-LALA-YTE (G3) are shown. hCD40 mice injected with PBS only were used as controls (G1). (a) represents inflammatory cell infiltration, (b) represents synovial hyperplasia, and (c) represents pannus.

[0079] Fig.24 The amino acid sequences discussed in this disclosure are listed. DETAILED DESCRIPTION

[0080] The present disclosure provides examples of antibodies, antigen-binding fragments thereof, that bind to CD40 (TNF receptor superfamily member 5).

[0081] CD40 and the immune system

[0082] The immune system can distinguish between normal cells in the body and cells that are considered "foreign," which allows the immune system to attack foreign cells while leaving normal cells alone. This mechanism sometimes involves proteins called immune checkpoints. Immune checkpoints are molecules in the immune system that either turn signals on (co-stimulatory molecules) or off.

[0083] Checkpoint inhibitors prevent the immune system from attacking normal tissues, thereby preventing autoimmune diseases. Many tumor cells also express checkpoint inhibitors. These tumor cells escape immune surveillance by "co-opting" certain immune checkpoint pathways (especially in T cells specific for tumor antigens) (Creelan, Benjamin C. "Update on immune checkpoint inhibitors in lung cancer", Cancer Control 21.1 (2014): pp. 80-89). Because many immune checkpoints are initiated by ligand-receptor interactions, they are easily blocked by antibodies against ligands and / or their receptors.

[0084] CD40 (also known as tumor necrosis factor receptor superfamily member 5 or TNFRSF5) is a member of the tumor necrosis factor receptor superfamily expressed on antigen presenting cells (APCs) such as dendritic cells (DCs), macrophages, B cells and monocytes, as well as many non-immune cells and a wide range of tumors. Interaction with its trimeric ligand CD154 (also known as CD40 ligand or CD40L) on activated T helper cells leads to APC activation, thereby inducing adaptive immunity.

[0085] Physiologically, signaling through CD40 on APCs is thought to represent a major component of T cell help and to mediate, in large part, the ability of helper T cells to license APCs. For example, CD40 ligation on DCs induces increased surface expression of co-stimulatory and MHC molecules, production of proinflammatory cytokines, and enhanced T cell triggering. CD40 ligation on resting B cells increases antigen presentation function and proliferation.

[0086] In preclinical models, rat anti-mouse CD40 mAbs have shown significant therapeutic activity in the treatment of CD40+ B-cell lymphomas (80%-100% of mice cured and immune rechallenged in a CD8 T cell-dependent manner) and are also effective against a variety of CD40-negative tumors. These mAbs were able to eliminate bulky tumors in mice with near-terminal disease. CD40 mAbs have been studied in clinical trials and are used to treat melanoma, pancreatic cancer, mesothelioma, hematological malignancies, especially non-Hodgkin lymphoma, lymphoma, chronic lymphocytic leukemia, and advanced solid tumors.

[0087] Therapeutic anti-CD40 antibodies show a variety of activities ranging from strong agonism to antagonism. Currently, there is no satisfactory explanation for this heterogeneity. The main mechanism of agonistic CD40 mAbs is to activate host APCs to induce clinically significant anti-tumor T cell responses in patients. These responses include tumor regression initiation that is independent of T cells but dependent on macrophages. CD40-activated macrophages can have tumor killing effects (at least in pancreatic cancer) and may also promote tumor stroma depletion, thereby inducing tumor shrinkage in vivo. Importantly, these mechanisms do not require tumors to express CD40, so there is reason to include patients with multiple tumors in many clinical trials. For these strategies aimed at activating DCs, macrophages, or both, the goal of CD40 mAbs is not necessarily to kill cells bound to them, such as via complement-mediated cytotoxicity (CMC) or antibody-dependent cell-mediated cytotoxicity (ADCC). Therefore, by design, strong agonistic antibodies do not mediate CMC or ADCC.

[0088] In contrast, other human CD40 mAbs can mediate CMC and ADCC against CD40+ tumors, such as nearly all B-cell malignancies, a subset of melanomas, and certain carcinomas. Finally, there is some evidence that binding to CD40 on tumor cells can promote tumor cell apoptosis, and that this can be accomplished without involving any immune effector pathways. This has been demonstrated for CD40+ B-cell malignancies and certain solid tumors, such as CD40+ carcinomas and melanomas.

[0089] Due to the central role of CD40 in generating an effective immune response, CD40 also plays an important role in the pathogenesis of autoimmune diseases. CD40 causes T cell-dependent autoimmune diseases in several ways. First, CD40 signaling can act at the level of T cell selection in the thymus. Medullary thymic epithelial cells (mTEC) mediate the negative selection of potential autoreactive T cells by expressing peripheral tissue-restricted antigens. Although RANK, a member of the TNFR family, is crucial in the development of embryonic mTEC, CD40 and RANK work together to promote postnatal mTEC development, thereby promoting self-tolerance. The destruction of CD40-CD154 interaction in mTEC can potentially lead to the failure of central tolerance. Secondly, CD40 signaling leads to the production of proinflammatory cytokines such as IL-6, which can affect the differentiation of T cells into Th17 cells. CD40 is also upregulated after activation of antigen presenting cells (APCs). An increase in the level of either constitutive or inducible CD40 can lead to an increase in the intensity of CD40-CD154 interaction. Another mechanism could be the abnormal expression of CD40 in tissues where it is not normally detected. It has been hypothesized that abnormal expression of MHC class II molecules on endocrine tissues could lead to the initiation of autoimmune diseases. Abnormal CD40 expression on such tissues is also thought to be a contributing factor to the initiation of autoimmunity in Graves' disease and to the production of inflammatory cytokines that lead to failure of islet cell transplantation. Finally, CD4+ T cells carrying CD40 play a role in type 1 diabetes in humans and mice. Therefore, CD40 is an attractive candidate receptor that can initiate various autoimmune processes, in which the activation of B cells and T cells plays a role in the pathogenesis.

[0090] For a detailed description of CD40 and its functions, see, for example, Vonderheide et al., "Agonistic CD40 antibodies and cancer therapy", (2013): pp. 1035-1043; Beatty et al., "CD40 agonists alter tumor stroma and show efficacy against pancreatic carcinoma in mice and humans", Science 331.6024 (2011): pp. 1612-1616; Vonderheide et al., "Clinical activity and immune modulation in cancer patients treated with CP-870,893, a novel CD40 agonist monoclonal antibody", Journal of Clinical Oncology 25.7 (2007): pp. 876-883; Peters et al., "CD40 and autoimmunity: the dark side of a great activator.", Seminars in immunology., Vol. 21, No. 5, Academic Press, 2009; each of these documents is incorporated herein by reference in its entirety.

[0091] The present disclosure provides several anti-CD40 antibodies, antigen-binding fragments thereof, and methods of using these anti-CD40 antibodies and antigen-binding fragments to inhibit tumor growth and treat cancer.

[0092] Antibodies and antigen-binding fragments

[0093] The present disclosure provides anti-CD40 antibodies and antigen-binding fragments thereof. Generally, antibodies (also known as immunoglobulins) are composed of two types of polypeptide chains, namely light chains and heavy chains. The non-limiting antibodies of the present disclosure may be complete tetra-immunoglobulin chain antibodies comprising two heavy chains and two light chains. The heavy chain of the antibody may be of any isotype (including IgM, IgG, IgE, IgA or IgD) or subisotype (including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc.). The light chain may be a kappa light chain or a lambda light chain. The antibody may include two identical copies of a light chain and two identical copies of a heavy chain. Each contains a variable domain (or variable region, V H) and multiple constant domains (or constant regions) are bound to each other through disulfide bonds within their constant domains to form the "backbone" of the antibody. L ) and a light chain of a constant domain (or constant region) each bind to a heavy chain through a disulfide bond. The variable region of each light chain is aligned with the variable region of the heavy chain to which it binds. The variable regions of both the light and heavy chains contain three hypervariable regions, which are sandwiched between more conserved framework regions (FR).

[0094] These hypervariable regions, called complementarity determining regions (CDRs), form loops that comprise the major antigen-binding surface of the antibody. The four framework regions largely adopt a beta-sheet conformation, while the CDRs form loops that connect and in some cases form part of the beta-sheet structure. The CDRs in each chain are held tightly together by the framework regions and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding region.

[0095] Methods for identifying the CDR regions of an antibody by analyzing its amino acid sequence are well known, and a number of definitions of CDRs are commonly used. The Kabat definition is based on sequence variability, and the Chothia definition is based on the location of structural loop regions. These methods and definitions are described, for example, in Martin, "Protein sequence and structure analysis of antibody variable domains", Antibody engineering, Springer Berlin Heidelberg, 2001, pp. 422-439; Abhinandan et al., "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains," Molecular immunology 45.14 (2008): pp. 3832-3839; Wu, TT and Kabat, EA, (1970) J. Exp. Med., Vol. 132: pp. 211-250; Martin et al., Methods Enzymol., Vol. 203: pp. 121-153 (1991); Morea et al., Biophys Chem., Vol. 68, No. 1-3: pp. 9-16 (October 1997); Morea et al., J Mol. Biol., Vol. 275, No. 2: pp. 269-294 (January 1998); Chothia et al., Nature, Vol. 342, No. 6252: pp. 877-883 (December 1989); Ponomarenko and Bourne, BMC Structural Biology, Vol. 7, No. 64 (2007), each of which is incorporated herein by reference in its entirety. Unless otherwise specified in the present disclosure, Kabat numbering is used in the present disclosure as the default numbering.

[0096] CDR is very important for recognizing the epitope of an antigen. As used herein, an "epitope" is the smallest part of a target molecule that can be specifically bound by the antigen-binding domain of an antibody. The minimum size of an epitope can be about three, four, five, six or seven amino acids, but these amino acids do not have to be in a continuous linear sequence of the primary structure of the antigen, because the epitope can depend on the three-dimensional configuration of the antigen based on the secondary and tertiary structures of the antigen.

[0097] In some embodiments, the antibody is a complete immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, but differ in their constant regions, particularly the hinge and upper CH2 domains. The sequences and differences of IgG subclasses are known in the art and are described in, for example, Vidarsson et al., "IgG subclasses and allotypes: from structure to effector functions.", Frontiers in immunology 5 (2014); Irani et al., "Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases.", Molecular immunology 67.2 (2015): 171-182; Shakib, Farouk, ed., "The human IgG subclasses: molecular analysis of structure, function and regulation.", Elsevier, 2016, each of which is incorporated herein by reference in its entirety.

[0098] Antibodies can also be immunoglobulin molecules derived from any species (e.g., humans, rodents, mice, camelids). Antibodies disclosed herein also include, but are not limited to, polyclonal antibodies, monoclonal antibodies, monospecific antibodies, multispecific antibodies, and chimeric antibodies comprising immunoglobulin binding domains fused to another polypeptide. The term "antigen binding domain" or "antigen binding fragment" is a part of the specific binding activity of an antibody that retains a complete antibody, that is, any part of an antibody that can specifically bind to an epitope on a target molecule of a complete antibody. It includes, for example, Fab, Fab', F(ab')2 and variants of these fragments. Therefore, in some embodiments, an antibody or its antigen binding fragment can be, for example, scFv, Fv, Fd, dAb, bispecific antibodies, bispecific scFv, double antibodies, linear antibodies, single-chain antibody molecules, multispecific antibodies formed by antibody fragments, and any polypeptide comprising an antibody binding domain or a binding domain homologous thereto. Non-limiting examples of antigen binding domains include, e.g., heavy and / or light chain CDRs of an intact antibody, heavy and / or light chain variable regions of an intact antibody, the full length heavy or light chain of an intact antibody, or a single CDR from a heavy or light chain of an intact antibody.

[0099] In some embodiments, the antigen binding fragment may form a part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor is a fusion of a single-chain variable fragment (scFv) fused to a CD3-ζ transmembrane domain and an intracellular domain as described herein. In some embodiments, the chimeric antigen receptor also includes intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS). In some embodiments, the chimeric antigen receptor includes multiple signaling domains, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to increase effectiveness. Therefore, on the one hand, the present disclosure also provides cells (e.g., T cells) expressing chimeric antigen receptors as described herein.

[0100] In some embodiments, the scFV has one heavy chain variable domain and one light chain variable domain. In some embodiments, the scFV has two heavy chain variable domains and two light chain variable domains.

[0101] In some embodiments, the sequences of the antibodies or antigen-binding fragments thereof described herein (e.g., CDR or VH / VL sequences) can be used to generate bispecific antibodies targeting CD40 and additional antigens (e.g., TNFRSF9 (4-1BB), MSLN, FAP, ALB, CTLA4, HER2, GPC3, MYC, EPCAM, TNFRSF14 (LIGHTR), or ITGAX (CD11c)).

[0102] Anti-CD40 antibodies and antigen-binding fragments

[0103] The present disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to CD40 (e.g., human CD40). The antibodies and antigen-binding fragments described herein are capable of binding to CD40. These antibodies can be agonists or antagonists. In some embodiments, these antibodies can promote the CD40 signaling pathway, thereby increasing the immune response. In some embodiments, the antibodies can block the CD40 signaling pathway, thereby reducing the immune response. In some embodiments, these antibodies can induce complement dependent cytotoxicity (CMC) or antibody dependent cellular cytotoxicity (ADCC).

[0104] The present disclosure provides, for example, mouse anti-CD40 antibodies 2F8, 6A4, 12B5, chimeric antibodies thereof, and human or humanized antibodies thereof.

[0105] The CDR sequences of 2F8 and 2F8-derived antibodies (e.g., human or humanized antibodies) include CDRs of the heavy chain variable domain (SEQ ID NOs: 1-3) and CDRs of the light chain variable domain (SEQ ID NOs: 4-6) as defined by Kabat numbering. CDRs may also be defined by the Chothia system. According to Chothia numbering, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 19-21, and the CDR sequences of the light chain variable domain are shown in SEQ ID NOs: 22-24.

[0106] Similarly, the CDR sequences of 6A4 and 6A4-derived antibodies include the CDRs of the heavy chain variable domain (SEQ ID NOs: 7-9) and the CDRs of the light chain variable domain (SEQ ID NOs: 10-12) as defined by Kabat numbering. According to Chothia numbering, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 25-27, and the CDRs of the light chain variable domain are shown in SEQ ID NOs: 28-30.

[0107] The CDR sequences of 12B5 and 12B5-derived antibodies include CDRs of the heavy chain variable domain (SEQ ID NOs: 13-15) and CDRs of the light chain variable domain (SEQ ID NOs: 16-18) as defined by Kabat numbering. According to Chothia numbering, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 31-33, and the CDRs of the light chain variable domain are shown in SEQ ID NOs: 34-36.

[0108] The amino acid sequence of the heavy chain variable region of the 2F8 antibody is shown in SEQ ID NO: 65. The amino acid sequence of the light chain variable region of the 2F8 antibody is shown in SEQ ID NO: 37.

[0109] The amino acid sequence of the heavy chain variable region of the 6A4 antibody is shown in SEQ ID NO: 38. The amino acid sequence of the light chain variable region of the 6A4 antibody is shown in SEQ ID NO: 39.

[0110] The amino acid sequence of the heavy chain variable region of the 12B5 antibody is shown in SEQ ID NO: 40. The amino acid sequence of the light chain variable region of the 12B5 antibody is shown in SEQ ID NO: 41.

[0111] Also provided are amino acid sequences of the heavy chain variable region and the light chain variable region of the modified antibodies. In some embodiments, the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 65, 38 or 40. In some embodiments, the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 37, 39 or 41. The heavy chain variable region sequence can be paired with the corresponding light chain variable region sequence, and together they bind CD40.

[0112] Humanization percentage means the percentage of identity of the heavy chain or light chain variable region sequence compared to the human antibody sequence in the International Immunogenetics Information System (IMGT) database. The best comparison (top hit) means that the heavy chain or light chain variable region sequence is closer to a specific species than to other species. For example, the best comparison with man means that the sequence is closer to people than to other species. The best comparison with humans and cynomolgus monkeys (Macaca fascicularis) means that the sequence has the same percentage of identity with humans and cynomolgus monkey sequences, and compared with other species sequences, these percentages of identity are the highest. In some embodiments, the humanization percentage is greater than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95%. Detailed descriptions of how to determine the percentage of humanization and how to determine the optimal alignment are known in the art and are described, for example, in Jones et al., "The INNs and outs of antibody nonproprietary names", MAbs. Vol. 8, No. 1, Taylor & Francis, 2016, which is incorporated herein by reference in its entirety. A high percentage of humanization generally has a variety of advantages, for example, being safer and more effective in humans, being more likely to be tolerated by human subjects, and / or having less likelihood of side effects. In some embodiments, the variable region is fully human, for example, derived from a human heavy chain immunoglobulin locus sequence (e.g., recombination of human IGHV, human IGHD, and human IGHJ genes), and / or a human kappa chain immunoglobulin locus sequence (e.g., recombination of human IGKV and human IGKJ genes).

[0113] In addition, in some embodiments, the antibodies or antigen-binding fragments thereof described herein may also contain one, two or three heavy chain variable region CDRs selected from the following items: SEQ ID NO: 1-3, SEQ ID NO: 7-9, SEQ ID NO: 13-15, SEQ ID NO: 19-21, SEQ ID NO: 25-27 and SEQ ID NO: 31-33; and / or one, two or three light chain variable region CDRs selected from the following items: SEQ ID NO: 4-6, SEQ ID NO: 10-12, SEQ ID NO: 16-18, SEQ ID NO: 22-24, SEQ ID NO: 28-30 and SEQ ID NO: 34-36.

[0114] In some embodiments, the antibody may have a heavy chain variable region (VH) comprising complementarity determining regions (CDR) 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VH CDR3 amino acid sequence. In some embodiments, the antibody may have a light chain variable region (VL) that includes CDR1, 2, and 3, wherein the CDR1 region includes or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR1 amino acid sequence, the CDR2 region includes or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR2 amino acid sequence, and the CDR3 region includes or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR3 amino acid sequence. The selected VH CDR1, 2, 3 amino acid sequences and the selected VL CDR1, 2, 3 amino acid sequences are shown in Fig. 9 (Kabat CDR) and Fig.10 (Chothia CDR).

[0115] In some embodiments, the antibodies or antigen-binding fragments described herein may comprise a heavy chain variable domain comprising one, two or three of the following CDRs: a CDR of SEQ ID NO: 1 having zero, one or two amino acid insertions, deletions or substitutions; a CDR of SEQ ID NO: 2 having zero, one or two amino acid insertions, deletions or substitutions; a CDR of SEQ ID NO: 3 having zero, one or two amino acid insertions, deletions or substitutions.

[0116] In some embodiments, the antibodies or antigen-binding fragments described herein may comprise a heavy chain variable domain comprising one, two or three of the following CDRs: a CDR of SEQ ID NO:7 having zero, one or two amino acid insertions, deletions or substitutions; a CDR of SEQ ID NO:8 having zero, one or two amino acid insertions, deletions or substitutions; a CDR of SEQ ID NO:9 having zero, one or two amino acid insertions, deletions or substitutions.

[0117] In some embodiments, the antibodies or antigen-binding fragments described herein may comprise a heavy chain variable domain comprising one, two or three of the following CDRs: a CDR of SEQ ID NO: 13 having zero, one or two amino acid insertions, deletions or substitutions; a CDR of SEQ ID NO: 14 having zero, one or two amino acid insertions, deletions or substitutions; a CDR of SEQ ID NO: 15 having zero, one or two amino acid insertions, deletions or substitutions.

[0118] In some embodiments, the antibodies or antigen-binding fragments described herein may comprise a heavy chain variable domain comprising one, two or three of the following CDRs: the CDR of SEQ ID NO: 19 having zero, one or two amino acid insertions, deletions or substitutions; the CDR of SEQ ID NO: 20 having zero, one or two amino acid insertions, deletions or substitutions; the CDR of SEQ ID NO: 21 having zero, one or two amino acid insertions, deletions or substitutions.

[0119] In some embodiments, the antibodies or antigen-binding fragments described herein may comprise a heavy chain variable domain comprising one, two or three of the following CDRs: a CDR of SEQ ID NO:25 having zero, one or two amino acid insertions, deletions or substitutions; a CDR of SEQ ID NO:26 having zero, one or two amino acid insertions, deletions or substitutions; a CDR of SEQ ID NO:27 having zero, one or two amino acid insertions, deletions or substitutions.

[0120] In some embodiments, the antibodies or antigen-binding fragments described herein may comprise a heavy chain variable domain comprising one, two or three of the following CDRs: the CDR of SEQ ID NO:31 having zero, one or two amino acid insertions, deletions or substitutions; the CDR of SEQ ID NO:32 having zero, one or two amino acid insertions, deletions or substitutions; the CDR of SEQ ID NO:33 having zero, one or two amino acid insertions, deletions or substitutions.

[0121] In some embodiments, the antibodies or antigen-binding fragments described herein may comprise a light chain variable domain comprising one, two or three of the following CDRs: a CDR of SEQ ID NO:4 having zero, one or two amino acid insertions, deletions or substitutions; a CDR of SEQ ID NO:5 having zero, one or two amino acid insertions, deletions or substitutions; a CDR of SEQ ID NO:6 having zero, one or two amino acid insertions, deletions or substitutions.

[0122] In some embodiments, the antibodies or antigen-binding fragments described herein may comprise a light chain variable domain comprising one, two or three of the following CDRs: the CDR of SEQ ID NO: 10 having zero, one or two amino acid insertions, deletions or substitutions; the CDR of SEQ ID NO: 11 having zero, one or two amino acid insertions, deletions or substitutions; the CDR of SEQ ID NO: 12 having zero, one or two amino acid insertions, deletions or substitutions.

[0123] In some embodiments, the antibodies or antigen-binding fragments described herein may comprise a light chain variable domain comprising one, two or three of the following CDRs: a CDR of SEQ ID NO: 16 having zero, one or two amino acid insertions, deletions or substitutions; a CDR of SEQ ID NO: 17 having zero, one or two amino acid insertions, deletions or substitutions; a CDR of SEQ ID NO: 18 having zero, one or two amino acid insertions, deletions or substitutions.

[0124] In some embodiments, the antibodies or antigen-binding fragments described herein may comprise a light chain variable domain comprising one, two or three of the following CDRs: a CDR of SEQ ID NO:22 having zero, one or two amino acid insertions, deletions or substitutions; a CDR of SEQ ID NO:23 having zero, one or two amino acid insertions, deletions or substitutions; a CDR of SEQ ID NO:24 having zero, one or two amino acid insertions, deletions or substitutions.

[0125] In some embodiments, the antibodies or antigen-binding fragments described herein may comprise a light chain variable domain comprising one, two or three of the following CDRs: the CDR of SEQ ID NO:28 having zero, one or two amino acid insertions, deletions or substitutions; the CDR of SEQ ID NO:29 having zero, one or two amino acid insertions, deletions or substitutions; the CDR of SEQ ID NO:30 having zero, one or two amino acid insertions, deletions or substitutions.

[0126] In some embodiments, the antibodies or antigen-binding fragments described herein may comprise a light chain variable domain comprising one, two or three of the following CDRs: a CDR of SEQ ID NO:34 having zero, one or two amino acid insertions, deletions or substitutions; a CDR of SEQ ID NO:35 having zero, one or two amino acid insertions, deletions or substitutions; a CDR of SEQ ID NO:36 having zero, one or two amino acid insertions, deletions or substitutions.

[0127] Insertions, deletions and substitutions may be within the CDR sequence or at one or both ends of the CDR sequence. In some embodiments, the CDR is determined based on the Kabat numbering convention. In some embodiments, the CDR is determined based on the Chothia numbering convention. In some embodiments, the CDR is determined based on a combination of the Kabat and Chothia numbering conventions.

[0128] The present disclosure also provides an antibody or antigen-binding fragment thereof that binds to CD40. The antibody or antigen-binding fragment thereof contains: a heavy chain variable region (VH), the VH comprising or consisting of an amino acid sequence having at least 80%, 85%, 90% or 95% identity with a selected VH sequence, and a light chain variable region (VL), the VL comprising or consisting of an amino acid sequence having at least 80%, 85%, 90% or 95% identity with a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 65, and the selected VL sequence is SEQ ID NO: 37. In some embodiments, the selected VH sequence is SEQ ID NO: 38, and the selected VL sequence is SEQ ID NO: 39. In some embodiments, the selected VH sequence is SEQ ID NO: 40, and the selected VL sequence is SEQ ID NO: 41.

[0129] The present disclosure also provides antibodies or antigen-binding fragments thereof that can compete with the antibodies described herein.In some aspects, the antibodies or antigen-binding fragments can bind to the same epitope as the antibodies described herein.

[0130] The present disclosure also provides antibodies or antigen-binding fragments thereof that cross-compete with any antibody or antigen-binding fragment as described herein. Cross-competition assays are known in the art and are described in, for example, Moore et al., "Antibody cross-competition analysis of the human immunodeficiency virus type 1gp120exterior envelope glycoprotein.", Journal of virology, 70.3 (1996): pp. 1863-1872, which is incorporated herein by reference in its entirety. On the one hand, the present disclosure also provides antibodies or antigen-binding fragments thereof that bind to the same epitope or region as any antibody or antigen-binding fragment as described herein. Epitope grouping assays are known in the art and are described in, for example, Estep et al., "High throughput solution-based measurement ofantibody-antigen affinity and epitope binning.", MAbs. Vol. 5 No. 2, Taylor & Francis, 2013, which is incorporated herein by reference in its entirety.

[0131] In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, these sequences are compared for the best comparison purpose (for example, a gap can be introduced in one or both of the first and second amino acid or nucleic acid sequences for the best comparison, and non-homologous sequences can be ignored for the purpose of comparison). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, these molecules are identical at that position. Considering the number of gaps and the length of each gap that need to be introduced to achieve the best comparison of the two sequences, the percent identity between the two sequences is a function of the number of identical positions shared by the sequences. For example, the comparison of the sequence and the determination of the percent identity between the two sequences can be completed using the Blossum 62 scoring matrix, wherein the gap penalty is 12, the gap extension penalty is 4, and the frameshift gap penalty is 5.

[0132] The present disclosure also provides nucleic acids comprising polynucleotides encoding polypeptides comprising an immunoglobulin heavy chain or an immunoglobulin light chain. An immunoglobulin heavy chain or an immunoglobulin light chain comprises Fig. 9 or Fig.10 The CDRs shown, or having Fig.11When the polypeptide is paired with a corresponding polypeptide (eg, a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to CD40 (eg, human CD40).

[0133] Anti-CD40 antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments and multispecific (e.g., bispecific) antibodies or antibody fragments. Other antibodies provided herein are polyclonal antibodies, monoclonal antibodies, multispecific (multimers, e.g., bispecific) antibodies, human antibodies, chimeric antibodies (e.g., human-mouse chimeras), single-chain antibodies, antibodies prepared in cells (i.e., intrabodies), and antigen-binding fragments thereof. The antibody or its antigen-binding fragment can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the antibody or its antigen-binding fragment is an IgG antibody or its antigen-binding fragment.

[0134] As long as the antibody fragment retains the desired affinity and specificity of the full-length antibody, it is suitable for the provided methods. Therefore, an antibody fragment that binds to CD40 will retain the ability to bind to CD40. The Fv fragment is an antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of a heavy chain variable domain and a light chain variable domain in tight association, which can be covalent in nature, such as scFv. In this configuration, it is the three CDRs of each variable domain that interact to define the antigen binding site on the surface of the VH-VL dimer. In general, the six CDRs or a subset thereof together confer antigen binding specificity to the antibody. However, even a single variable domain (or half an Fv that includes only three CDRs specific for a certain antigen) has the ability to recognize and bind to an antigen, but the affinity is usually lower than that of the entire binding site.

[0135] Single-chain Fv (scFv) antibody fragments comprise the VH and VL domains (or regions) of an antibody, wherein these domains are present in a single polypeptide chain. Typically, the scFv polypeptide also includes a polypeptide linker between the VH and VL domains so that the scFv forms a desired structure for antigen binding.

[0136] The Fab fragment contains the variable domain and constant domain of the light chain and the variable domain and the first constant domain (CH1) of the heavy chain. The F(ab')2 antibody fragment comprises a pair of Fab fragments, which are usually covalently linked near their carboxyl termini by hinge cysteines between them. Other chemical couplings of antibody fragments are also known in the art.

[0137] Diabodies are small antibody fragments with two antigen-binding sites that contain VH and VL linked on the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

[0138] Linear antibodies include a pair of tandem Fd segments (VH-CH1-VH-CH1), which together with complementary light chain polypeptides form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

[0139] The antibodies and antibody fragments of the disclosure may be modified in the Fc region to provide desired effector function or serum half-life.

[0140] The multimerization of antibodies can be achieved by natural aggregation of antibodies or by chemical or recombinant connection techniques known in the art. For example, a certain percentage of purified antibody preparations (e.g., purified IgG1 molecules) spontaneously form protein aggregates containing antibody homodimers and other higher-order antibody multimers.

[0141] Alternatively, antibody homodimers can be formed by chemical connection techniques known in the art. For example, heterologous bifunctional cross-linking agents include, but are not limited to, SMCC (succinimidyl 4-(maleimidomethyl) cyclohexane-1-carboxylate) and SATA (N-succinimidyl S-acetylthioacetate) can be used to form antibody polymers. An exemplary scheme for forming antibody homodimers is described in Ghetie et al. (Proc. Natl. Acad. Sci. USA, Vol. 94: pp. 7509-7514, 1997). By pepsin digestion, antibody homodimers can be converted into Fab'2 homodimers. Another method for forming antibody homodimers is by using an autophagic T15 peptide, which is described in Zhao et al. (J. Immunol. Vol. 25: pp. 396-404, 2002).

[0142] In some embodiments, multispecific antibodies are bispecific antibodies. Bispecific antibodies can be prepared by engineering the interface between a pair of antibody molecules so that the percentage of the heterodimer recovered from the recombinant cell culture is maximized. For example, the interface can include at least a portion of the CH3 domain of the antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced by larger side chains (for example, tyrosine or tryptophan). By replacing the large amino acid side chains with smaller amino acid side chains (for example, alanine or threonine), a compensation "cavity" identical or similar to the large side chain size is produced on the interface of the second antibody molecule. This provides a mechanism that can make the yield of heterodimers higher than other unwanted end products (such as homodimers). The method is described in, for example, WO 96 / 27011, which is incorporated herein by reference in its entirety.

[0143] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other antibody can be coupled to biotin. Heteroconjugate antibodies can also be prepared using any convenient cross-linking method. Suitable cross-linking agents and cross-linking techniques are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, which is incorporated herein by reference in its entirety.

[0144] Methods for producing bispecific antibodies from antibody fragments are also known in the art. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al. (Science, Vol. 229: p. 81, 1985) described a method in which an intact antibody is proteolytically cleaved to produce F(ab')2 fragments. These fragments are reduced in the presence of a dithiol complexing agent, sodium arsenite, to stabilize adjacent dithiols and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted into thionitrobenzoate (TNB) derivatives. One of the Fab'TNB derivatives is then converted into a Fab'thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of another Fab'TNB derivative to form a bispecific antibody.

[0145] Any of the antibodies or antigen-binding fragments described herein may be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in a subject or solution). Non-limiting examples of stabilizing molecules include: polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin, such as human serum albumin). Conjugation of stabilizing molecules can increase the half-life of the antibody or antigen-binding fragment in vitro (e.g., in tissue culture or when stored in a pharmaceutical composition) or in vivo (e.g., in the human body) or extend its biological activity.

[0146] In some embodiments, the antibodies or antigen binding fragments described herein may be conjugated to a therapeutic agent. Antibody drug conjugates including antibodies or antigen binding fragments thereof may be covalently or non-covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic agent or a cell growth inhibitor (e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin, maytansinoid alkaloids (such as DM-1 and DM-4), diketones, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin and cyclophosphamide and the like).

[0147] In some embodiments, the antibodies or antigen-binding fragments thereof described herein recognize endogenous CD40 or recombinant CD40. In some embodiments, the antibodies or antigen-binding fragments thereof described herein recognize human CD40.

[0148] In some embodiments, the half-life of an antibody or antigen-binding fragment thereof described herein in a wild-type mouse (e.g., a C57BL / 6 mouse) is at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, or at least 18 days. In some embodiments, the half-life of an antibody or antigen-binding fragment thereof described herein in a CD40 gene humanized mouse (e.g., an hCD40 mouse) is at least 1 day, at least 2 days, at least 3 days, at least 4 days, or at least 5 days.

[0149] In some embodiments, the clearance rate (CL) of the antibody or antigen-binding fragment thereof described herein in wild-type mice (e.g., C57BL / 6 mice) is less than 7 ml / day / kg, less than 6 ml / day / kg, less than 5 ml / day / kg, or less than 4 ml / day / kg. In some embodiments, the clearance rate (CL) of the antibody or antigen-binding fragment thereof described herein in CD40 gene humanized mice (e.g., hCD40 mice) is less than 15 ml / day / kg, less than 14 ml / day / kg, less than 13 ml / day / kg, or less than 12 ml / day / kg.

[0150] In some embodiments, the half-life of an antibody or antigen-binding fragment thereof described herein in a humanized FcRn mouse (e.g., hFcRn mouse) is at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days. In some embodiments, the clearance rate (CL) of an antibody or antigen-binding fragment thereof described herein in a humanized FcRn mouse (e.g., hFcRn mouse) is less than 16 ml / day / kg, less than 15 ml / day / kg, less than 14 ml / day / kg, less than 13 ml / day / kg, less than 12 ml / day / kg, less than 11 ml / day / kg, less than 10 ml / day / kg, less than 9 ml / day / kg, less than 8 ml / day / kg, or less than 7 ml / day / kg.

[0151] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can enter cells expressing endogenous, recombinant or human CD40. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can enter cells by endocytosis. In some embodiments, the antibodies or antigen-binding fragments described herein enter at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of cells (e.g., cells expressing CD40) by endocytosis.

[0152] In some embodiments, antibodies or antigen binding fragments thereof as described herein show immunostimulatory effects. In some embodiments, antibodies or antigen binding fragments thereof as described herein show immunosuppressive effects. In some embodiments, antibodies or antigen binding fragments thereof as described herein suppress one or more immune functions (e.g., antigen-induced antibody production) to less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1%.

[0153] In some embodiments, the immunosuppressive effect of the antibody or its antigen-binding fragment described herein is reversible. In some embodiments, the immunosuppressive effect of the antibody or its antigen-binding fragment described herein is irreversible. In some embodiments, at least 10 days, at least 15 days, at least 20 days, at least 25 days, at least 30 days, at least 32 days, at least 35 days, at least 40 days, at least 45 days or at least 60 days after the subject (e.g., mouse) is administered with the antibody or its antigen-binding fragment, the immune function (e.g., T cell-dependent humoral immune function) of the subject is restored. In some embodiments, compared with the same immune function before the subject (e.g., mouse) is administered with the antibody or its antigen-binding fragment, the immune function (e.g., T cell-dependent humoral immune function) of the subject is restored to at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or higher.

[0154] In some embodiments, the immunosuppressive effect of the antibody or antigen-binding fragment thereof does not reduce CD20 in an immune system organ (e.g., spleen) of a subject (e.g., mouse). + / CD19 + The percentage of cells.

[0155] In some embodiments, the antibody or antigen-binding fragment thereof saturates the CD40 receptor at a concentration of about or less than 0.1 μg / mL, about 0.2 μg / mL, about 0.3 μg / mL, 0.4 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL.

[0156] In some embodiments, the antibodies or antigen-binding fragments thereof described herein reduce the binding of CD154 to CD40 to less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.

[0157] In some embodiments, the antibody or antigen binding fragment thereof has a CD40 receptor occupancy percentage (RO%) of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%. In some embodiments, the antibody or antigen binding fragment thereof reduces the percentage of activated B cells to less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%, compared to the percentage of activated B cells when the antibody or antigen binding fragment thereof is not administered.

[0158] Antibody characteristics

[0159] The antibodies or antigen-binding fragments thereof described herein can block the binding between CD40 and a CD40 ligand (eg, CD154).

[0160] As described herein, the antibodies or antigen-binding fragments thereof may be CD40 agonists or antagonists. In some embodiments, by binding to CD40, the antibodies may inhibit the CD40 signaling pathway. In some embodiments, the antibodies may upregulate an immune response or downregulate an immune response.

[0161] In some embodiments, antibodies or antigen-binding fragments thereof as described herein can increase the activity or number of immune responses, immune cells (e.g., T cells, CD8+T cells, CD4+T cells, macrophages, antigen-presenting cells) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold or 20-fold. In some embodiments, antibodies or antigen-binding fragments thereof as described herein can reduce the activity or number of immune cells (e.g., T cells, CD8+T cells, CD4+T cells, macrophages, antigen-presenting cells) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold or 20-fold.

[0162] In some embodiments, the antibody (or antigen-binding fragment thereof) is activated at a rate of less than 0.1 s -1 Less than 0.01s -1 , less than 0.001s -1 , less than 0.0001s -1 or less than 0.00001s -1The dissociation rate (koff) of the antibody specifically binds to CD40 (e.g., human CD40, monkey CD40 (e.g., macaque, cynomolgus monkey (Macaca fascicularis)), dog CD40, mouse CD40 and / or chimeric CD40). In some embodiments, the dissociation rate (koff) is greater than 0.01 s -1 , greater than 0.001s -1 , greater than 0.0001s -1 , greater than 0.00001s -1 or greater than 0.000001s -1 .

[0163] In some embodiments, the kinetic binding rate (kon) is greater than 1×10 2 / Ms, greater than 1×10 3 / Ms, greater than 1×10 4 / Ms, greater than 1×10 5 / Ms or greater than 1×10 6 / Ms. In some embodiments, the kinetic binding rate (kon) is less than 1×10 5 / Ms, less than 1×10 6 / Ms or less than 1×10 7 / Ms.

[0164] Affinity can be derived from the quotient of the kinetic rate constants (KD = koff / kon). In some embodiments, KD is less than 1×10 -6 M, less than 1×10 -7 M, less than 1×10 -8 M, less than 1×10 -9 M or less than 1×10 -10 In some embodiments, the KD is less than 50 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, the KD is greater than 1×10 -7 M, greater than 1×10 -8 M, greater than 1×10 -9 M, greater than 1×10 -10 M, greater than 1×10 -11 M or greater than 1×10 -12 M.

[0165] General techniques for measuring the affinity of an antibody for an antigen include, for example, ELISA, RIA, and surface plasmon resonance (SPR). In some embodiments, the antibody binds to human CD40 (SEQ ID NO: 42), mouse CD40 (SEQ ID NO: 50), and / or chimeric CD40 (SEQ ID NO: 49). In some embodiments, the antibody does not bind to human CD40 (SEQ ID NO: 42), mouse CD40 (SEQ ID NO: 50), and / or chimeric CD40 (SEQ ID NO: 49). In some embodiments, the antibody binds to FcRn (SEQ ID NO: 51).

[0166] In some embodiments, thermal stability is determined. In some embodiments, the antibodies or antigen-binding fragments described herein may have a thermal stability of greater than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, 100°C, 101°C, 102°C, 103°C, 104°C, 105°C, 106°C, 107°C, 108°C, 109°C In some embodiments, the Tm is less than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C or 95°C.

[0167] In some embodiments, the antibody has a tumor growth inhibition percentage (TGI%) greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or 200%. In some embodiments, the antibody has a tumor growth inhibition percentage less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or 200%. TGI% can be determined, for example, at 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days or 30 days after the start of treatment, or at 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months after the start of treatment. As used herein, tumor growth inhibition percentage (TGI%) is calculated using the formula:

[0168] TGI(%)=[1-(Ti-T0) / (Vi-V0)]×100%

[0169] Ti is the mean tumor volume in the treatment group at day i. T0 is the mean tumor volume in the treatment group at day zero. Vi is the mean tumor volume in the control group at day i. V0 is the mean tumor volume in the control group at day zero.

[0170] In some embodiments, an antibody or antigen-binding fragment thereof as described herein is a CD40 antagonist. In some embodiments, the antibody or antigen-binding fragment reduces CD40 signaling in a target cell expressing CD40.

[0171] In some embodiments, the antibody or antigen-binding fragment can enhance APC (eg, DC cell) function, for example, inducing surface expression of co-stimulatory and MHC molecules, inducing the production of pro-inflammatory cytokines, and / or enhancing T cell triggering function.

[0172] In some embodiments, the antibody or antigen binding fragment can bind to tumor cells expressing CD40. In some embodiments, the antibody or antigen binding fragment can induce complement-mediated cytotoxicity (CMC) and / or antibody-dependent cellular cytotoxicity (ADCC) and kill tumor cells.

[0173] In some embodiments, the antibody or antigen binding fragment has a functional Fc region. In some embodiments, the effector function of the functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector function of the functional Fc region is ADCC and phagocytosis.

[0174] In some embodiments, the antibody or antigen binding fragment can induce antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the antibody or antigen binding fragment cannot induce antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the antibody or antigen binding fragment can induce complement-mediated cytotoxicity (CMC). In some embodiments, the antibody or antigen binding fragment cannot induce complement-mediated cytotoxicity (CMC).

[0175] In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3 or human IgG4. In some embodiments, the antibody is a human IgG1 antibody, optionally with an SI mutation, a LALA mutation, a N297A mutation, a YTE mutation and / or a FLAA mutation. In some embodiments, the antibody is a human IgG4 antibody, optionally with an SI mutation, a LALA mutation, a N297A mutation, a YTE mutation and / or a FLAA mutation.

[0176] In some embodiments, the antibody or antigen binding fragment does not have a functional Fc region. For example, the antibody or antigen binding fragment is Fab, Fab', F(ab')2 and Fv fragment. In some embodiments, the Fc region has LALA mutation (according to the L234A and L235A mutations of EU numbering), or LALA-PG mutation (according to the L234A, L235A, P329G mutations of EU numbering). In some embodiments, the Fc region has FLAA mutation (according to the F234A and L235A of EU numbering). In some embodiments, Fc has SI mutation (according to the S239D and I332E mutations of EU numbering). In some embodiments, Fc has N297A mutation according to EU numbering. In some embodiments, Fc has YTE mutation (according to the M252Y, S254T and T256E of EU numbering).

[0177] In some embodiments, the antibodies or antigen-binding fragments described herein include an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of SEQ ID NOs: 52-59.

[0178] In some embodiments, the antibodies or antigen-binding fragments described herein can inhibit PMBC proliferation, for example, to less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, or less than 30% compared to an isotype control antibody (e.g., hIgG1).

[0179] In some embodiments, antibody or antigen binding fragment as described herein can suppress B cell proliferation and / or B cell activation.For example, compared with after isotype control antibody (such as hIgG1) is processed, the percentage of activated B cell subpopulation in PMBC cells after antibody or antigen binding fragment as described herein is processed can be reduced to less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10% or less than 5%.

[0180] Method for preparing anti-CD40 antibodies

[0181] The isolated human CD40 fragment can be used as an immunogen to produce antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies can be produced in animals by multiple injections (e.g., subcutaneous or intraperitoneal injections) of antigenic peptides or proteins. In some embodiments, the antigenic peptides or proteins are injected together with at least one adjuvant. In some embodiments, the antigenic peptides or proteins can be conjugated to an agent that is immunogenic in the species to be immunized. Animals can be injected with more than one (e.g., two, three, or four) antigenic peptides or proteins.

[0182] The full-length polypeptide or protein, or an antigenic peptide fragment thereof, can be used as an immunogen. The antigenic peptide of the protein includes at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acid sequence of CD40, and covers the epitope of the protein, so that antibodies raised against the peptide form a specific immune complex with the protein. As described above, the full-length sequence of human CD40 (SEQ ID NO: 42) is known in the art. In some embodiments, a human CD40 protein with an Fc tag or a His tag is used as an immunogen.

[0183] Immunogens are typically used to prepare antibodies by immunizing a suitable subject (e.g., a human or transgenic animal expressing at least one human immunoglobulin locus). Suitable immunogenic preparations may contain, for example, recombinantly expressed or chemically synthesized polypeptides (e.g., fragments of human CD40). The preparations may also include an adjuvant (such as Freund's complete or incomplete adjuvant) or a similar immunostimulant.

[0184] Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a CD40 polypeptide or an antigenic peptide thereof (e.g., a portion of CD40) as an immunogen. Antibody titers in the immunized subject can be monitored over time by standard techniques, such as by enzyme-linked immunosorbent assay (ELISA), using immobilized CD40 polypeptide or peptide. If necessary, the antibody molecules can be isolated from a mammal (e.g., from blood) and further purified by well-known techniques (such as protein A or protein G chromatography) to obtain an IgG fraction. At an appropriate time after immunization, for example, when specific antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler et al. (Nature, Vol. 256, pp. 495-497, 1975), human B cell hybridoma technique (Kozbor et al., Immunol. Today, Vol. 4, p. 72, 1983), EBV-hybridoma technique (Cole et al., "Monoclonal Antibodies and Cancer Therapy", Alan R. Liss, Inc., pp. 77-96, 1985) or trioma technique. The techniques for producing hybridomas are well known (see generally "Current Protocols in Immunology", 1994, edited by Coligan et al., John Wiley & Sons, Inc., New York, NY). Hybridoma cells producing monoclonal antibodies are detected by screening hybridoma culture supernatants for antibodies that bind the polypeptide or epitope of interest, for example, using a standard ELISA assay.

[0185] Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding the human, humanized or chimeric antibodies or antigen-binding fragments thereof described herein, or by peptide synthesis. Such variants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence constituting the antigen-binding site of the antibody or antigen-binding domain. In a population of such variants, some antibodies or antigen-binding fragments will have increased affinity for the target protein (e.g., CD40). Any combination of deletions, insertions and / or combinations may be performed to obtain antibodies or antigen-binding fragments thereof with increased binding affinity for the target. Amino acid changes introduced into antibodies or antigen-binding fragments may also change or introduce new post-translational modifications into antibodies or antigen-binding fragments, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation sites (e.g., changing the amino acid sequence so that different sugars are linked by enzymes present in the cell), or introducing new glycosylation sites.

[0186] The antibodies disclosed herein can be derived from animals of any species, including mammals. Non-limiting examples of natural antibodies include antibodies derived from humans, primates (e.g., monkeys and apes), cattle, pigs, horses, sheep, camelids (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits, including transgenic rodents genetically engineered to produce human antibodies).

[0187] Human and humanized antibodies include antibodies having variable and constant regions derived from human germline immunoglobulin sequences (or having the same amino acid sequence as antibodies derived from human germline immunoglobulin sequences). Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or somatic mutations in vivo), such as in CDRs.

[0188] Humanized antibodies are usually non-human CDRs transplanted onto human frameworks (FRs). Therefore, humanized antibodies have one or more amino acid sequences introduced therein from non-human sources. These non-human amino acid residues are generally referred to as "import" residues, which are generally taken from the "import" variable domains. Humanization can be performed essentially by, for example, replacing the corresponding sequence of a human antibody with a rodent CDR or CDR sequence. These methods are described in, for example, Jones et al., Nature, Vol. 321: pp. 522-525 (1986); Riechmann et al., Nature, Vol. 332: pp. 323-327 (1988); Verhoeyen et al., Science, Vol. 239: pp. 1534-1536 (1988), each of which is incorporated herein by reference in its entirety. Therefore, "humanized" antibodies are chimeric antibodies in which a small portion of a complete human V domain is replaced by a corresponding sequence from a non-human species. In practice, humanized antibodies are typically mouse antibodies in which some CDR residues and some FR residues are substituted by residues from analogous sites in human antibodies.

[0189] The selection of human VH and VL domains for preparing humanized antibodies is very important for reducing immunogenicity. According to the so-called "best fit" method, the V domain sequence of mouse antibodies is screened for the entire library of known human domain sequences. Then, the human sequence closest to the mouse sequence is accepted as the human FR of humanized antibodies (Sims et al., J.Immunol., Vol. 151: p. 2296 (1993); Chothia et al., J.Mol.Biol., Vol. 196: p. 901 (1987)).

[0190] More importantly, the antibody humanization retains high specificity and affinity to antigen and other favorable biological properties.In order to achieve this goal, humanized antibodies can be prepared by using the three-dimensional model analysis of parental sequence and various conceptual humanized products of parental and humanized sequences.Three-dimensional immunoglobulin model is usually available and familiar to those skilled in the art.The computer program of the possible three-dimensional conformational structure of the candidate immunoglobulin sequence of explanation and display selection can be obtained.The inspection of these displays allows the analysis of the possible effect of residues in the function of the candidate immunoglobulin sequence, i.e., analyzes the residues that affect the ability of the candidate immunoglobulin to bind its antigen.By this method, can select and combine FR residues from receptor and imported sequence, so as to obtain desired antibody characteristics, such as the affinity to the target antigen increased.

[0191] Typically, amino acid sequence variants of human, humanized or chimeric anti-CD40 antibodies will contain an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence present in the light or heavy chain of the original antibody.

[0192] In some embodiments, a mouse (e.g., RenMab) having a humanized heavy chain immunoglobulin locus and a humanized kappa chain immunoglobulin locus is used. TM Mouse) produces antibodies. The heavy chain immunoglobulin locus is a region on the chromosome that contains antibody heavy chain genes. The locus can include, for example, human IGHV (variable) genes, human IGHD (diversity) genes, human IGHJ (joining) genes, and mouse heavy chain constant domain genes. The κ chain immunoglobulin locus is a region on the chromosome that contains genes encoding antibody light chains (κ chains). The κ chain immunoglobulin locus can include, for example, human IGKV (variable) genes, human IGKJ (joining) genes, and mouse light chain constant domain genes. About RenMab TM A detailed description of mice can be found in PCT / CN2020 / 075698, the entire text of which is incorporated herein by reference.

[0193] In some embodiments, mice (e.g., RenLite TM Mouse) produces antibodies. The heavy chain immunoglobulin locus is a region on the chromosome that contains antibody heavy chain genes. The locus can include, for example, human IGHV (variable) genes, human IGHD (diversity) genes, human IGHJ (joining) genes, and mouse heavy chain constant domain genes. The κ chain immunoglobulin locus is a region on the chromosome that contains genes encoding common light chains. The κ chain immunoglobulin locus can include, for example, human IGKV (variable) genes, human IGKJ (joining) genes, and mouse light chain constant domain genes. About RenLiteTM A detailed description of mice can be found in PCT / CN2021 / 097652, which is incorporated herein by reference in its entirety.

[0194] The antibodies produced by the mouse have complete human VH, complete human VL and mouse constant regions. In some embodiments, human VH and human VL are connected to human IgG constant regions (e.g., IgG1, IgG2, IgG3 and IgG4). In some embodiments, the constant region has a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 52, 53, 54, 55, 56, 57, 58 or 59.

[0195] Identity or homology relative to a native sequence is typically the percentage of amino acid residues present in a candidate sequence that are identical to the sequence present in a human, humanized or chimeric anti-CD40 antibody or fragment, after aligning the candidate sequence with the native sequence and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.

[0196] The anti-CD40 antibody or antigen-binding fragment may be further modified. For example, cysteine ​​residues may be introduced into the Fc region, thereby allowing interchain disulfide bonds to form in this region. The homodimeric antibodies so produced may have any increased in vitro and / or in vivo half-life. Homodimeric antibodies with increased in vitro and / or in vivo half-life may also be prepared using heterobifunctional cross-linkers, as described, for example, by Wolff et al. (Cancer Res., Vol. 53: pp. 2560-2565, 1993). Alternatively, the antibody may be engineered to have dual Fc regions (see, e.g., Stevenson et al., Anti-Cancer Drug Design, Vol. 3: pp. 219-230, 1989).

[0197] In some embodiments, the anti-CD40 antibodies or antigen-binding fragments thereof may be covalently modified. These covalent modifications may be performed by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of the antibodies or antibody fragments are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with an organic derivatizing agent that is capable of reacting with selected side chains or N-terminal or C-terminal residues.

[0198] In some embodiments, antibody variants are provided, which have a carbohydrate structure lacking fucose (directly or indirectly) connected to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65% or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the sugar chain at Asn297 relative to the sum of all sugar structures (e.g., complex, hybrid and high mannose structures) connected to Asn297, as measured by MALDI-TOF mass spectrometry (e.g., as described in WO 2008 / 077546). Asn297 refers to an asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues; or position 314 in Kabat numbering); however, due to minor sequence variations in antibodies, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function.In some embodiments, to reduce glycan heterogeneity, the Fc region of the antibody may be further engineered to replace the asparagine at position 297 with alanine (N297A).

[0199] In some embodiments, to promote production efficiency by avoiding Fab arm exchange, the Fc region of the antibody is further engineered to replace the serine at position 228 (EU numbering) of IgG4 with proline (S228P). For a detailed description of the S228 mutation, see, for example, Silva et al., "The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation.", Journal of Biological Chemistry 290.9 (2015): 5462-5469, which is incorporated herein by reference in its entirety.

[0200] Recombinant vector

[0201] The present disclosure also provides recombinant vectors (e.g., expression vectors) comprising the isolated polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein), host cells into which these recombinant vectors are introduced (i.e., such that these host cells contain the polynucleotides and / or vectors comprising the polynucleotides), and recombinant antibody polypeptides or fragments thereof produced by recombinant technology.

[0202] As used herein, "vector" is any construct capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. "Expression vector" can deliver one or more polynucleotides of interest in a host cell into which the expression vector has been introduced and express them as encoded polypeptides. Therefore, in an expression vector, by being operably connected to a regulatory element (such as a promoter, enhancer and / or poly-A tail) at or near or on both sides of the integration site of the polynucleotide of interest in the vector or in the genome of the host cell, the polynucleotide of interest is positioned to be expressed in the vector so that the polynucleotide of interest will be translated in the host cell into which the expression vector is introduced.

[0203] The vector can be introduced into the host cell by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection and infection and / or transduction (e.g., with a recombinant virus). Therefore, non-limiting examples of vectors include viral vectors (which can be used to produce recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0204] In some embodiments, a polynucleotide disclosed herein (e.g., a polynucleotide encoding a polypeptide disclosed herein) is introduced using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), which may involve the use of non-pathogenic (defective), replication-competent viruses, or replication-defective viruses may be used. In the latter case, viral propagation typically occurs only in complementing virus packaging cells. Suitable systems are disclosed, for example, in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA, Vol. 86: 317-321; Flexner et al., 1989, Ann. NY Acad Sci., Vol. 569: 86-103; Flexner et al., 1990, Vaccine, Vol. 8: 17-21; U.S. Pat. Nos. 4,603,112, 4,769,330 and 5,017,487; WO 89 / 01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91 / 02805; Berkner-Biotechniques, Vol. 6: pp. 616-627, 1988; Rosenfeld et al., 1991, Science, Vol. 252: pp. 431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, Vol. 91: pp. 215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, Vol. 90: pp. 11498-11502; Guzman et al., 1993, Circulation, Vol. 88: pp. 2838-2848; and Guzman et al., 1993, Cir. Res., Vol. 73: pp. 1202-1207. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be "naked," as described, for example, in Ulmer et al., 1993, Science, Vol. 259: pp. 1745-1749, and Cohen, 1993, Science, Vol. 259: pp. 1691-1692. The uptake of naked DNA may be increased by coating the DNA on biodegradable beads that are efficiently transported into cells.

[0205] For expression, the DNA insert comprising the polynucleotide encoding the antibody or polypeptide disclosed herein can be operably linked to a suitable promoter (e.g., a heterologous promoter), such as the bacteriophage λPL promoter, E. coli (E. coli) lac, trp and tac promoters, SV40 early and late promoters, and promoters of retroviral LTRs, etc. Other suitable promoters are known to those skilled in the art. The expression construct may also contain transcription initiation and termination sites, and a ribosome binding site for translation in the transcribed region. The coding portion of the mature transcript expressed by the construct may include a translation initiation codon at the start of the polypeptide to be translated and a termination codon (UAA, UGA or UAG) at an appropriate position at the end of the polypeptide to be translated.

[0206] As described above, the expression vector may include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance genes for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culture in Escherichia coli and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells, such as Escherichia coli (E.coli), Streptomyces (Streptomyces) and Salmonella typhimurium (Salmonella typhimurium) cells; fungal cells, such as yeast cells; insect cells, such as fruit fly S2 and fall armyworm Sf9 cells; animal cells, such as CHO, COS, Bowes melanoma and HK 293 cells; and plant cells. Suitable culture media and conditions for host cells described herein are known in the art.

[0207] Non-limiting vectors for bacteria include pQE70, pQE60, and pQE-9 from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG from Stratagene; and pSVK3, pBPV, pMSG, and pSVL from Pharmacia. Other suitable vectors will be apparent to those skilled in the art.

[0208] Suitable non-limiting bacterial promoters include E. coli lacI and lacZ promoters, T3 and T7 promoters, gpt promoter, lambda PR and PL promoters and trp promoter. Suitable eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, promoters of retroviral LTR (such as the promoter of Rous sarcoma virus (RSV)) and metallothionein promoters (such as mouse metallothionein-I promoter).

[0209] In Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters are available, such as alpha factor, alcohol oxidase, and PGH. For review, see Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, and Grant et al., Methods Enzymol., Vol. 153: 516-544 (1997).

[0210] The construct is introduced into the host cell by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection or other methods. These methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), which is incorporated herein by reference in its entirety.

[0211] The transcription of the DNA encoding the antibodies of the present disclosure by higher eukaryotes can be increased by inserting an enhancer sequence into the vector. An enhancer is a cis-acting element of DNA, usually about 10 bp-300 bp, that acts to increase the transcriptional activity of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer located on the late side of the replication origin at base pairs 100-270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancer.

[0212] In order to secrete the translated protein into the endoplasmic reticulum, the periplasmic space or the extracellular environment, an appropriate secretion signal can be incorporated into the expressed polypeptide. The signal can be an endogenous signal of the polypeptide or a heterologous signal.

[0213] Polypeptides (e.g., antibodies) can be expressed in a modified form, such as a fusion protein (e.g., a GST fusion protein) or with a histidine tag, and can include not only a secretion signal, but also additional heterologous functional regions. For example, a region of additional amino acids (particularly charged amino acids) can be added to the N-terminus of the polypeptide to improve stability and durability in a host cell, during purification, or during subsequent processing and storage. In addition, peptide moieties can be added to the polypeptide to promote purification. These regions can be removed before the final preparation of the polypeptide. Adding peptide moieties to a polypeptide to cause secretion or excretion, improve stability, and promote purification, etc., is a conventional technique well known in the art.

[0214] Treatment

[0215] The antibodies or antigen-binding fragments thereof disclosed herein can be used for a variety of therapeutic purposes.

[0216] In one aspect, the present disclosure provides methods for treating cancer in a subject, methods for reducing the rate of increase of tumor volume over time in a subject, methods for reducing the risk of forming metastases, or methods for reducing the risk of forming additional metastases in a subject. In some embodiments, treatment may terminate, slow, delay, or inhibit the progression of cancer. In some embodiments, treatment may reduce the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0217] In one aspect, the disclosure features a method comprising administering a therapeutically effective amount of an antibody disclosed herein or an antigen-binding fragment thereof to a subject in need thereof (e.g., a subject suffering from or identified or diagnosed as suffering from cancer), such as breast cancer (e.g., triple-negative breast cancer), carcinoid, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or hematological malignancies. In some embodiments, the cancer is unresectable melanoma or metastatic melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone-refractory prostate cancer. In some embodiments, the subject suffers from a solid tumor. In some embodiments, the cancer is squamous cell carcinoma of the head and neck (SCCHN), renal cell carcinoma (RCC), triple-negative breast cancer (TNBC), or colorectal cancer. In some embodiments, the subject suffers from Hodgkin's lymphoma. In some embodiments, the subject has triple negative breast cancer (TNBC), gastric cancer, urothelial carcinoma, Merkel cell carcinoma, or head and neck cancer. In some embodiments, the cancer is melanoma, pancreatic cancer, mesothelioma, a hematological malignancy, particularly non-Hodgkin's lymphoma, lymphoma, chronic lymphocytic leukemia, or an advanced solid tumor.

[0218] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk for cancer.Cancer patients can be identified using various methods known in the art.

[0219] In one aspect, the present disclosure provides methods for treating, preventing, or reducing the risk of developing diseases associated with abnormal or unwanted immune responses (e.g., autoimmune diseases), such as by affecting the functional properties of APC cells (e.g., by blocking the interaction between CD40 and CD40L). These autoimmune diseases include, but are not limited to, alopecia areata, lupus, ankylosing spondylitis, Meniere's disease, antiphospholipid syndrome, mixed connective tissue disease, autoimmune Addison's disease, disease), multiple sclerosis, autoimmune hemolytic anemia, myasthenia gravis, autoimmune hepatitis, pemphigus vulgaris, Behcet's disease, pernicious anemia, bullous pemphigoid, polyarthritis nodularis, cardiomyopathy, polychondritis, celiac dermatitis, polyglandular syndrome, chronic fatigue syndrome (CFIDS), polymyalgia rheumatica, chronic inflammatory demyelinating, polymyositis and dermatomyositis, chronic inflammatory polyneuropathy, primary agammaglobulinemia, Churg-Strauss syndrome, primary biliary cirrhosis, cicatricial pemphigoid , psoriasis, CREST syndrome, Raynaud's phenomenon, cold agglutinin disease, Reiter's syndrome, Crohn's disease, rheumatic fever, discoid lupus, rheumatoid arthritis, cryoglobulinemia, sarcoidosis, fibromyalgia, scleroderma, Graves' disease, Sjögren's syndrome, Guillain-Barre syndrome, stiff-man syndrome, Hashimoto's thyroiditis, Takayasu arteritis, idiopathic pulmonary fibrosis, temporal arteritis / giant cell arteritis, idiopathic thrombocytopenic purpura (ITP), ulcerative colitis, IgA nephropathy, uveitis, diabetes (e.g., type I diabetes), vasculitis, lichen planus, and vitiligo. Anti-CD40 antibodies or antigen-binding fragments thereof can also be administered to a subject to treat, prevent, or reduce the risk of developing a disease associated with an abnormal or unwanted immune response associated with cell, tissue, or organ transplantation (e.g., kidney, liver, and heart transplantation (e.g., graft-versus-host disease (GVHD)), or to prevent allogeneic transplant rejection. In some embodiments, the subject suffers from skin disease, liver disease (e.g., cirrhosis), hidradenitis, experimental autoimmune encephalomyelitis. In some embodiments, the subject suffers from nephropathy, lupus, Sjögren's syndrome, ulcerative colitis, psoriasis, allergic dermatitis, atopic dermatitis, suppurative hidradenitis, immune thrombocytopenia (ITP) or other inflammatory arthritis. In some embodiments, the subject suffers from multiple sclerosis or myasthenia gravis. In some embodiments, the subject suffers from Crohn's disease, ulcerative colitis or type 1 diabetes. In some embodiments, the subject suffers from autoimmune thyroid disease, Graves' disease, multiple sclerosis, psoriasis, inflammatory bowel disease (e.g., Crohn's disease (CD) and ulcerative colitis), rheumatoid arthritis, Sjögren's syndrome, autoimmune nephritis or systemic lupus erythematosus.The relationship between CD40 and various autoimmune diseases is described in, for example, Peters et al., "CD40 and autoimmunity: the dark side of a great activator.", Seminars in immunology., Vol. 21, No. 5, Academic Press, 2009, and Karnell, Jodi L. et al., "Targeting the CD40-CD40L pathway in autoimmune diseases: Human immunity and beyond.", Advanced drug delivery reviews (2018); Albach et al., "Safety, pharmacokinetics and pharmacodynamics of single rising doses of BI655064, an antagonistic anti-CD40 antibody in healthy subjects: a potential novel treatment for autoimmune diseases.", European journal of clinical pharmacology 74.2 (2018): pp. 161-169; the entire text of each of these documents is incorporated herein by reference.

[0220] As used herein, "effective amount" refers to an amount or dosage sufficient to achieve beneficial or desired results, including terminating, slowing, delaying or inhibiting the progression of a disease (e.g., an autoimmune disease or cancer). The effective amount will vary depending on, for example, the age and weight of the subject to whom the antibody, antigen-binding fragment, antibody encoding polynucleotide, vector comprising the polynucleotide and / or composition thereof is to be administered, the severity of the symptoms and the route of administration, and therefore can be determined on an individual basis.

[0221] The effective amount can be applied in one or more administrations. For example, the effective amount of an antibody or antigen binding fragment is enough to improve, terminate, stabilize, reverse, inhibit, slow down and / or delay the amount of the progress of an autoimmune disease or cancer in a patient, or is enough to improve, terminate, stabilize, reverse, slow down and / or delay the amount of a cell (e.g., a biopsy cell, any one of the cancer cells described herein or a cell line (e.g., a cancer cell line)) in vitro propagation. As understood in the art, the effective amount of an antibody or antigen binding fragment can be based on, especially the patient's medical history and other factors (such as the type (and / or dosage) of the antibody used) change.

[0222] The effective amount and dosage regimen of the antibodies, polynucleotides encoding antibodies and / or compositions disclosed herein can be determined empirically, and making such determinations is within the technical scope of the art. It will be understood by those skilled in the art that the dosage that must be applied will vary according to, for example, the mammals that will receive the antibodies, polynucleotides encoding antibodies and / or compositions disclosed herein, the route of administration, the specific type of the antibodies, polynucleotides encoding antibodies, antigen-binding fragments and / or compositions disclosed herein used, and other drugs administered to mammals. Guidance on selecting the appropriate dosage of antibodies or antigen-binding fragments can be found in the literature on the therapeutic use of antibodies and antigen-binding fragments, such as Handbook of Monoclonal Antibodies, Ferrone et al., ed., Noges Publications, Park Ridge, NJ, 1985, Chapter 22, pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., ed., Raven Press, New York, 1977, pp. 365-389.

[0223] The typical daily dose of an effective amount of an antibody is 0.01 mg / kg to 100 mg / kg. In some embodiments, the dosage may be less than 100 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, or 0.1 mg / kg. In some embodiments, the dosage may be greater than 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.1 mg / kg, 0.05 mg / kg, or 0.01 mg / kg. In some embodiments, the dosage is about 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.9 mg / kg, 0.8 mg / kg, 0.7 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, or 0.1 mg / kg.

[0224] In any of the methods described herein, at least one antibody, its antigen binding fragment or pharmaceutical composition (e.g., any of the antibodies, antigen binding fragments or pharmaceutical compositions described herein) and optionally at least one additional therapeutic agent may be administered to a subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day or three times a day). In some embodiments, at least two different antibodies and / or antigen binding fragments are administered with the same composition (e.g., a liquid composition). In some embodiments, at least one antibody or antigen binding fragment and at least one additional therapeutic agent are administered with the same composition (e.g., a liquid composition containing at least one antibody or antigen binding fragment and a solid oral composition containing at least one additional therapeutic agent). In some embodiments, at least one additional therapeutic agent is administered in the form of a pill, tablet or capsule. In some embodiments, at least one additional therapeutic agent is administered with a sustained release oral formulation.

[0225] In some embodiments, one or more additional therapeutic agents may be administered to a subject before or after administration of at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein). In some embodiments, one or more additional therapeutic agents and at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) are administered to a subject such that there is overlap in the biologically active periods of the one or more additional therapeutic agents and at least one antibody or antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein) in the subject.

[0226] In some embodiments, at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) may be administered to a subject over an extended period of time (e.g., over a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years). A skilled medical professional may determine the length of the treatment period using any of the methods described herein to diagnose or track the effectiveness of the treatment (e.g., to observe at least one symptom of cancer). As described herein, a skilled medical professional may also change the type and amount (e.g., increase or decrease) of the antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) administered to the subject, and may also adjust (e.g., increase or decrease) the dose or frequency of at least one antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) administered to the subject based on an assessment of the effectiveness of the treatment (e.g., using any of the methods described herein and known in the art).

[0227] In some embodiments, one or more additional therapeutic agents may be administered to the subject. The additional therapeutic agent may include one or more inhibitors, and the one or more inhibitors are selected from the group consisting of: B-Raf inhibitors, EGFR inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c-Met inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, Akt inhibitors, mTOR inhibitors, PI3K / mTOR dual inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, and isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2) inhibitors. In some embodiments, the additional therapeutic agent is an indoleamine 2,3-dioxygenase-1 (IDO1) inhibitor (e.g., epacadostat).

[0228] In some embodiments, the additional therapeutic agent may include one or more inhibitors selected from the group consisting of: a HER3 inhibitor, a LSD1 inhibitor, a MDM2 inhibitor, a BCL2 inhibitor, a CHK1 inhibitor, an inhibitor of activated hedgehog signaling pathway, and an agent that selectively degrades the estrogen receptor.

[0229] In some embodiments, the additional therapeutic agent may include one or more therapeutic agents selected from the group consisting of trabectedin, nab-paclitaxel, trebananib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, sorafenib , Votrient, pazopanib, IMA-901, AGS-003, cabozantinib, vinflunine, Hsp90 inhibitors, Ad-GM-CSF, temazolomide, IL-2, IFNa, vinblastine, thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, bortezomid, amrubicine, carfilzomib, pralatrexate, and enzastaurin.

[0230] In some embodiments, the additional therapeutic agent may include one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, a tumor necrosis factor (TNF) α, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a CX3CL1-targeted therapy, a CXCL9-targeted therapy, a CXCL10-targeted therapy, a CCL5-targeted therapy, an LFA-1 agonist, an ICAM1 agonist, and a selectin agonist.

[0231] In some embodiments, the subject is administered carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI.

[0232] In some embodiments, the additional therapeutic agent is an anti-OX40 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, an anti-ICOS antibody, an anti-CD27 antibody, an anti-4-1BB antibody, and / or an anti-GITR antibody. In some embodiments, the anti-CD40 antibody or antigen-binding fragment thereof described herein may be administered together with an anti-PD-1 antibody and an anti-TIGIT antibody.

[0233] Pharmaceutical compositions and routes of administration

[0234] Also provided herein are pharmaceutical compositions comprising at least one (e.g., one, two, three, or four) antibody or antigen-binding fragment described herein. Two or more (e.g., two, three, or four) of any of the antibodies or antigen-binding fragments described herein may be present in any combination in a pharmaceutical composition. The pharmaceutical composition may be formulated in any manner known in the art.

[0235] Pharmaceutical compositions are formulated to be compatible with their intended routes of administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous or intraperitoneal). Compositions may include sterile diluents (e.g., sterile water or saline), fixed oils, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents, antibacterial or antifungal agents (such as benzyl alcohol or methyl paraben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.), antioxidants (such as ascorbic acid or sodium bisulfite), chelating agents (such as ethylenediaminetetraacetic acid), buffers (such as acetate, citrate or phosphate) and isotonic agents (such as sugars (e.g., glucose), polyols (e.g., mannitol or sorbitol) or salts (e.g., sodium chloride)), or any combination thereof. Liposomal suspensions may also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Patent No. 4,522,811). The preparation of the composition may be formulated and encapsulated in ampoules, disposable syringes or multi-dose vials. When needed (as, for example, in injectable formulations), suitable fluidity can be maintained by, for example, using a coating (such as lecithin) or a surfactant. Absorption of the antibody or its Fab can be prolonged by including agents that delay absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene-vinyl acetate copolymers, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza and Nova Pharmaceuticals).

[0236] Compositions containing one or more of any of the antibodies or antigen-binding fragments described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing predetermined quantities of active compound to facilitate administration and uniformity of dosage).

[0237] Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and produced under good manufacturing practice (GMP) conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., a single dose). Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients or adjuvants. The formulation depends on the selected route of administration. For injection, the antibody can be formulated in an aqueous solution, preferably in a physiologically compatible buffer to reduce discomfort at the injection site. The solution may contain a preparatant, such as a suspending agent, a stabilizer and / or a dispersant. Alternatively, the antibody can be in a lyophilized form so that it can be formulated with a suitable solvent (e.g., sterile pyrogen-free water) before use.

[0238] The toxicity and therapeutic efficacy of the composition can be determined by standard pharmaceutical methods in cell culture or experimental animals (e.g., monkeys). For example, the LD50 (the dose that is lethal to 50% of the population) and the ED50 (the dose that is therapeutically effective to 50% of the population) can be determined: the therapeutic index is the ratio of LD50:ED50. Agents that exhibit high therapeutic indices are preferred. When an agent exhibits adverse side effects, care should be taken to minimize potential damage (i.e., reduce adverse side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical methods.

[0239] The data obtained from cell culture assays and animal studies can be used to formulate any given agent for a subject (e.g., a human). The therapeutically effective amount of one or more (e.g., one, two, three, or four) antibodies or their antigen-binding fragments (e.g., any one of the antibodies or antibody fragments described herein) will be a treatment subject (e.g., a human subject identified as having cancer) or a subject identified as having a risk of illness (e.g., a subject previously suffering from cancer but now cured) in a disease (e.g., killing cancer cells), reducing the severity, frequency, and / or duration of one or more symptoms of the disease in a subject (e.g., a human). The effectiveness and dosage of any one of the antibodies or antigen-binding fragments described herein can be determined by a health care professional or veterinary professional using methods known in the art and by observing one or more symptoms of the disease in a subject (e.g., a human). Certain factors can affect the dosage and timing required for the effective treatment of a subject (e.g., the severity of the disease or condition, previous treatment, the general health and / or age of the subject, and the presence of other diseases).

[0240] Exemplary dosages include milligram or microgram amounts of any of the antibodies or antigen-binding fragments described herein per kilogram of subject body weight (e.g., about 1 μg / kg to about 500 mg / kg; about 100 μg / kg to about 500 mg / kg; about 100 μg / kg to about 50 mg / kg; about 10 μg / kg to about 5 mg / kg; about 10 μg / kg to about 0.5 mg / kg; or about 1 μg / kg to about 50 μg / kg). Although these dosages cover a wide range, it will be appreciated by those of ordinary skill in the art that the efficacy of therapeutic agents (including antibodies and antigen-binding fragments thereof) varies, and that effective amounts can be determined by methods known in the art. Typically, a relatively low dose is first administered, after which the attending healthcare professional or veterinary professional (in the case of therapeutic applications) or researcher (when still working in the development phase) can gradually increase the dose until an appropriate response is obtained. Furthermore, it should be understood that the specific dosage level for any particular subject will depend on a variety of factors, including the activity of the specific compound used, the age, weight, general health, sex and diet of the subject, the time of administration, the route of administration, the rate of excretion and the half-life of the antibody or antibody fragment in vivo.

[0241] The pharmaceutical composition may be included in a container, pack, or dispenser together with instructions for administration. The present disclosure also provides methods for preparing antibodies or antigen-binding fragments thereof for various uses as described herein.

[0242] Example

[0243] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0244] Example 1. Generation of human anti-CD40 antibodies

[0245] To generate antibodies against human CD40, RenMab was immunized with human CD40. TM Mouse or RenLite TM Mouse. Anti-CD40 antibody was prepared as described below.

[0246] RenMab TM The mouse has a humanized heavy chain immunoglobulin locus and a humanized kappa chain immunoglobulin locus. The heavy chain immunoglobulin locus is the region on the chromosome that contains the antibody heavy chain genes. The locus includes IGHV (variable), IGHD (diversity), IGHJ (joining), and heavy chain constant domain genes. The kappa chain immunoglobulin locus is the region on the chromosome that contains the genes encoding the antibody light chain (kappa chain). The kappa chain immunoglobulin locus includes IGKV (variable), IGKJ (joining), and light chain constant domain genes. About RenMab TMA detailed description of mice can be found in PCT / CN2020 / 075698, the entire text of which is incorporated herein by reference.

[0247] RenLite TM The mouse can be used as a genetic engineering model for strategic engineering of antibody genes that are fully humanized in the heavy chain variable region while maintaining a fully humanized common light chain. TM A detailed description of mice can be found in, for example, PCT / CN2021 / 097652, which is incorporated herein by reference in its entirety.

[0248] Mouse immunization

[0249] RenMab was immunized with Fc-tagged human CD40 protein (hCD40-Fc, Beijing Biopsies Biotechnology Co., Ltd., catalog number: CD0-H5253) or dog CD40 (dCD40-Fc, Beijing Sino Biological Technology Co., Ltd., catalog number: 70105-D02H) TM Mouse or RenLite TM Mice. Specifically, hCD40-Fc or dCD40-Fc was emulsified with an adjuvant and injected into the heel, neck or tail base of the mouse. The mice were immunized 3 times: complete Freund's adjuvant (CFA) was used for the first immunization, and incomplete Freund's adjuvant (IFA) was used for the second and third immunizations. The interval between the first and second immunizations was two weeks, and the interval between the second and third immunizations was two weeks. Orbital blood was collected 1 week after the third immunization, and the antibody titer was analyzed using ELISA. Two weeks later, mice with high titers were selected and injected with hCD40-His (human CD40 protein with His tag, Beijing Biopsies Biotechnology Co., Ltd., catalog number: CD0-H5228) or dCD40-His (CD40 protein, canine, recombinant (His tag), Beijing Yiqiao Shenzhou Technology Co., Ltd., catalog number: 70105-D08H) through the tail vein.

[0250] In another experiment, RenMab was immunized by injecting (by intramuscular (im) injection) expression plasmids encoding human CD40 and / or dog CD40 into the tibialis anterior muscle of mice. TM Mouse or RenLite TM Mice. Mice were injected at least four times, with at least 14 days between each injection. Blood was collected seven days after the last immunization, and the serum was tested for antibody titers by ELISA.

[0251] A booster step (by injection of expression plasmid or by injection of protein) was also performed at least seven days after immunization. In addition, mice were injected (by intraperitoneal administration) with CHO-S-hCD40 (transfected CHO-S cells expressing human CD40 protein) or CHO-S-dCD40 (transfected CHO-S cells expressing dog CD40 protein) for pulse immunization. Immune system organs (e.g., bone marrow, lymph nodes, and spleen) were then collected five days after injection.

[0252] Antigen-specific B cells are also isolated directly from immunized mice without fusion with myeloma cells. Antibody light and heavy chain variable region sequences are obtained directly from antigen-specific B cells. For example, using single cell technology (e.g., Plasma cells secreting antigen-specific monoclonal antibodies were screened using an optofluidic system (Berkeley Lights Inc.). Antibody variable region sequences were obtained using reverse transcription and PCR-based sequencing. Antibodies were expressed by transfecting cells with vectors containing antibody variable region sequences. FACS was used to verify binding between the antibody and CD40. Exemplary anti-CD40 antibodies obtained by this method include: 2F8, 6A4, 15B4, 16D5, and 12B5.

[0253] The antibodies are named according to the following rules. For example, when the heavy chain variable region (VH) and light chain variable region (VL) of the 2F8 antibody are linked to the human IgG1 constant region, the antibody is named 2F8-IgG1. Similarly, 2F8-IgG2 and 2F8-IgG4 were produced, which include the same VH and VL sequences of 2F8, but the constant regions are from human IgG2 and IgG4 subtypes, respectively.

[0254] The constant region may also include mutations. For example, LALA mutations (L234A and L235A according to EU numbering), N297A, YTE mutations (M252Y, S254T and T256E according to EU numbering), FLAA mutations (F234A and L235A according to EU numbering) or SI mutations (S239D and I332E according to EU numbering) are introduced into the Fc region of 2F8-IgG1, generating 2F8-IgG1-LALA, 2F8-IgG1-N297A, 2F8-IgG1-YTE, 2F8-IgG1-FLAA and 2F8-IgG1-SI, respectively. YTE mutations are also introduced into the Fc region of 2F8-IgG1-LALA or 2F8-IgG1-N297A, generating 2F8-IgG1-LALA-YTE or 2F8-IgG1-N297A-YTE, respectively. Antibodies with different constant region sequences were also prepared for 6A4, 15B4, 16D5 and 12B5.

[0255] The amino acid sequences of the heavy chain CDR1, 2, 3 and light chain CDR1, 2, 3 of 2F8 are shown in SEQ ID NO: 1-6 (Kabat numbering) or SEQ ID NO: 19-24 (Chothia numbering), respectively. The human heavy chain variable region and human light chain variable region of 2F8 are shown in SEQ ID NO: 65 and SEQ ID NO: 37, respectively.

[0256] The amino acid sequences of the heavy chain CDR1, 2, 3 and light chain CDR1, 2, 3 of 6A4 are shown in SEQ ID NO: 7-12 (Kabat numbering) or SEQ ID NO: 25-30 (Chothia numbering), respectively. The human heavy chain variable region and human light chain variable region of 6A4 are shown in SEQ ID NO: 38 and SEQ ID NO: 39, respectively.

[0257] The amino acid sequences of the heavy chain CDR1, 2, 3 and light chain CDR1, 2, 3 of 12B5 are shown in SEQ ID NO: 13-18 (Kabat numbering) or SEQ ID NO: 31-36 (Chothia numbering), respectively. The human heavy chain variable region and human light chain variable region of 12B5 are shown in SEQ ID NO: 40 and SEQ ID NO: 41, respectively.

[0258] Antibody preparation

[0259] The positive antibody sequences in the sequence verification stage were extracted with plasmids and transfected into a 100 ml system. The expression supernatant was collected after 10-13 days of cell culture and then subjected to affinity chromatography. The obtained antibody samples were used for subsequent in vitro testing and screening.

[0260] Example 2. Binding affinity of anti-CD40 antibodies

[0261] Bleselumab (VH SEQ ID NO:43, VL SEQ ID NO:44; HC SEQ ID NO:66, LC SEQ ID NO:60) is a fully human IgG4 monoclonal antibody targeting CD40 that is approved for the treatment of lupus.

[0262] A Biacore was used with a pre-immobilized protein A sensor chip. TM(Biacore, Inc., Piscataway, NJ, USA) 8K biosensor, using surface plasmon resonance (SPR) to measure the binding affinity of anti-CD40 antibodies 2F8-IgG4-FLAA, 6A4-IgG4-FLAA, 12B5-IgG4-FLAA, and blelumab analogs to human CD40 (hCD40), monkey (cynomolgus macaque) CD40 (fasCD40), or dog CD40 (dCD40).

[0263] Specifically, hCD40-His, fasCD40-His (cynomolgus CD40 / TNFRSF5 protein, His tag, Beijing Biopsies Biotechnology Co., Ltd., catalog number: CD0-C52H6) or dCD40-His was diluted to 200 nM, 100 nM, 50 nM, 25 nM, 6.25 nM, 3.125 nM or 0 nM with 1× HBS-EP+ buffer (pH 7.4) and then injected into the Biacore TM 8K biosensor for about 50 seconds to meet the desired capture level (e.g., about 100 response units (RU)). Purified anti-CD40 antibody at a concentration of 1 μg / ml in 1×HBS-EP+ buffer (pH 7.4) was then injected at 1 μl / min for 50 seconds. Dissociation was monitored for 400 seconds. After the last injection of each round of titration, the chip was regenerated with glycine solution (pH 2.0) at a rate of 30 μl / min for 30 seconds.

[0264] By using Biacore TM 8K Evaluation Software 3.0 fits the data to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994, Methods Enzymology, Vol. 6, pp. 99-110), thereby simultaneously obtaining the kinetic association rate (kon) and dissociation rate (koff). Affinity is derived from the quotient of the kinetic rate constant (KD = koff / kon).

[0265] As will be appreciated by one of ordinary skill in the art, the same methodology is used for each antibody tested, with appropriate adjustments made to parameters such as antibody concentration.

[0266] The results of the tested antibodies are summarized in the table below. The results show that the anti-CD40 antibodies 2F8-IgG4-FLAA and 12B5-IgG4-FLAA specifically bind to human, monkey and dog CD40. 6A4-IgG4-FLAA specifically binds to human and monkey CD40.

[0267] Table 1

[0268] Anti-CD40 antibody Analyte 1 solution <![CDATA[K on (1 / Ms)]]> <![CDATA[K off (1 / s)]]> KD(M) 2F8-IgG4-FLAA hCD40-His 6.54E+05 2.29E-04 3.51E-10 2F8-IgG4-FLAA fasCD40-His 9.89E+05 2.97E-04 3.01E-10 2F8-IgG4-FLAA dCD40-His 5.13E+05 2.66E-03 5.18E-09 6A4-IgG4-FLAA hCD40-His 3.09E+05 5.11E-04 1.66E-09 6A4-IgG4-FLAA fasCD40-His 2.88E+05 1.49E-02 5.17E-08 6A4-IgG4-FLAA dCD40-His — — — 12B5-IgG4-FLAA hCD40-His 5.29E+05 3.89E-04 7.35E-10 12B5-IgG4-FLAA fasCD40-His 5.35E+05 2.65E-04 4.95E-10 12B5-IgG4-FLAA dCD40-His 4.01E+05 1.29E-02 3.21E-08 Blelumab analogs hCD40-His 4.29E+05 6.82E-04 1.59E-09 Blelumab analogs fasCD40-His 2.70E+05 9.07E-04 3.35E-09 Blelumab analogs dCD40-His — — —

[0269] Example 3. Jurkat-luc-hCD40 reporter cell activation assay

[0270] Iscalimab (VH SEQ ID NO:45, VL SEQ ID NO:46; HC SEQ ID NO:61, LC SEQ ID NO:62) is a fully human IgG1 antibody that blocks the CD40 signaling pathway and is being developed by Novartis and XOMA for the treatment of Sjögren's syndrome.

[0271] BI655064 (VH SEQ ID NO:47, VL SEQ ID NO:48; HC SEQ ID NO:63, LC SEQ ID NO:64) is a humanized pure antagonistic anti-CD40 IgG1 monoclonal antibody developed by Boehringer Ingelheim for the treatment of rheumatoid arthritis (RA) and lupus nephritis, among others.

[0272] Experiments were performed to test whether the anti-CD40 antibodies 2F8-IgG4-FLAA, 6A4-IgG4-FLAA, 15B4-IgG4-FLAA, 12B5-IgG4-FLAA, BI655064 analogs, icalizumab analogs, and blelumab analogs could block the CD40 signaling pathway.

[0273] CHO-K1-FcγRIIB cells (Promega, catalog number: CS1979A09) were seeded in 96-well plates (cell density: 4×10 4 cells / well) and incubated at 37°C overnight. The test antibody was serially (3-fold) diluted from the highest concentration of 6 μg / mL. Human CD40 ligand CD40L-His (human CD40 ligand / TNFSF5 protein, His, Flag tag (active trimer) (MALS verification), Beijing Biopsies Biotechnology Co., Ltd., catalog number: CDL-H52Db) was serially diluted from the highest concentration of 3 μg / ml. Jurkat-Luc-hCD40 cells (transfected Jurkat-Luc cells expressing human CD40 protein) were seeded in 96-well plates (cell density: 5×10 5Cells / well), then add 25μl of serially diluted antibody solution and / or human CD40 ligand to each well. The working concentration of the antibody is 0.5μg / ml, 1μg / ml and 2μg / ml. Incubate the above 96-well plate in a 37°C incubator for 6 hours. After incubation, remove the culture plate and add 75μl Bio-lite TM Luciferase detection reagent (Nanjing Novozyme Biotech Co., Ltd., catalog number: DD1201-02-AB). The culture plate was incubated at room temperature for 5-10 minutes and then placed in a luminescence detector to detect the fluorescence signal.

[0274] As the concentration of all anti-CD40 antibodies decreased, the fluorescent signal (indicating cell binding to CD40 ligand) increased, indicating that the binding between human CD40 and human CD40 ligand was blocked by the anti-CD40 antibodies.

[0275] Example 4: In vivo immunosuppressive effects of anti-CD40 antibodies

[0276] Immunosuppressive effects of anti-CD40 antibodies in hCD40 mice

[0277] A CD40 gene humanized mouse model is generated to express a chimeric CD40 protein (SEQ ID NO: 49), in which a portion of the extracellular region of the mouse CD40 protein is replaced by the corresponding human CD40 extracellular region. Specifically, amino acids 20-192 (SEQ ID NO: 50) of mouse CD40 are replaced by amino acids 20-192 (SEQ ID NO: 42) of human CD40. The humanized mouse model (hCD40 mouse) provides a tool for testing new treatment methods in a clinical setting by significantly reducing the difference between the clinical outcomes of humans and experimental mice expressing mouse CD40. A detailed description of the CD40 gene humanized mouse model can be found in PCT / CN2018 / 091845, which is incorporated herein by reference in its entirety.

[0278] Experiments were conducted to analyze the effects of human anti-CD40 antibodies on immune responses. Ovalbumin (OVA) was used as an antigen to stimulate the immune response of hCD40 mice. In brief, hCD40 mice (6-8 weeks old) were divided into a control group and six treatment groups (5 mice per group). The treatment group mice were randomly selected to be administered anti-CD40 antibody blelumab analogs (G2), 12B5-IgG4-FLAA (G3), 16D5-IgG4-FLAA (G4), 2F8-IgG4-FLAA (G5), 6A4-IgG4-FLAA (G6) and 15B4-IgG4-FLAA (G7) at a dose level of 2 mg / kg intraperitoneally (ip). The control group mice (G1) were injected with an equal volume of phosphate buffered saline (PBS). A total of two doses were administered on day 0 (grouping day) and day 4 (4 days after grouping). Ovalbumin (OVA) was diluted to 1 mg / ml with PBS and mixed with CFA at a volume ratio of 1:1, and the immunization volume was 200 μl / mouse. The OVA / CFA mixture was intraperitoneally administered to mice on day 1 (1 day after grouping). Serum was collected from each animal on days 10 and 17, and the collected serum was subjected to ELISA analysis. Figure 1 The experimental scheme is shown. OVA was pre-coated on ELISA plates. ELISA analysis was performed using goat anti-mouse IgG H&L (HRP) (Abcam, catalog number: ab97265). The detailed dosing regimen is shown in the table below.

[0279] Table 2

[0280]

[0281] like Figure 2 As shown, the ELISA results of sera collected on day 10 showed that the anti-CD40 antibodies blelumab analogs (G2), 12B5-IgG4-FLAA (G3), 16D5-IgG4-FLAA (G4), 2F8-IgG4-FLAA (G5), 6A4-IgG4-FLAA (G6) and 15B4-IgG4-FLAA (G7) reduced the immune response of hCD40 mice compared with PBS (G1). Figure 3 ELISA results of serum collected on day 17 are shown. The results on day 17 are similar to those obtained on day 10. Specifically, breklumab analogs (G2), 12B5-IgG4-FLAA (G3), 2F8-IgG4-FLAA (G5) and 6A4-IgG4-FLAA (G6) reduced the immune response, with OD lower than 16D5-IgG4-FLAA (G4) and 15B4-IgG4-FLAA (G7). It is expected that more antibodies will show reduced immune responses on day 10, because antibody degradation increases on day 17.

[0282] Immunosuppressive effect of anti-CD40 antibody in hCD40 / hFcRn mice

[0283] By hybridizing hCD40 mice with FcRn gene humanized mice (hFcRn mice), a CD40 / FcRn double gene humanized mouse model was also generated. The FcRn gene humanized mice were engineered to express human FcRn protein (SEQ ID NO: 51). A detailed description of FcRn gene humanized mice and CD40 / FcRn double gene humanized mice (hCD40 / hFcRn mice) can be found in PCT / CN2022 / 075057, which is incorporated herein by reference in its entirety.

[0284] Similar to the above experiments, the pharmacokinetics (PK) and in vivo efficacy of anti-CD40 antibody blelumab analogs, 12B5-IgG1-LALA-YTE and 2F8-IgG1-LALA-YTE were tested in hCD40 / hFcRn mice. In the treatment groups (G2-G10), hCD40 / hFcRn mice were injected with 1 mg / kg-4 mg / kg (i.e., 1 mg / kg, 2 mg / kg or 4 mg / kg) of anti-CD40 antibody blelumab analogs, 12B5-IgG1-LALA-YTE and 2F8-IgG1-LALA-YTE, respectively, by intraperitoneal (ip) administration on day 0 (grouping day). The control group mice (G1) were injected with an equal volume of PBS. On day 1 (1 day after grouping), mice were intraperitoneally administered with an OVA / CFA mixture. Serum was collected from each animal 4 hours after antibody administration, and on days 1, 5, 10, 17, and 21. The collected serum was then subjected to PK analysis. ELISA analysis was also performed on serum collected from each animal on days 10, 17, and 21. The detailed dosing schedule is shown in the table below.

[0285] Table 3

[0286]

[0287] The pharmacokinetic results showed that after injection of different antibodies, the antibody concentration in the serum of hCD40 / hFcRn mice decreased over time, which was consistent with the pharmacokinetic characteristics. The ELISA results of the serum collected on days 10, 17 and 21 are shown in the following table and Figures 6 to 8In summary, anti-OVA antibodies in all mice in the control and treatment groups showed a trend of continuous increase throughout the experiment. In addition, mice in the treatment group produced less anti-OVA antibodies than mice in the control group. The results also showed a dose-relevance, that is, the higher the dose level of the administered antibody, the fewer anti-OVA antibodies were produced and the better the immunosuppressive effect obtained. In general, the immunosuppressive effect of 2F8-IgG1-LALA-YTE was better than that of 12B5-IgG1-LALA-YTE, and the immunosuppressive effect of 12B5-IgG1-LALA-YTE was better than that of the brekuzumab analog.

[0288] Table 4

[0289]

[0290] CV: Coefficient of variation

[0291] Example 5. Pharmacokinetic Analysis

[0292] PK analysis in hCD40 mice and C57BL / 6 mice

[0293] The pharmacokinetic clearance rate of anti-CD40 antibodies in hCD40 mice was determined. Specifically, mice were divided into 4 groups (3 mice per group), and 3 mg / kg of brekumab analogs (G1), 12B5-IgG4-FLAA (G2), 6A4-IgG4-FLAA (G3) and 2F8-IgG4-FLAA (G4) were administered by intravenous injection. Blood samples were collected at 15 minutes, 1 day, 3 days, 7 days, 10 days and 14 days after administration and 4 days before administration.

[0294] Serum levels of human antibodies were determined by sandwich ELISA. Briefly, goat anti-human IgG (H+L) (Jackson ImmunoResearch, catalog number: 109-005-088) was diluted to a final concentration of 2000 ng / mL, added to a 96-well plate (ELISA plate) at 100 μl / well, and then incubated overnight at 4°C. After the incubation, the cells were washed with PBS-T buffer (supplemented with Tween TM20% PBS) was used to wash the plate 4 times. The unbound area of ​​the antibody was blocked with 2% BSA (bovine serum albumin) at 37°C for 2 hours. The plate was then washed 4 times with PBS-T buffer. After washing, 100 μL serum (1 / 20 dilution (MRD) in 1% BSA) was added to each well. The wells were sealed and incubated at 37°C for 1 hour. After washing the plate using a plate washer, 100 μl / well of peroxidase AffiniPure F (ab') 2 fragment goat anti-human IgG, Fcγ specific fragment (Jackson ImmunoResearch, catalog number: 109-036-098) was added to each well of the plate and incubated at 37°C for 1 hour. After washing the plate, tetramethylbenzidine (TMB) solution was added to a 96-well plate as a substrate in an amount of 100 μl / well. After incubation in the dark at room temperature, 100 μl of stop solution (Beyotime, catalog number: P0215) was added to each well. The absorbance of each well was read at 450 nm and 630 nm using a microplate reader. TM The data were analyzed using the data analysis software. The absorbance values ​​and corresponding concentrations of the calibration samples prepared for each test product were used to establish a calibration curve with four parameters (i.e., T 1 / 2 , C max , AUC 0天-14天 The standard curve was used to calculate the antibody concentration of each serum sample. The drug concentration-time curve was established using the sample concentration calculated at each time point. Phoenix TM WinNolin8.3 was used to calculate the pharmacokinetic parameters. The results are shown in the table below.

[0295] Similar to the above experiments, the pharmacokinetic clearance of anti-CD40 antibody blelumab analogs, 12B5-IgG4-FLAA, 6A4-IgG4-FLAA and 2F8-IgG4-FLAA were also determined in C57BL / 6 mice. The detailed dosing regimen is shown in the table below.

[0296] Table 5

[0297]

[0298] Four days before antibody administration, the antibody concentration tested was 0 μg / ml (results not shown). Figure 4As shown in the table below, the results are consistent with typical pharmacokinetic characteristics, indicating that after injection of different antibodies, the antibody concentration in the serum of hCD40 mice and C57BL / 6 mice decreases over time. The half-life of anti-CD40 antibodies in C57BL / 6 (B6) mice ranged from 12.37 days to 17.70 days, while the half-life of anti-CD40 antibodies in hCD40 mice ranged from 1.87 days to 4.84 days. The clearance rate (CL) of anti-CD40 antibodies in C57BL / 6 mice ranged from 4.65 ml / day / kg to 6.35 ml / day / kg, while the CL of anti-CD40 antibodies in hCD40 mice ranged from 12.92 ml / day / kg to 14.69 ml / day / kg. The results showed that the concentrations of all antibodies were similar in wild-type C57BL / 6 mice. In contrast, in hCD40 mice, the concentrations of all antibodies decreased faster, which can be explained by TMDD (target-mediated drug disposition).

[0299] Table 6

[0300]

[0301] T 1 / 2 : Terminal half-life

[0302] C max : Maximum concentration

[0303] AUC 0天-14天 :Area under the blood concentration-time curve from 0 days to 14 days

[0304] CL: Clearance

[0305] PK analysis in hFcRn mice

[0306] Similar to the above experiment, the pharmacokinetic clearance of anti-CD40 antibody 12B5 was determined in hFcRn mice, and the mice were divided into 4 groups (5 mice per group). 2 mg / kg 12B5-IgG1-LALA-YTE (G1), 12B5-IgG1-N297A-YTE (G2), 12B5-IgG4-YTE (G3) or 12B5-IgG4-FLAA (G4) were administered by intravenous injection. Blood samples were collected at 2 hours, 1 day, 3 days, 7 days, 10 days, 14 days, 21 days, 28 days and 35 days after administration and 4 days before administration. The detailed dosing regimen is shown in the table below.

[0307] Table 7

[0308]

[0309]

[0310] Four days before antibody administration, the antibody concentration tested was 0 μg / ml (results not shown). Figure 5 As shown in the table below, the results are consistent with typical pharmacokinetic characteristics, indicating that the antibody concentration in the serum of hFcRn mice decreases over time after injection of different antibodies. The half-life (T) of 12B5-IgG1-LALA-YTE (G1) and 12B5-IgG4-YTE (G3) in mice 1 / 2 ) were 11.99 days and 13.72 days respectively. 12B5-IgG1-N297A-YTE (G2) showed the shortest half-life of 6.58 days. The results showed that 12B5-IgG1-N297A-YTE (G2) had the shortest half-life. The half-lives of 12B5-IgG1-LALA-YTE (G1) and 12B5-IgG4-YTE (G3) were longer than that of 12B5-IgG4-FLAA (G4).

[0311] Table 8

[0312]

[0313] T 1 / 2 : Terminal half-life;

[0314] C max : maximum concentration;

[0315] AUC 0天-35天 : Area under the blood concentration-time curve from 0 days to 35 days

[0316] CL: Clearance

[0317] Example 6. Binding affinity of anti-CD40 antibodies to human FcRn

[0318] A Biacore was used with a pre-immobilized protein A sensor chip. TM The binding affinity of anti-CD40 antibodies 12B5-IgG1-LALA-YTE, 12B5-IgG1-N297A-YTE, 12B5-IgG4-YTE, and 12B5-IgG4 to human FcRn was measured using surface plasmon resonance (SPR) using a Biacore 8K or T200 biosensor (Biacore, Inc., Piscataway, NJ, USA).

[0319] hFcRn-His (FCGRT & B2M heterodimer protein, His tag & Strep II tag (SPR & BLI & MALS verification, Beijing Biopsies Biotechnology Co., Ltd., catalog number: FCM-H5286)) was diluted to 1 μg / ml with 1×HBS-EP+ buffer (PH7.4) and then injected into BiacoreTM8K or T200 biosensor at 10 μl / min for about 50 seconds to meet the required capture level (e.g., about 200 response units (RU)). Then, purified anti-CD40 antibodies (with 1×HBS-EP+ buffer (PH 7.4)) at a concentration of 1250 nM, 625 nM, 312.5 nM, 156.25 nM, 78.125 nM or 0 nM were injected at a rate of 30 μl / min for 50 seconds. Dissociation was monitored for 200 seconds. After the last injection of each round of titration, the chip was regenerated with glycine solution (pH 1.7) at a rate of 30 μl / min for 30 seconds. The binding curve was obtained. The data was analyzed by analysis software, and the binding and dissociation curves were fitted using a steady-state affinity binding model to obtain affinity kinetic data.

[0320] The above experiment was repeated except that 1×HBS-EP+ buffer (pH 7.4) was replaced with 1×HBS-EP+ buffer (pH 6.0).

[0321] As will be appreciated by one of ordinary skill in the art, the same approach with appropriate adjustment of parameters (eg, antibody concentration) was employed for each antibody tested. The results for the antibodies tested are summarized in the table below.

[0322] The results are shown in the table below. When the pH value is 7.4, 12B5-IgG4-FLAA does not bind to hFcRn, while the other antibodies bind to hFcRn. At pH 6.0, all antibodies show high binding affinity to hFcRn. The results show that under acidic conditions, the affinity of all antibodies to hFcRn increases. In addition, 12B5-IgG1-LALA-YTE and 12B5-IgG4-YTE have higher binding affinity to hFcRn than 12B5-IgG1-N297A-YTE.

[0323] Table 9

[0324]

[0325] Example 7. Determination of antibody-dependent cellular cytotoxicity (ADCC)

[0326] Rituximab (VH SEQ ID NO:67, VL SEQ ID NO:68) is a chimeric monoclonal antibody that targets CD20 and was first introduced in 1997 as an intravenous treatment for relapsed or refractory low-grade or follicular CD20-positive B-cell non-Hodgkin lymphoma (NHL).

[0327] In this experiment, FcR-TANK cells (Immune Onco Biopharmaceutical Technology (Shanghai) Co., Ltd.) were used as effector cells, and Raji cells (ATCC, catalog number: CCL-86) were used as target cells. The cells were incubated with each antibody (final concentration 50 μg / mL) at an E:T (effector: target) ratio of 3:1 for 2 hours or 4 hours to determine ADCC activity.

[0328] The results are shown in Fig.12 2F8-IgG1 and rituximab analogs had ADCC activity, but 2F8-IgG1-LALA-YTE did not show ADCC effect.

[0329] Example 8. Complement-dependent cytotoxicity (CDC) assay

[0330] Experiments were performed to evaluate the CDC effect of anti-CD40 antibodies. In the experiments, Raji cells (cell density 5×10 4 Cells / well) were seeded in 96-well plates, and then the antibody 2F8-IgG1-LALA-YTE, rituximab analogs, or human IgG1 were added at a final concentration of 50 μg / mL. The cells were incubated with each antibody at 37°C for 30 minutes, and then diluted normal human serum complement (QIDEL, catalog number: A113) was added. After incubation at 37°C with 5% CO2 for 1.5 hours, PrestoBlue TM Cell viability reagent (Invitrogen, catalog number: 2413466) was added to each well, and the plate was incubated at 37° C. with 5% CO 2 for 2 hours.

[0331] The results are shown in Fig.13 2F8-IgG1-LALA-YTE did not show CDC effect, while the positive control rituximab analogue showed strong CDC effect.

[0332] Example 9. Inhibitory effect of anti-CD40 antibody on PBMC proliferation

[0333] Peripheral blood mononuclear cells (PBMC) were seeded in 96-well plates (5×10 4Cells / well). Serial dilutions of antibody 2F8-IgG1-LALA-YTE or hIgG1 (100 ng / mL, 10 ng / mL, 8 ng / mL, 5 ng / mL, 4 ng / mL, 2 ng / mL, 1 ng / mL, 0.5 ng / mL, or 0.25 ng / mL) were added to each well and incubated at 37°C for 1 hour. Then 10 μl of anti-FLAG antibody (Sigma, catalog number: F1804) conjugated to shCD154 (human CD40 ligand / TNFSF5 protein, His, Flag tag (active trimer MALS verification), ACRO, catalog number: CDL-H52Db) was added to each well, and the plate was incubated at 37°C for 64 hours. Novozyme Biotech 2.0 Luminescent Cell Viability Assay (Novozyme, catalog number: DD1101-02) was used to measure the fluorescence signal by a microplate reader.

[0334] The results are shown in Fig.14 , which showed that 2F8-IgG1-LALA-YTE effectively inhibited PBMC proliferation.

[0335] Example 10. Inhibitory effect of anti-CD40 antibodies on B cell proliferation

[0336] In peripheral blood, B cell subsets can be distinguished according to different stages of differentiation, maturation and activation, and are characterized by the expression of different surface markers, such as CD19, CD20, CD27, CD23, CD69, CD80 and CD86, among which CD23, CD69, CD80 and CD86 are specific surface markers of active B cells.

[0337] In this experiment, human PBMC cells were cultured at 1 × 10 5 The cells were seeded at a density of 10 cells / well in a 96-well plate. Anti-CD40 antibodies 2F8-IgG1-LALA-YTE and hIgG1 were serially diluted (3 times) from the highest concentration of 1000 ng / mL. 10 μL of antibody was added to the 96-well plate. After incubation at 37°C for 1 hour, 10 μL of antibody conjugated to shCD154 was added. M2 antibody (Sigma, catalog number: F1804) and incubated at 37°C for 18 hours. Then, cells were incubated with PE anti-human CD20 (BioLegend, catalog number: 302306), PerCP / Cyanine5.5 anti-human CD69 antibody (BioLegend, catalog number: 310926), Brilliant Violet 421 TMAnti-human CD86 antibody (BioLegend, catalog number: 305426), FITC anti-human CD80 antibody (BioLegend, catalog number: 375406), APC / Cy7 anti-human CD23 (BioLegend, catalog number: 338502), human CD40 ligand / TNFSF5 protein, His, Flag tag (active trimer) (MALS verified) (ACRO, catalog number: CDL-H52Db-100ug) and monoclonal antibody M2 antibody (Sigma, catalog number: F1804-1MG) was incubated at 4°C in the dark for 30 minutes. FIG. 15A to FIG. 15B middle.

[0338] As the concentration of 2F8-IgG1-LALA-YTE increased, the percentages of activated B cell subsets all decreased, indicating that 2F8-IgG1-LALA-YTE effectively inhibited the proliferation and activation of B cells.

[0339] Example 11. Evaluation of the effect of anti-CD40 antibodies using the EAE model of hCD40 / hFcRn mice

[0340] Experimental autoimmune encephalomyelitis (EAE) is the most commonly used experimental model of multiple sclerosis (MS), a human inflammatory demyelinating disease. In this experiment, an EAE model was established with hCD40 / hFcRn mice to analyze the effect of human anti-CD40 antibodies. Specifically, hCD40 / hFcRn mice were divided into a control group (G1) and two treatment groups (G2 and G3). Then, on the first day (one day after grouping), hCD40 / hFcRn mice were immunized by injecting 100 μL MOG (myelin oligodendrocyte glycoprotein (35-55)) (300 μg / mouse dosage level) to two points of the posterior ribs, and 250 μL PTX (pertussis toxin) was injected intraperitoneally 2 hours and 48 hours after immunization to obtain an EAE model. Control group (G1) mice were injected with an equal volume of PBS.

[0341] On day 0 (grouping day), day 4 (4 days after grouping) and day 8 (8 days after grouping), mice in groups G2 and G3 were administered 10 mg / kg hIgG1 or 2F8-IgG1-LALA-YTE by intraperitoneal injection, respectively. The control group mice (G1) were not injected. The mice were weighed and examined for neurological symptoms of EAE every day, and the scores were scored according to the following grades: 0 = no symptoms; 1 = floppy tail; 2 = hind limb weakness; 3 = hind limb paralysis; 4 = forelimb paralysis; 5 = mice dying or dead; 0.5 grades represent intermediate scores. At the end of the experiment (30 days after grouping), the spinal cord was collected for histopathological analysis. The detailed dosing regimen is shown in the table below.

[0342] Table 10

[0343]

[0344] like Figures 16 to 19 As shown, 2F8-IgG1-LALA-YTE (G3) can prevent weight loss induced by animal modeling ( Fig.16 ) and reduced clinical scores ( Fig.17 In EAE mice administered with 2F8-IgG1-LALA-YTE, myelin inflammatory cell infiltration ( Fig.18 ) and spinal cord demyelination ( Fig.19 ) also has a certain improvement effect.

[0345] Example 12. Evaluation of the effect of anti-CD40 antibodies using the CIA model of hCD40 mice

[0346] The collagen-induced arthritis (CIA) model is a commonly used model because of its immunological and pathological similarities to human rheumatoid arthritis (RA). The hCD40 mouse model was used to establish the CIA model and analyze the effects of human anti-CD40 antibodies.

[0347] Specifically, hCD40 mice were divided into a control group (G1) and two model groups (G2 and G3). Then, on day 0 (the first day of immunization), hCD40 mice were immunized by injecting 50 μL of type II collagen (CII) emulsion (composed of equal volumes of CFA and collagen from chicken sternal cartilage (SIGMA catalog number: C9301)) into two points at the tail base. On day 21 (21 days after the first immunization), a second immunization was performed with the same dose to obtain a CIA model. The control group (G1) was injected with an equal volume of PBS.

[0348] G3 mice were administered 3 mg / kg 12B5-IgG1-LALA-YTE by intraperitoneal injection on days 0, 4, 7, 20, 24, and 27. G2 mice were administered an equal volume of PBS. G1 mice were not injected. All mice were weighed and monitored daily for signs of erythema and swelling in the following areas: (A) interphalangeal joints, (B) metacarpophalangeal joints, and (C) forepaw wrist joints and hind paw metatarsophalangeal joints and ankle joints. The severity of clinical arthritis in a single claw was scored on a scale of 0-4 as follows:

[0349] 0 = normal. No significant difference in appearance relative to healthy mice;

[0350] 1 = erythema and swelling of one type of joint (A, B, or C);

[0351] 2 = erythema and swelling of two types of joints (A, B, or C);

[0352] 3 = erythema and swelling of all three types of joints (A, B, or C); and

[0353] 4 = Erythema and swelling of the entire paw.

[0354] The animals were scored as the sum of the scores of all four limbs on a scale of 0-16. At the end of the experiment (56 days after the first immunization), the limbs were collected for histopathological analysis. The detailed dosing regimen is shown in the table below.

[0355] Table 11

[0356] Group Antibody Dosage (mg / kg) way Number of mice Total number of medications G1 - - - 10 - G2 PBS - ip 10 6 G3 12B5-IgG1-LALA-YTE 3 ip 10 6

[0357] like Figure 20 to Figure 23 As shown, 12B5-IgG1-LALA-YTE (G3) can prevent weight loss caused by animal modeling compared with the control group G2 ( Fig. 20 ) and significantly reduced clinical severity, as reflected by lower clinical scores and morbidity ( Figure 21 to Figure 22 ).like Fig.23 As shown, the results of histopathological analysis of joint tissues showed that inflammatory cell influx (a), synovial hyperplasia (b), pannus formation (c), and spinal cord demyelination were also improved in the 12B5-IgG1-LALA-YTE-treated group compared with the control group.

[0358] Example 13. In vivo efficacy of anti-CD40 antibodies

[0359] Atopic dermatitis (AD) is an inflammatory disorder of the skin characterized by erythema, pruritus, scaling, lichenification, and papulovesicles.

[0360] In this experiment, the in vivo efficacy of the anti-CD40 antibody 2F8-IgG1-LALA-YTE in dogs diagnosed with AD was evaluated. Prior to the experiment, the dog had recurrent AD symptoms for more than 1 year and received repeated glucocorticoid treatment. Specifically, the antibody was administered by intravenous (iv) injection. The injection volume was calculated based on the dog's weight. The frequency of administration was once every 2 weeks (6 administrations in total). After two injections, it was observed that the erythema on the dog's skin was significantly reduced. Peripheral blood was also collected to test blood biochemistry and complete blood cell count (CBC). The results showed that the tested antibody 2F8-IgG1-LALA-YTE was well tolerated and had no toxicity to dogs. No adverse reactions were observed.

[0361] Other Implementations

[0362] It should be understood that although the invention has been described in conjunction with the specific embodiments of the invention, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

Claims

1. An antibody or antigen-binding fragment thereof that binds to CD40 (TNF receptor superfamily member 5), the antibody or antigen-binding fragment thereof comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VLCDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VLCDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VLCDR3 amino acid sequence, wherein the selected VH CDR1, 2 and 3 amino acid sequence and the selected VL CDR1, 2 and 3 amino acid sequence are one of the following: (1) the selected VH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 1, 2, 3, respectively, and the selected VLCDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 4, 5, 6, respectively; (2) the selected VH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 7, 8, 9, respectively, and the selected VLCDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 10, 11, 12, respectively; (3) the selected VH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 13, 14, 15, respectively, and the selected VLCDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 16, 17, 18, respectively; (4) the selected VH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 19, 20, 21, respectively, and the selected VLCDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 22, 23, 24, respectively; (5) the selected VH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 25, 26, 27, respectively, and the selected VLCDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 28, 29, 30, respectively; and (6) The selected VH CDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 31, 32, 33, respectively, and the selected VLCDR1, 2, 3 amino acid sequences are shown in SEQ ID NOs: 34, 35, 36, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, respectively.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 7, 8 and 9, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 10, 11 and 12, respectively.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 13, 14 and 15, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 16, 17 and 18, respectively.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 19, 20 and 21, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 22, 23 and 24, respectively.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 25, 26 and 27, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 28, 29 and 30, respectively.

7. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 31, 32 and 33, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences shown in SEQ ID NOs: 34, 35 and 36, respectively.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof specifically binds to human, monkey or dog CD40.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof, a single-chain variable fragment (scFv) and / or a multispecific antibody (eg, a bispecific antibody).

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the antibody or antigen-binding fragment thereof is a human IgG1 antibody or antigen-binding fragment thereof (optionally having a YTE and / or LALA mutation) or a human IgG4 antibody or antigen-binding fragment thereof (optionally having a YTE).

11. A nucleic acid comprising a polynucleotide encoding a polypeptide comprising: (1) an immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH), wherein the VH comprises complementarity determining regions (CDRs) 1, 2 and 3, wherein the CDRs 1, 2 and 3 comprise the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3, respectively, and wherein the VH binds to CD40 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 37; (2) an immunoglobulin light chain or a fragment thereof, comprising a VL, wherein the VL comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein CDRs 1, 2, and 3 comprise the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, respectively, and wherein the VL binds to CD40 when paired with a VH comprising the amino acid sequence shown in SEQ ID NO: 65; (3) an immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH), wherein the VH comprises complementarity determining regions (CDRs) 1, 2 and 3, wherein CDRs 1, 2 and 3 comprise the amino acid sequences shown in SEQ ID NOs: 7, 8 and 9, respectively, and wherein the VH binds to CD40 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 39; (4) an immunoglobulin light chain or a fragment thereof, comprising a VL, wherein the VL comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein CDRs 1, 2, and 3 comprise the amino acid sequences shown in SEQ ID NOs: 10, 11, and 12, respectively, and wherein the VL binds to CD40 when paired with a VH comprising the amino acid sequence shown in SEQ ID NO: 38; (5) an immunoglobulin heavy chain or a fragment thereof, comprising a VH, wherein the VH comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein CDRs 1, 2, and 3 comprise the amino acid sequences shown in SEQ ID NOs: 13, 14, and 15, respectively, and wherein the VH binds to CD40 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 41; or (6) an immunoglobulin light chain or a fragment thereof, comprising a VL, wherein the VL comprises CDR1, 2, and 3, wherein the CDR1, 2, and 3 comprise the amino acid sequences shown in SEQ ID NOs: 16, 17, and 18, respectively, and wherein the VL binds to CD40 when paired with a VH comprising the amino acid sequence shown in SEQ ID NO:

40.

12. The nucleic acid of claim 11, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide, the polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, the immunoglobulin heavy chain or a fragment thereof comprising a VH, the VH comprising CDR1, 2, 3, and the CDR1, 2, 3 comprising the amino acid sequences shown in SEQ ID NO: 1, 2, and 3, respectively.

13. A nucleic acid according to claim 11, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide, the polypeptide comprises an immunoglobulin light chain or a fragment thereof, the immunoglobulin light chain or the fragment thereof comprises a VL, the VL comprises CDR1, 2, 3, and the CDR1, 2, 3 comprise the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6, respectively.

14. The nucleic acid of claim 11, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide, the polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, the immunoglobulin heavy chain or a fragment thereof comprising a VH, the VH comprising CDR1, 2, 3, and the CDR1, 2, 3 comprising the amino acid sequences shown in SEQ ID NOs: 7, 8, and 9, respectively.

15. The nucleic acid of claim 11, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide, the polypeptide comprising an immunoglobulin light chain or a fragment thereof, the immunoglobulin light chain or a fragment thereof comprising a VL, the VL comprising CDR1, 2, 3, and the CDR1, 2, 3 comprising the amino acid sequences shown in SEQ ID NOs: 10, 11, and 12, respectively.

16. The nucleic acid of claim 11, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide, the polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, the immunoglobulin heavy chain or a fragment thereof comprising a VH, the VH comprising CDR1, 2, 3, and the CDR1, 2, 3 comprising the amino acid sequences shown in SEQ ID NOs: 13, 14, and 15, respectively.

17. A nucleic acid according to claim 11, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide, the polypeptide comprises an immunoglobulin light chain or a fragment thereof, the immunoglobulin light chain or the fragment thereof comprises a VL, the VL comprises CDR1, 2, 3, and the CDR1, 2, 3 comprise the amino acid sequences shown in SEQ ID NOs: 16, 17 and 18, respectively.

18. The nucleic acid of any one of claims 11-17, wherein the VH specifically binds to human, monkey or dog CD40 when paired with the VL, or the VL specifically binds to human, monkey or dog CD40 when paired with the VH.

19. The nucleic acid according to any one of claims 11-18, wherein the immunoglobulin heavy chain or fragment thereof is a human immunoglobulin heavy chain or fragment thereof (e.g., a human IgG1 heavy chain or fragment thereof, optionally with a YTE and / or LALA mutation; or a human IgG4 heavy chain or fragment thereof, optionally with a YTE), and the immunoglobulin light chain or fragment thereof is a human immunoglobulin light chain or fragment thereof.

20. The nucleic acid of any one of claims 11-19, wherein the nucleic acid encodes a single-chain variable fragment (scFv) or a multispecific antibody (eg, a bispecific antibody).

21. The nucleic acid according to any one of claims 11-20, wherein the nucleic acid is cDNA.

22. A vector comprising one or more nucleic acids according to any one of claims 11-21.

23. A vector comprising two nucleic acids according to any one of claims 11 to 21, wherein the vector encodes a VL region and a VH region, and the VL region and the VH region together bind to CD40.

24. A pair of vectors, wherein each vector comprises a nucleic acid as described in any one of claims 11-21, wherein the pair of vectors together encode a VL region and a VH region, and the VL region and VH region together bind to CD40.

25. A cell comprising the vector of claim 22 or 23, or the vector pair of claim 24.

26. The cell according to claim 25, wherein the cell is a CHO cell.

27. A cell comprising one or more nucleic acids of any one of claims 11-21.

28. A cell comprising two nucleic acids as described in any one of claims 11-21.

29. The cell of claim 28, wherein the two nucleic acids together encode a VL region and a VH region that together bind CD40.

30. A method for producing an antibody or an antigen-binding fragment thereof, the method comprising (a) culturing the cell under conditions sufficient for the cell to produce an antibody or antigen-binding fragment as described in any one of claims 25 to 29; and (b) collecting the antibodies or antigen-binding fragments produced by the cells.

31. An antibody or antigen-binding fragment thereof that binds to CD40, the antibody or antigen-binding fragment thereof comprising: A heavy chain variable region (VH), the VH comprising an amino acid sequence that is at least 90% identical to a selected VH sequence, and a light chain variable region (VL), the VL comprising an amino acid sequence that is at least 90% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) the selected VH sequence is SEQ ID NO: 65, and the selected VL sequence is SEQ ID NO: 37; (2) the selected VH sequence is SEQ ID NO: 38, and the selected VL sequence is SEQ ID NO: 39; and (3) The selected VH sequence is SEQ ID NO: 40, and the selected VL sequence is SEQ ID NO:

41.

32. The antibody or antigen-binding fragment thereof of claim 31 , wherein the VH comprises the sequence of SEQ ID NO: 65 and the VL comprises the sequence of SEQ ID NO:

37.

33. The antibody or antigen-binding fragment thereof of claim 31 , wherein the VH comprises the sequence of SEQ ID NO: 38 and the VL comprises the sequence of SEQ ID NO:

39.

34. The antibody or antigen-binding fragment thereof of claim 31, wherein the VH comprises the sequence of SEQ ID NO: 40 and the VL comprises the sequence of SEQ ID NO:

41.

35. The antibody or antigen-binding fragment thereof according to any one of claims 31-34, wherein the antibody or antigen-binding fragment thereof specifically binds to human, monkey or dog CD40.

36. The antibody or antigen-binding fragment thereof according to any one of claims 31-35, wherein the antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof, a single-chain variable fragment (scFv) and / or a multispecific antibody (eg, a bispecific antibody).

37. The antibody or antigen-binding fragment thereof according to any one of claims 31-36, wherein the antibody or antigen-binding fragment thereof is a human IgG1 antibody or antigen-binding fragment thereof (optionally having a YTE and / or LALA mutation) or a human IgG4 antibody or antigen-binding fragment thereof (optionally having a YTE).

38. An antibody or antigen-binding fragment thereof, comprising VH CDR1, 2, 3 and VL CDR1, 2, 3 of the antibody or antigen-binding fragment thereof according to any one of claims 1-10 and 31-37.

39. An antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof of any one of claims 1-10 and 31-38.

40. An antibody drug conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-10 and 31-39 covalently bound to a therapeutic agent.

41. The antibody drug conjugate of claim 40, wherein the therapeutic agent is a cytotoxic agent or a cytostatic agent.

42. A method of treating a subject suffering from cancer, the method comprising: A therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-10 and 31-39, or the antibody drug conjugate of claim 40 or 41 is administered to the subject.

43. The method of claim 42, wherein the subject has a solid tumor.

44. The method of claim 42, wherein the cancer is melanoma, pancreatic cancer, mesothelioma, or a hematological malignancy.

45. The method of claim 42, wherein the cancer is non-Hodgkin lymphoma, lymphoma, or chronic lymphocytic leukemia.

46. ​​A method for reducing tumor growth rate, the method comprising The tumor cells are contacted with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-10 and 31-39, or the antibody drug conjugate according to claim 40 or 41.

47. A method for killing tumor cells, the method comprising The tumor cells are contacted with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-10 and 31-39, or the antibody drug conjugate according to claim 40 or 41.

48. A method of suppressing an immune response in a subject, the method comprising An effective amount of a composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-10 and 31-39, or the antibody drug conjugate according to claim 40 or 41 is administered to a subject.

49. The method of claim 48, wherein the subject suffers from an autoimmune disease.

50. A method for treating an autoimmune disease, the method comprising An effective amount of a composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-10 and 31-39, or the antibody drug conjugate according to claim 40 or 41 is administered to a subject.

51. The method of claim 50, wherein the autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, atopic dermatitis, or multiple sclerosis.

52. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-10 and 31-39 and a pharmaceutically acceptable carrier.

53. A pharmaceutical composition comprising the antibody drug conjugate of claim 40 or 41 and a pharmaceutically acceptable carrier.

54. An antibody or antigen-binding fragment thereof that binds to CD40 (TNF receptor superfamily member 5), the antibody or antigen-binding fragment thereof comprising an Fc region, wherein the Fc region lacks ADCC activity or has reduced ADCC activity compared to a wild-type Fc region.

55. The antibody or antigen-binding fragment thereof of claim 54, wherein the KD between the antibody or antigen-binding fragment thereof and FcRn (eg, human FcRn) is less than 1×10 -5 M, less than 5×10 -6 M, less than 1×10 -6 M, less than 5×10 -7 M, less than 1×10 -7 M or less than 5×10 -8 M.

56. The antibody or antigen-binding fragment thereof according to claim 54 or 55, wherein the Fc region is of IgGl or IgG4 subtype.

57. The antibody or antigen-binding fragment thereof of any one of claims 54-56, wherein the Fc region comprises a YTE mutation.

58. The antibody or antigen-binding fragment thereof of any one of claims 54, 55 and 57, wherein the Fc region comprises a LALA mutation.

59. The antibody or antigen-binding fragment thereof of any one of claims 1-10, 31-37, and 54-58, wherein when administered to a subject, the antibody or antigen-binding fragment thereof has a half-life of at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, or at least 18 days.

60. The antibody or antigen-binding fragment thereof of claim 59, wherein the subject is a mouse. The antibody or antigen-binding fragment thereof according to claim 59 or 60, wherein the subject is genetically modified to express human or humanized CD40.

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