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

[Technical field]

[0001] The present disclosure relates to anti-CD40 (TNF receptor superfamily member 5) antibodies and uses thereof. [Background technology]

[0002] Claiming priority This application claims the benefit of PCT Application No. PCT / CN2022 / 087003, filed April 15, 2022. The entire contents of the above-mentioned application are incorporated herein by reference.

[0003] Autoimmune disease is a condition caused by abnormal immune response against normal body parts. There are at least 80 kinds of autoimmune disease. The cause of autoimmune disease is generally not fully understood. Some autoimmune diseases, such as lupus, run in families, and some other autoimmune diseases can be triggered by infection or other environmental factors. Some common autoimmune diseases include, for example, celiac disease, type I diabetes mellitus, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.

[0004] The recent clinical and commercial success of therapeutic antibodies has generated great interest in using antibodies to treat a variety of immune-related disorders, and there is a need for the development of antibodies for use in a variety of antibody-based therapeutic approaches to treat autoimmune diseases. Summary of the Invention

[0005] The present disclosure relates to anti-CD40 antibodies, antigen-binding fragments thereof, and uses thereof.

[0006] In one aspect, the disclosure provides a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein 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) comprising CDRs 1, 2, and 3, wherein 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 CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence. and a light chain variable region comprising an amino acid sequence that is at least 80% identical to a CDR3 amino acid sequence of at least one of the following: (1) the selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 1, 2, and 3, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 4, 5, and 6, respectively; (2) the selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 7, 8, and 9, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 10, 11, and 12, respectively; (3) the selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 13, 14, and 15, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, and 18, respectively; (4) the selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 19, 20, and 21, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 22, 23, and 24, respectively; (5) the selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 25, 26, and 27, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 28, 29, and 30, respectively; and (6) The selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 31, 32, and 33, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 34, 35, and 36, respectively; It is one of them.

[0007] In some embodiments, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively. In some embodiments, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively. In some embodiments, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively. In some embodiments, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively. In some embodiments, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 25, 26, and 27, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively. In some embodiments, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 34, 35, and 36, respectively.

[0008] 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 thereof 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 thereof 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).

[0009] In one aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: (1) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, wherein in some embodiments, the VH binds to CD40 when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 37; (2) An immunoglobulin light chain or fragment thereof comprising a VL comprising complementarity determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively, wherein in some embodiments, the VL binds to CD40 when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 65; (3) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, wherein in some embodiments, the VH binds to CD40 when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 39; (4) An immunoglobulin light chain or fragment thereof comprising a VL comprising complementarity determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively, wherein in some embodiments, the VL binds to CD40 when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 38; (5) An immunoglobulin heavy chain or fragment thereof comprising a VH comprising complementarity determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively, wherein in some embodiments, the VH binds to CD40 when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 41; or (6) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively, wherein in some embodiments, the VL binds to CD40 when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 40; The present invention relates to a nucleic acid comprising a polynucleotide encoding a polypeptide comprising:

[0010] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDR1, 2, and 3 comprising the amino acid sequences set forth 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 comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth 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 comprising a VH comprising CDR1, 2, and 3 comprising the amino acid sequences set forth 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 comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth 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 comprising a VH comprising CDR1, 2, and 3 comprising the amino acid sequences set forth 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, comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively.

[0011] In some embodiments, the VH, when paired with the VL, specifically binds human, monkey, or dog CD40, or the VL, when paired with the VH, specifically binds 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.

[0012] In one aspect, the disclosure relates to a vector comprising one or more of the nucleic acids described herein. In one aspect, the disclosure relates to a vector comprising two of the nucleic acids described herein, in some embodiments, the vectors encoding a VL region and a VH region that both bind to CD40. In one aspect, the disclosure relates to a pair of vectors, in some embodiments, each vector comprises one of the nucleic acids described herein, and in some embodiments, together the pair of vectors encode a VL region and a VH region that both bind to CD40.

[0013] In one aspect, the disclosure relates to a cell comprising a vector or a pair of vectors 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 described herein. In one aspect, the disclosure relates to a cell comprising two of the nucleic acids described herein. In some embodiments, the two nucleic acids both encode a VL region and a VH region that both bind to CD40.

[0014] In one aspect, the disclosure relates to a method of producing an antibody or antigen-binding fragment thereof, comprising: (a) culturing a cell described herein under conditions sufficient for the cell to produce the antibody or antigen-binding fragment; and (b) harvesting the antibody or antigen-binding fragment produced by the cell.

[0015] In one aspect, the disclosure relates to an antibody or antigen-binding fragment thereof that binds to CD40, comprising a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90% identical to a selected VH sequence, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90% identical to a selected VL sequence, wherein 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.

[0016] In some embodiments, the VH comprises the sequence of SEQ ID NO: 65 and the VL comprises the sequence of SEQ ID NO: 37. In some embodiments, the VH comprises the sequence of SEQ ID NO: 38 and the VL comprises the sequence of SEQ ID NO: 39. In some embodiments, the VH comprises the sequence of SEQ ID NO: 40 and the VL comprises the sequence of SEQ ID NO: 41.

[0017] 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 thereof 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).

[0018] In one aspect, the disclosure relates to 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 described herein.

[0019] In one aspect, the disclosure relates to antibodies, or antigen-binding fragments thereof, that cross-compete with the antibodies, or antigen-binding fragments thereof, described herein.

[0020] In one aspect, the present disclosure relates to an antibody drug conjugate comprising an antibody or antigen-binding fragment thereof described herein covalently linked to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent.

[0021] In one aspect, the present disclosure relates to a method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof, 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.

[0022] In one aspect, the 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 described herein, or an antibody drug conjugate. In one aspect, the disclosure relates to a method of killing tumor cells, the method comprising contacting tumor cells with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein, or an antibody drug conjugate. In one aspect, the disclosure relates to a method of inhibiting an immune response in a subject, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof, or an antibody drug conjugate described herein. In some embodiments, the subject suffers from an autoimmune disease.

[0023] In one aspect, the disclosure relates to a method of treating an autoimmune disease, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof, or a composition comprising an antibody-drug conjugate described herein, hi some embodiments, the autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, allergic dermatitis, or multiple sclerosis.

[0024] In one aspect, the present disclosure relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described herein and a pharma- ceutically acceptable carrier.In one aspect, the present disclosure relates to a pharmaceutical composition comprising an antibody drug conjugate described herein and a pharma- ceutically acceptable carrier.

[0025] In one aspect, the disclosure relates to an antibody or antigen-binding fragment thereof that binds to CD40 comprising an Fc region, in some embodiments, the Fc region lacks ADCC efficacy or has reduced ADCC efficacy 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 1×10-5M, less than 5×10-6M, less than 1×10-6M, less than 5×10-7M, less than 1×10-7M, or less than 5×10-8M. In some embodiments, the Fc region is an 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, the half-life of the antibodies or antigen-binding fragments thereof described herein, when administered to a subject, 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 engineered to express human or humanized CD40.

[0026] As used herein, the term "cancer" refers to cells with autonomous growth potential. Examples of such cells include cells with an abnormal state or condition characterized by rapid proliferation of cell growth. The term is meant to include cancerous growths, e.g., tumors, oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, regardless of the type of tissue change or stage of invasiveness. Also included are malignant tumors of various organ systems, e.g., respiratory, cardiovascular, renal, reproductive, hematological, nervous system, hepatic, gastrointestinal, and endocrine systems, as well as most colon cancers, renal cell carcinoma, prostate and / or testicular cancer, non-small cell lung cancer, and small intestine cancer. "Naturally occurring" cancers include any cancer that is not experimentally induced by implanting cancer cells in a subject, e.g., naturally occurring cancers, cancers caused by exposing a patient to a carcinogen, cancers caused by the insertion of a transgenic oncogene or knockout of a tumor suppressor gene, and cancers caused by infectious diseases, e.g., viral infections. The term "carcinoma" is art-recognized and refers to a malignant tumor of epithelial or endocrine tissue. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissue. "Adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term "sarcoma" is art-recognized and refers to a malignant tumor of mesenchymal origin. The term "hematopoietic neoplastic disorder" includes disorders involving hyperplastic / neoplastic cells of hematopoietic origin. Hematopoietic neoplastic disorders may arise from the myeloid, lymphoid, or erythroid lineages, or from their precursor cells.

[0027] 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) complementarity determining regions (CDRs) (e.g., any of the three CDRs from an immunoglobulin light chain or any 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 (bispecific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, an antibody can contain an Fc region of a human antibody. The term antibody also includes derivatives, such as bispecific antibodies, single-chain antibodies, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments.

[0028] As used herein, the term "antigen-binding fragment" refers to a portion of a full-length antibody, which 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.

[0029] As used herein, the term "human antibody" refers to an antibody encoded by endogenous nucleic acid present in a human (e.g., a rearranged human immunoglobulin heavy or light chain locus). In some embodiments, a human antibody is recovered from a human or produced in human cell culture (e.g., in human hybridoma cells). In some embodiments, a human antibody is produced in a non-human cell (e.g., a mouse or hamster cell line). In some embodiments, a human antibody is produced in a bacterial cell or a yeast cell. In some embodiments, a human antibody is produced in a transgenic non-human animal (e.g., a cow) that contains unrearranged or rearranged human immunoglobulin loci (e.g., a heavy or light chain human immunoglobulin locus).

[0030] As used herein, the term "chimeric antibody" refers to an antibody that contains 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 that contains variable domain sequences (e.g., all or a portion of the light and / or heavy chain variable domain sequences) of a non-human (e.g., mouse) antibody and the constant domain of a human antibody. Further examples of chimeric antibodies are described herein and are well known in the art.

[0031] As used herein, the term "humanized antibody" refers to a non-human antibody that contains minimal sequence derived from non-human (e.g., murine) immunoglobulin and contains sequence derived from human immunoglobulin. In a non-limiting example, a humanized antibody is a human antibody (recipient antibody) in which residues from hypervariable (e.g., CDR) regions of the recipient antibody are replaced by residues from hypervariable (e.g., CDR) regions of a non-human antibody (e.g., donor antibody), such as a mouse, rat, or rabbit antibody, that has the desired specificity, affinity, and capacity. In some embodiments, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human (e.g., murine) immunoglobulin residues. In some embodiments, a humanized antibody can contain residues that are not found in the recipient antibody or in the donor antibody. These modifications can be made to further refine antibody performance. In some embodiments, a humanized antibody contains nearly all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops (CDRs) corresponding to those of a non-human (e.g., murine) immunoglobulin and all or substantially all of the framework regions being those of a human immunoglobulin sequence. The humanized antibody can also contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Humanized antibodies can be produced using molecular biology techniques well known in the art. Non-limiting examples of methods for generating humanized antibodies are described herein.

[0032] As used herein, the term "single chain antibody" means a single polypeptide containing at least two immunoglobulin variable domains (e.g., the variable domains of a mammalian immunoglobulin heavy or light chain) capable of specifically binding to an antigen. Non-limiting examples of single chain antibodies are described herein.

[0033] As used herein, the term "multimeric antibody" refers to an antibody that contains four or more (e.g., six, eight, or ten) immunoglobulin variable domains. In some embodiments, a multimeric antibody is capable of cross-linking one target molecule (e.g., CD40) to at least one second target molecule (e.g., CTLA-4) on the surface of a mammalian cell (e.g., a human T cell).

[0034] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification and refer to an animal, human, or non-human to which treatment according to the methods of the invention is provided. Veterinary and non-veterinary applications are contemplated by the present invention. Human patients can be adult humans or juvenile humans (e.g., humans 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. For example, patients include non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, porcines (e.g., pigs, minipigs), equines, canines, felines, bovines, and other domestic, livestock, and zoo animals.

[0035] As used herein, the phrases "specifically bind" and "specifically bind" when referring to an antibody means that the antibody interacts with its target molecule (e.g., CD40) preferably in preference to other molecules because the interaction is dependent on the presence of a particular structure (i.e., an antigenic determinant or epitope) on the target molecule; in other words, the reagent generally recognizes and binds to a molecule that contains a particular structure rather than to the entire molecule. An antibody that specifically binds to a target molecule may also be referred to as a target-specific antibody. For example, an antibody that specifically binds to the CD40 molecule may be referred to as a CD40-specific antibody or an anti-CD40 antibody.

[0036] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to polymers of amino acids of any length, of at least two amino acids.

[0037] As used herein, the terms "polynucleotide," "nucleic acid molecule," and "nucleic acid sequence" are used interchangeably herein and refer to polymers of nucleotides of any length, of at least two nucleotides, including, but not limited to, DNA, RNA, DNA / RNA hybrids, and modifications thereof.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Methods and materials for use in the present invention are described herein, and other suitable methods and materials known in the art can also be used. 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 by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0039] Other features and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims. [Brief description of the drawings]

[0040] [Figure 1] Figure 1 shows an experimental scheme for analyzing the effect of anti-CD40 antibodies on immune responses in hCD40 mice. Anti-CD40 antibodies were administered on days 0 and 4. OVA and CFA were administered on day 1. Anti-OVA antibody concentrations were measured by ELISA on days 10 and 17. [Diagram 2] Anti-OVA antibody concentrations measured by ELISA are shown on day 10. hCD40 mice were administered PBS (G1), bleselumab analogs (G2), 12B5-IgG4-FLAA (G3), 16D5-IgG4-FLAA (G4), 2F8-IgG4-FLAA (G5), 6A4-IgG4-FLAA (G6), or 15B4-IgG4-FLAA (G7). [Diagram 3] Anti-OVA antibody concentrations on day 17 measured by ELISA are shown. [Figure 4] 1 shows the concentration of bleselumab analogs (G1), 12B5-IgG4-FLAA (G2), 6A4-IgG4-FLAA (G3), and 2F8-IgG4-FLAA (G4) over time in the serum of hCD40 mice and the concentration of bleselumab analogs (G5), 12B5-IgG4-FLAA (G6), 6A4-IgG4-FLAA (G7), and 2F8-IgG4-FLAA (G8) over time in the serum of C57BL / 6 mice. [Diagram 5] FIG. 1 shows 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. [Figure 6]Anti-OVA antibody concentrations measured by ELISA on day 10 are shown. hCD40 / hFcRn mice were administered PBS (G1), bleselumab 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. [Figure 7] Anti-OVA antibody concentrations on day 17 measured by ELISA are shown. [Figure 8] Anti-OVA antibody concentrations on day 21, as measured by ELISA, are shown. [Figure 9] The CDR sequences of the anti-CD40 antibodies (2F8, 6A4, and 12B5) and their related anti-CD40 antibodies are listed, as defined by the Kabat numbering. [Figure 10] The CDR sequences of the anti-CD40 antibodies (2F8, 6A4, and 12B5) and their related anti-CD40 antibodies are listed, as defined by the Chothia numbering. [Figure 11] The amino acid sequences of the heavy chain variable region and light chain variable region of anti-CD40 antibodies (2F8, 6A4, and 12B5) are listed. [Figure 12] 1 shows cytotoxicity data for 12B5-IgG1-LALA-YTE, 12B5-IgG1, rituximab analogs, and human IgG1. [Figure 13] 1 shows cytotoxicity data for 12B5-IgG1-LALA-YTE, rituximab analogs, and human IgG1. [Figure 14] Shows the inhibitory effect of 12B5-IgG1-LALA-YTE and human IgG1 on PBMC proliferation. [Figure 15-1] 1 shows the inhibitory effect of 12B5-IgG1-LALA-YTE on B cell proliferation. [Figure 15-2] 1 shows the inhibitory effect of hIgG1 on B cell proliferation. [Figure 16]The body weights of hCD40 / hFcRn mice injected with MOG to induce the EAE model and subsequently treated with hIgG1 (G2) or anti-hCD40 antibody 2F8-IgG1-LALA-YTE (G3) are shown. PBS-injected hCD40 / hFcRn mice were used as control (G1). [Figure 17] Clinical scores of hCD40 / hFcRn mice injected with MOG to induce the EAE model and subsequently treated with hIgG1 (G2) or anti-hCD40 antibody 2F8-IgG1-LALA-YTE (G3). PBS-injected hCD40 / hFcRn mice were used as control (G1). [Figure 18] H&E (hematoxylin and eosin) stained images of spinal cords from hCD40 / hFcRn mice injected with MOG to induce the EAE model and subsequently treated with hIgG1 (G2) or anti-hCD40 antibody 2F8-IgG1-LALA-YTE (G3). PBS-injected hCD40 / hFcRn mice were used as controls (G1). Images were used to investigate inflammatory cell infiltration. [Figure 19] LFB (Luxol Fast Blue) staining 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). PBS-injected hCD40 / hFcRn mice were used as controls (G1). Images were used to investigate spinal cord demyelination. [Figure 20] Body weights of hCD40 mice injected with CII emulsion to induce the CIA model and subsequently treated with PBS (G2) or anti-hCD40 antibody 12B5-IgG1-LALA-YTE (G3) are shown. PBS-injected hCD40 mice alone were used as control (G1). [Figure 21] Clinical scores of hCD40 mice injected with CII emulsion to induce the CIA model and subsequently treated with PBS (G2) or anti-hCD40 antibody 12B5-IgG1-LALA-YTE (G3). PBS-injected hCD40 mice alone were used as control (G1). [Figure 22]The incidence of CIA in hCD40 mice injected with CII emulsion to induce the CIA model and subsequently treated with PBS (G2) or anti-hCD40 antibody 12B5-IgG1-LALA-YTE (G3) is shown. PBS-injected hCD40 mice alone were used as control (G1). [Diagram 23] H&E staining images of joint tissues harvested from the limbs of hCD40 mice injected with CII emulsion to induce the CIA model and then treated with PBS (G2) or anti-hCD40 antibody 12B5-IgG1-LALA-YTE (G3). PBS-injected hCD40 mice alone were used as control (G1). (a) represents inflammatory cell infiltration, (b) represents synovial hyperplasia, and (c) represents pannus. [Figure 24-1] The amino acid sequences discussed in this disclosure are listed below. [Figure 24-2] The amino acid sequences discussed in this disclosure are listed below. [Figure 24-3] The amino acid sequences discussed in this disclosure are listed below. [Figure 24-4] The amino acid sequences discussed in this disclosure are listed below. [Figure 24-5] The amino acid sequences discussed in this disclosure are listed below. [Figure 24-6] The amino acid sequences discussed in this disclosure are listed below. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] The present disclosure provides examples of antibodies, antigen-binding fragments thereof, that bind to CD40 (TNF receptor superfamily member 5).

[0042] CD40 and the immune system The immune system can distinguish between healthy cells in the body and cells it considers "foreign," allowing it to attack the foreign cells rather than just targeting healthy cells. This mechanism sometimes involves proteins called immune checkpoints, which are molecules within the immune system (co-stimulatory molecules) that either strengthen or weaken signals.

[0043] Checkpoint inhibitors can prevent the immune system from attacking healthy tissues, thereby preventing autoimmune diseases. Many tumor cells also express checkpoint inhibitors. These tumor cells escape immune surveillance by incorporating 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): 80-89). Because many immune checkpoints are initiated by ligand-receptor interactions, they can be easily blocked by antibodies against the ligands and / or their receptors.

[0044] 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 on 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 and induction of adaptive immunity.

[0045] Physiologically, signaling through CD40 on APCs is thought to be a major component of T cell help and in large part mediates the ability of helper T cells to license APCs. Ligation of CD40 on DCs induces, for example, increased surface expression of costimulatory and MHC molecules, production of proinflammatory cytokines, and enhanced T cell triggering. Ligation of CD40 on resting B cells increases antigen-presenting function and proliferation.

[0046] In preclinical models, rat anti-mouse CD40 mAbs have shown remarkable therapeutic activity in the treatment of CD40+ B-cell lymphoma (80-100% of mice were cured and immunized against rechallenge, dependent on CD8 T cells), and are also effective in a variety of CD40-negative tumors. These mAbs can clear tumor masses from mice with near-endstage disease. CD40 mAbs are being investigated in clinical trials to treat melanoma, pancreatic cancer, mesothelioma, hematological malignancies, particularly non-Hodgkin's lymphoma, lymphoma, chronic lymphocytic leukemia, and advanced solid tumors.

[0047] Therapeutic anti-CD40 antibodies display a variety of activities, ranging from strong agonism to antagonism. Currently, there is no satisfactory explanation for this heterogeneity. The main mechanistic basis for agonistic CD40 mAbs is to activate host APCs to induce clinically meaningful antitumor T cell responses in patients. These include induction of tumor regression, independent of T cells but dependent on macrophages. CD40-activated macrophages can be tumor-destructive and, at least in pancreatic cancer, may also promote destruction of the tumor stroma, inducing tumor lysis in vivo. Importantly, these mechanisms do not require expression of CD40 by the tumor, which has justified the inclusion of patients with a wide range of tumors in many clinical trials. Insofar as these strategies aim to activate DCs, macrophages, or both, the goal is not necessarily to kill the cells to which the CD40 mAb binds, for example, by complement-mediated cytotoxicity (CMC) or antibody-dependent cellular cytotoxicity (ADCC). Thus, by design, potent agonist antibodies do not mediate CMC or ADCC.

[0048] In contrast, other human CD40 mAbs can mediate CMC and ADCC against CD40+ tumors, such as nearly all B-cell malignancies, some melanomas, and certain carcinomas. Finally, there is some evidence that ligation of CD40 on tumor cells promotes apoptosis, and this can be achieved without any involvement of immune effector pathways. This has been shown for CD40+ B-cell malignancies, as well as certain solid tumors, such as CD40+ carcinomas and melanomas.

[0049] Due to the central role of CD40 in generating effective immune responses, CD40 also plays an important role in the pathogenesis of autoimmune diseases. CD40 contributes to T cell-dependent autoimmune diseases in several ways. First, CD40 signaling can function at the level of T cell selection in the thymus. Medullary thymic epithelial cells (mTECs) mediate the negative selection of potentially self-reactive T cells by expressing peripheral tissue-restricted antigens. While RANK, a TNFR family member, is crucial in the development of embryonic mTECs, CD40 cooperates with RANK in promoting the development of postnatal mTECs and thus self-tolerance. Disruption of CD40-CD154 interactions in mTECs may potentially contribute to the dysfunction of central tolerance. Second, CD40 signaling leads to the production of proinflammatory cytokines such as IL-6, which may affect cell differentiation into Th17 cells. CD40 is also upregulated upon activation of antigen-presenting cells (APCs). Increased levels of CD40, either constitutive or induced, may contribute to increased strength of CD40-CD154 interactions. Another mechanism may be abnormal expression of CD40 in tissues where it is normally undetectable. It has been hypothesized that abnormal expression of MHC class II molecules in endocrine tissues may contribute to the initiation of autoimmune diseases. Abnormal CD40 expression in such tissues has also been proposed as a contributing factor to the initiation of autoimmunity in Graves' disease and in the production of inflammatory cytokines that contribute to the dysfunction of islet cell transplants. Finally, CD40 bearing CD4+ T cells plays a role in type 1 diabetes in humans and mice. Thus, CD40 is an attractive candidate receptor for contributing to a variety of autoimmune processes in which B and T cell activation plays a role in the pathogenesis.

[0050] For detailed explanations of CD40 and its functions, see, for example, Vonderheide et al., "Agonistic CD40 antibodies and cancer therapy." (2013): 1035-1043; Beatty, et al., "CD40 agonists alter tumor stroma and show efficacy against pancreatic carcinoma in mice and humans." Science 331.6024 (2011): 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): 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 which is incorporated by reference in its entirety.

[0051] 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.

[0052] Antibodies and antigen-binding fragments The present disclosure provides anti-CD40 antibodies, and antigen-binding fragments thereof. Generally, antibodies (also called immunoglobulins) are composed of two classes of polypeptide chains, light chains and heavy chains. Non-limiting antibodies of the present disclosure can be intact four immunoglobulin chain antibodies, including two heavy chains and two light chains. The heavy chains of the antibodies can be of any isotype, including IgM, IgG, IgE, IgA, or IgD, or subisotypes, including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chains can be kappa or lambda light chains. The antibodies can include two identical copies of the light chains and two identical copies of the heavy chains. The heavy chains, each of which contains one variable domain (or variable region, VH) and multiple constant domains (or constant regions), are bound to each other via disulfide bonds within their constant domains to form the "stem" of the antibody. Each light chain, which contains one variable domain (or variable region, VL) and one constant domain (or constant region), is bound to one heavy chain via disulfide bonds. The variable region of each light chain aligns with the variable region of the heavy chain to which it is bound. The variable regions of both the light and heavy chains contain three hypervariable regions sandwiched between more conserved framework regions (FR).

[0053] The hypervariable regions, known as complementarity determining regions (CDRs), form the loops that comprise the principal antigen-binding surface of an antibody. The four framework regions largely conform to a β-sheet structure, and the CDRs form loops that connect, and in some cases form part of, the β-sheet structure. The CDRs of each chain are held in close proximity by the framework regions and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding region.

[0054] Methods for identifying CDR regions of an antibody by analyzing the amino acid sequence of the antibody are well known, and several definitions of CDR are commonly used: the Kabat definition is based on sequence variability, and the Chothia definition is based on the location of the 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. 422-439; Abhinandan, et al., "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains," Molecular immunology 45.14 (2008): 3832-3839; Wu, TT and Kabat, EA (1970) J. Exp. Med. 132: 211-250; Martin et al., Methods Enzymol. 203: 121-53 (1991); Morea et al., Biophys Chem. 68 (1-3): 9-16 (Oct. 1997); Morea et al., J Mol Biol. 275(2):269-94 (Jan. 1998), Chothia et al., Nature 342(6252):877-83 (Dec. 1989), Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007), each of which is incorporated by reference in its entirety. Kabat numbering is used by default in this disclosure unless otherwise indicated in this disclosure.

[0055] CDRs are 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 3, 4, 5, 6, or 7 amino acids, but these amino acids do not need to be in a contiguous linear sequence in the primary structure of the antigen, since the epitope can depend on the three-dimensional structure of the antigen based on the secondary and tertiary structure of the antigen.

[0056] In some embodiments, antibodies are intact immunoglobulin molecules (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved and differ in their constant regions, particularly the hinge and upper CH2 domains. The sequences and differences of IgG subclasses are well known in the art and are described, for example, in 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 by reference herein in its entirety.

[0057] An antibody can also be an immunoglobulin molecule from any species (e.g., human, rodent, mouse, camelid). Antibodies disclosed herein include, but are not limited to, polyclonal, monoclonal, monospecific, multispecific antibodies, and chimeric antibodies comprising an immunoglobulin binding domain fused to another polypeptide. The term "antigen-binding domain" or "antigen-binding fragment" refers to any portion of an antibody that retains the specific binding activity of the intact antibody, i.e., that is capable of specifically binding to an epitope on the target molecule of the intact antibody. This includes, for example, Fab, Fab', F(ab')2, and variants of these fragments. Thus, in some embodiments, an antibody or antigen-binding fragment thereof can be, for example, an scFv, Fv, Fd, dAb, bispecific antibody, bispecific scFv, diabody, linear antibody, single chain antibody molecule, multispecific antibody formed from antibody fragments, and any polypeptide that includes a binding domain that is an antibody binding domain, or is homologous thereto. Non-limiting examples of antigen-binding domains include, for example, the heavy and / or light chain CDRs of an intact antibody, the heavy and / or light chain variable regions of an intact antibody, the full-length heavy or light chain of an intact antibody, or individual CDRs derived from either the heavy or light chain of an intact antibody.

[0058] In some embodiments, the antigen-binding fragment can form part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor is a fusion of a single chain variable fragment (scFv) described herein fused to the CD3ζ transmembrane and endodomain. In some embodiments, the chimeric antigen receptor also comprises intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS). In some embodiments, the chimeric antigen receptor comprises multiple signaling domains, e.g., CD3z-CD28-41BB, or CD3z-CD28-OX40, for increased potency. Thus, in one aspect, the present disclosure further provides a cell (e.g., a T cell) expressing a chimeric antigen receptor described herein.

[0059] In some embodiments, an scFv has one heavy chain variable domain and one light chain variable domain, hi some embodiments, an scFv has two heavy chain variable domains and two light chain variable domains.

[0060] 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 an additional antigen (e.g., TNFRSF9 (4-1BB), MSLN, FAP, ALB, CTLA4, HER2, GPC3, MYC, EPCAM, TNFRSF14 (LIGHTR), or ITGAX (CD11c)).

[0061] Anti-CD40 antibodies and antigen-binding fragments 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. The antibodies can be agonists or antagonists. In some embodiments, the antibodies can promote the CD40 signaling pathway and thus increase the immune response. In some embodiments, the antibodies can block the CD40 signaling pathway and thus decrease the immune response. In some embodiments, the antibodies can initiate complement-dependent cytotoxicity (CMC) or antibody-dependent cellular cytotoxicity (ADCC).

[0062] The disclosure provides, for example, the murine anti-CD40 antibodies 2F8, 6A4, 12B5, chimeric antibodies thereof, and human or humanized antibodies thereof.

[0063] CDR sequences for 2F8 and antibodies derived from 2F8 (e.g., human or humanized antibodies) include the heavy chain variable domain CDR sequences SEQ ID NOs: 1-3 and light chain variable domain CDR sequences SEQ ID NOs: 4-6, as defined by Kabat numbering. CDRs can also be defined by the Chothia system. According to Chothia numbering, the heavy chain variable domain CDR sequences are set forth in SEQ ID NOs: 19-21 and the light chain variable domain CDR sequences are set forth in SEQ ID NOs: 22-24.

[0064] Similarly, CDR sequences for 6A4 and antibodies derived from 6A4 include the heavy chain variable domain CDR sequences SEQ ID NOs: 7 to 9 and the light chain variable domain CDR sequences SEQ ID NOs: 10 to 12, as defined by Kabat numbering. According to Chothia numbering, the heavy chain variable domain CDR sequences are set forth in SEQ ID NOs: 25 to 27 and the light chain variable domain CDR sequences are set forth in SEQ ID NOs: 28 to 30.

[0065] CDR sequences for 12B5 and antibodies derived from 12B5 include the heavy chain variable domain CDR sequences SEQ ID NOs: 13 to 15 and the light chain variable domain CDR sequences SEQ ID NOs: 16 to 18, as defined by Kabat numbering. According to Chothia numbering, the heavy chain variable domain CDR sequences are set forth in SEQ ID NOs: 31 to 33 and the light chain variable domain CDR sequences are set forth in SEQ ID NOs: 34 to 36.

[0066] The amino acid sequence for the heavy chain variable region of the 2F8 antibody is set forth in SEQ ID NO: 65. The amino acid sequence for the light chain variable region of the 2F8 antibody is set forth in SEQ ID NO: 37.

[0067] The amino acid sequence for the heavy chain variable region of the 6A4 antibody is set forth in SEQ ID NO: 38. The amino acid sequence for the light chain variable region of the 6A4 antibody is set forth in SEQ ID NO: 39.

[0068] The amino acid sequence for the heavy chain variable region of the 12B5 antibody is set forth in SEQ ID NO: 40. The amino acid sequence for the light chain variable region of the 12B5 antibody is set forth in SEQ ID NO: 41.

[0069] Also provided are amino acid sequences for the heavy and light chain variable regions 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, both of which bind to CD40.

[0070] Percent humanization refers to the percent identity of a heavy or light chain variable region sequence compared to human antibody sequences in the International Immunogenetics Database (IMGT). A top hit means that a heavy or light chain variable region sequence is closer to a particular species than to other species. For example, a top hit to human means that the sequence is closer to human than to other species. A top hit to human and cynomolgus monkey means that the sequence has the same percent identity to human sequence and cynomolgus monkey sequence, and these percent identity are the highest compared to sequences of other species. In some embodiments, the percent humanization 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 humanization percentage and how to determine the top hits are well 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 by reference in its entirety herein. A high humanization percentage often has various advantages, such as being safer and more effective in humans, more likely to be tolerated by human subjects, and / or less likely to have side effects. In some embodiments, the variable regions are fully human, for example, derived from human heavy chain immunoglobulin locus sequences (e.g., a combination of human IGHV, human IGHD, and human IGHJ genes) and / or human kappa chain immunoglobulin locus sequences (e.g., a combination of human IGKV and human IGKJ genes).

[0071] Furthermore, 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 group consisting of SEQ ID NOs: 1 to 3, SEQ ID NOs: 7 to 9, SEQ ID NOs: 13 to 15, SEQ ID NOs: 19 to 21, SEQ ID NOs: 25 to 27, and SEQ ID NOs: 31 to 33, and / or one, two, or three light chain variable region CDRs selected from the group consisting of SEQ ID NOs: 4 to 6, SEQ ID NOs: 10 to 12, SEQ ID NOs: 16 to 18, SEQ ID NOs: 22 to 24, SEQ ID NOs: 28 to 30, and SEQ ID NOs: 34 to 36.

[0072] In some embodiments, an antibody may have a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2 and 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) comprising CDR1, 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 VL 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 VL 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 VL CDR3 amino acid sequence. Selected VH CDR1, 2, 3 amino acid sequences and selected VL CDR1, 2, 3 amino acid sequences are shown in Figure 9 (Kabat CDRs) and Figure 10 (Chothia CDRs).

[0073] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO:1 with 0, 1, or 2 amino acid insertions, deletions, or substitutions, SEQ ID NO:2 with 0, 1, or 2 amino acid insertions, deletions, or substitutions, or SEQ ID NO:3 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0074] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO:7 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO:8 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO:9 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0075] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 13 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 14 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 15 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0076] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 19 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 20 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 21 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0077] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO:25 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO:26 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO:27 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0078] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 31 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 32 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 33 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0079] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two or three of the CDRs of SEQ ID NO:4 with 0, 1 or 2 amino acid insertions, deletions or substitutions, SEQ ID NO:5 with 0, 1 or 2 amino acid insertions, deletions or substitutions, or SEQ ID NO:6 with 0, 1 or 2 amino acid insertions, deletions or substitutions.

[0080] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 10 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 11 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 12 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0081] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 16 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 17 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 18 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0082] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two or three of the CDRs of SEQ ID NO: 22 with 0, 1 or 2 amino acid insertions, deletions or substitutions, SEQ ID NO: 23 with 0, 1 or 2 amino acid insertions, deletions or substitutions, or SEQ ID NO: 24 with 0, 1 or 2 amino acid insertions, deletions or substitutions.

[0083] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO:28 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO:29 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO:30 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0084] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 34 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 35 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 36 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0085] Insertions, deletions, and substitutions can occur within the CDR sequences or at either or both ends of the CDR sequences. In some embodiments, the CDRs are determined according to the Kabat numbering scheme. In some embodiments, the CDRs are determined according to the Chothia numbering scheme. In some embodiments, the CDRs are determined according to a combination of the Kabat and Chothia numbering schemes.

[0086] The present disclosure also provides an antibody or antigen-binding fragment thereof that binds to CD40. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) that comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH sequence, and a light chain variable region (VL) that comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to 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.

[0087] 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.

[0088] The present disclosure also provides an antibody or antigen-binding fragment thereof that cross-competes with any of the antibodies or antigen-binding fragments thereof described herein. Cross-competition assays are well known in the art and are described, for example, in Moore et al., "Antibody cross-competition analysis of the human immunodeficiency virus type 1 gp120 exterior envelope glycoprotein." Journal of Virology 70.3(1996):1863-1872, which is incorporated herein by reference in its entirety. In one aspect, the present disclosure also provides an antibody or antigen-binding fragment thereof that binds to the same epitope or region as any of the antibodies or antigen-binding fragments described herein. Epitope binding assays are well known in the art and are described, for example, in Estep et al., "High throughput solution-based measurement of antibody-antigen affinity and epitope binning." MAbs.Vol.5.No.2.Taylor&Francis,2013, which is incorporated herein by reference in its entirety.

[0089] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment for comparison, and non-homologous sequences may be ignored). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced to optimally align the two sequences and the length of each gap. For example, sequence comparison and percent identity determination between two sequences can be performed using the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0090] The disclosure also provides a nucleic acid comprising a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or an immunoglobulin light chain, the immunoglobulin heavy chain or immunoglobulin light chain comprising the CDRs shown in Figure 9 or Figure 10, or having the sequence shown in Figure 11. When the polypeptide is paired with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to CD40 (e.g., human CD40).

[0091] The anti-CD40 antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments, as well as multispecific (e.g., bispecific) antibodies or antibody fragments. Additional antibodies provided herein are polyclonal, monoclonal, multispecific (multimeric, e.g., bispecific), human antibodies, chimeric antibodies (e.g., human-mouse chimeras), single chain antibodies, intracellularly produced antibodies (i.e., intrabodies), and antigen-binding fragments thereof. The antibodies or antigen-binding fragments thereof 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 antibodies or antigen-binding fragments thereof are IgG antibodies or antigen-binding fragments thereof.

[0092] Antibody fragments are suitable for use in the methods provided, so long as they retain the desired affinity and specificity of the full-length antibody. Thus, antibody fragments that bind to CD40 retain the ability to bind to CD40. Fv fragments are antibody fragments that contain a complete antigen recognition and binding site. This region consists of a tightly associated dimer of one heavy and one light chain variable domain, which can be essentially covalent, for example in scFv. In this configuration, the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Overall, the six CDRs or a subset thereof confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) can have the ability to recognize and bind to an antigen, but usually with a lower affinity than the entire binding site.

[0093] Single-chain Fv or (scFv) antibody fragments comprise the VH and VL domains (or regions) of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

[0094] Fab fragments contain the variable and constant domains of the light chain and the variable and first constant domain (CH1) of the heavy chain. F(ab')2 antibody fragments comprise a pair of Fab fragments, generally covalently linked near their carboxy termini by hinge cysteines between them. Other chemical linkages of antibody fragments are well known in the art.

[0095] Diabodies are small antibody fragments that contain two antigen-binding sites, comprising a VH connected to a VL (VH and VL) in 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 can be forced to pair with the complementary domains of another chain to generate two antigen-binding sites.

[0096] Linear antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) that, together with complementary light chain polypeptides, form a pair of antigen-binding regions. Linear antibodies may be bispecific or monospecific.

[0097] The antibodies and antibody fragments of the disclosure can be modified within the Fc region to confer desired effector functions or serum half-life.

[0098] Antibody multimerization can be achieved by natural aggregation of antibodies or by chemical or recombinant conjugation techniques well known in the art. For example, a percentage of purified antibody preparations (e.g., purified IgG1 molecules) naturally form protein aggregates containing antibody homodimers and other higher order antibody multimers.

[0099] Alternatively, antibody homodimers can be formed by chemical coupling techniques well known in the art. For example, heterobifunctional crosslinkers including, but not limited to, SMCC (succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate) and SATA (N-succinimidyl S-acetylthio-acetate) can be used to form antibody multimers. An exemplary procedure for forming antibody homodimers is described in Ghetie et al. (Proc. Natl. Acad. Sci. USA 94:7509-7514, 1997). Antibody homodimers can be converted to Fab'2 homodimers by pepsin digestion. Another method for forming antibody homodimers is to use the autophilic T15 peptide described in Zhao et al. (J. Immunol. 25:396-404, 2002).

[0100] In some embodiments, the multispecific antibody is a bispecific antibody. Bispecific antibodies can be produced by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers recovered from recombinant cell culture. For example, the interface can contain 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 a first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller ones (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the large side chain is created in the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers over other unwanted end products such as homodimers. This method is described, for example, in WO 96 / 27011, which is incorporated by reference in its entirety.

[0101] Bispecific antibodies include cross-linked, or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be bound to avidin and the other to biotin. Heteroconjugate antibodies can be made using any convenient cross-linking method. Suitable cross-linking agents and 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.

[0102] Methods for generating bispecific antibodies from antibody fragments are also well known in the art. For example, bispecific antibodies can be prepared using chemical coupling. Brennan et al. (Science 229:81, 1985) describe a procedure in which intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab' TNB derivatives is then reconverted to a Fab' thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of another Fab' TNB derivative to form the bispecific antibody.

[0103] Any of the antibodies or antigen-binding fragments described herein can 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 in 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 a stabilizing molecule can increase the half-life or extend the biological activity of the antibody or antigen-binding fragment in vitro (e.g., when stored in tissue culture medium or as a pharmaceutical composition) or in vivo (e.g., in humans).

[0104] In some embodiments, the antibodies or antigen-binding fragments described herein can be conjugated to a therapeutic agent. The antibody drug conjugates comprising the antibodies or antigen-binding fragments thereof can be covalently or non-covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracene, maytansinoids (such as DM-1 and DM-4), dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide, and analogs).

[0105] 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.

[0106] 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.

[0107] In some embodiments, the clearance rate (CL) of an antibody or antigen-binding fragment thereof described herein in a wild-type mouse (e.g., a C57BL / 6 mouse) 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 an antibody or antigen-binding fragment thereof described herein in a CD40 gene humanized mouse (e.g., a hCD40 mouse) 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.

[0108] In some embodiments, the half-life of an antibody or antigen-binding fragment thereof described herein in an FcRn gene humanized mouse (e.g., an 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 an FcRn gene humanized mouse (e.g., an hFcRn mouse) is less than 16 L / 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.

[0109] 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., CD40-expressing cells) by endocytosis.

[0110] In some embodiments, the antibodies or antigen-binding fragments thereof described herein exhibit an immunostimulatory effect. In some embodiments, the antibodies or antigen-binding fragments thereof described herein exhibit an immunosuppressive effect. In some embodiments, the antibodies or antigen-binding fragments thereof described herein suppress one or more immune functions (e.g., antigen-induced antibody production) by 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 same immune function when the antibody or antigen-binding fragment thereof is not administered.

[0111] In some embodiments, the immunosuppressive effect of the antibodies or antigen-binding fragments thereof described herein is reversible. In some embodiments, the immunosuppressive effect of the antibodies or antigen-binding fragments thereof described herein is irreversible. In some embodiments, the immune function (e.g., T cell-dependent humoral immune function) of a subject (e.g., a mouse) is restored 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 administering the antibody or antigen-binding fragment thereof to the subject. In some embodiments, the immune function (e.g., T cell-dependent humoral immune function) of a subject (e.g., a mouse) is restored to at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more, compared to the same immune function before administering the antibody or antigen-binding fragment thereof to the subject.

[0112] In some embodiments, the immunosuppressive effect of the antibody or antigen-binding fragment thereof does not reduce the ratio of CD20+ / CD19+ cells in an organ of the immune system (eg, spleen) of a subject (eg, a mouse).

[0113] In some embodiments, the antibody or antigen-binding fragment thereof saturates CD40 receptors at a concentration of about 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 or less.

[0114] In some embodiments, the antibodies or antigen-binding fragments thereof described herein reduce binding of CD154 to CD40 by 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%.

[0115] In some embodiments, the CD40 receptor occupancy (RO%) of the antibody or antigen-binding fragment thereof is 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.

[0116] Antibody characteristics The antibodies or antigen-binding fragments thereof described herein can block the binding between CD40 and a CD40 ligand (eg, CD154).

[0117] The antibodies or antigen-binding fragments thereof described herein can be CD40 agonists or antagonists. In some embodiments, by binding to CD40, the antibodies can inhibit the CD40 signaling pathway. In some embodiments, the antibodies can upregulate an immune response or downregulate an immune response.

[0118] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can increase an immune response, 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. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can decrease an immune cell (e.g., T cells, CD8+ T cells, CD4+ T cells, macrophages, antigen presenting cells) activity or number by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold.

[0119] In some embodiments, the antibody (or antigen-binding fragment thereof) specifically binds to CD40 (e.g., human CD40, monkey CD40 (e.g., rhesus monkey, cynomolgus monkey), canine CD40, mouse CD40, and / or chimeric CD40) with a dissociation rate (koff) of less than 0.1 s-1, less than 0.01 s-1, less than 0.001 s-1, less than 0.0001 s-1, or less than 0.00001 s-1. In some embodiments, the dissociation rate (koff) is greater than 0.01 s-1, greater than 0.001 s-1, greater than 0.0001 s-1, greater than 0.00001 s-1, or greater than 0.000001 s-1.

[0120] In some embodiments, the kinetic association rate (k) is greater than 1×10 / Ms, greater than 1×10 / Ms, greater than 1×10 / Ms, greater than 1×10 / Ms, or greater than 1×10 / Ms. In some embodiments, the kinetic association rate (k) is less than 1×10 / Ms, less than 1×10 / Ms, or less than 1×10 / Ms.

[0121] Affinity can be estimated from the quotient of the kinetic rate constants (KD=koff / kon). In some embodiments, KD is less than 1×10-6M, less than 1×10-7M, less than 1×10-8M, less than 1×10-9M, or less than 1×10-10M. In some embodiments, KD is less than 50nM, 30nM, 20nM, 15nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, or 1nM. In some embodiments, KD is more than 1×10-7M, more than 1×10-8M, more than 1×10-9M, more than 1×10-10M, more than 1×10-11M, or more than 1×10-12M.

[0122] Common techniques for measuring the affinity of an antibody to 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).

[0123] In some embodiments, thermal stability is measured. The antibodies or antigen-binding fragments described herein can have a Tm of greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95° C. In some embodiments, the Tm is less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95°C.

[0124] In some embodiments, the antibody has a tumor growth inhibition percentage (TGI%) of more 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 of less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. TGI% can be measured, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the start of treatment, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the start of treatment. As used herein, tumor growth inhibition percentage (TGI%) is calculated using the following formula: TGI(%)=[1-(Ti-T0) / (Vi-V0)]×100% Ti is the mean tumor volume in the treatment group on day i. T0 is the mean tumor volume in the treatment group on day 0. Vi is the mean tumor volume in the control group on day i. V0 is the mean tumor volume in the control group on day 0.

[0125] In some embodiments, the antibodies or antigen-binding fragments thereof described herein are CD40 antagonists. In some embodiments, the antibodies or antigen-binding fragments reduce CD40 signaling in target cells that express CD40.

[0126] In some embodiments, the antibody or antigen-binding fragment can enhance APC (e.g., DC cell) function, e.g., inducing surface expression of costimulatory and MHC molecules, inducing proinflammatory cytokine production, and / or enhancing T cell triggering function.

[0127] In some embodiments, the antibody or antigen-binding fragment is capable of binding to tumor cells expressing CD40, hi some embodiments, the antibody or antigen-binding fragment is capable of inducing complement-mediated cytotoxicity (CMC) and / or antibody-dependent cellular cytotoxicity (ADCC) and killing the tumor cells.

[0128] 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 cellular 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.

[0129] In some embodiments, the antibody or antigen-binding fragment is capable of inducing antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the antibody or antigen-binding fragment is not capable of inducing antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the antibody or antigen-binding fragment is capable of inducing complement-mediated cytotoxicity (CMC). In some embodiments, the antibody or antigen-binding fragment is not capable of inducing complement-mediated cytotoxicity (CMC).

[0130] 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, an 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, an N297A mutation, a YTE mutation, and / or a FLAA mutation.

[0131] 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 a Fab, Fab', F(ab')2, or Fv fragment. In some embodiments, the Fc region has a LALA mutation (L234A and L235A mutations according to EU numbering) or a LALA-PG mutation (L234A, L235A, P329G mutations according to EU numbering). In some embodiments, the Fc region has a FLAA mutation (F234A and L235A mutations according to EU numbering). In some embodiments, the Fc has a SI mutation (S239D and I332E mutations according to EU numbering). In some embodiments, the Fc has a N297A mutation according to EU numbering. In some embodiments, the Fc has a YTE mutation (M252Y, S254T, and T256E according to EU numbering).

[0132] In some embodiments, the antibodies or antigen-binding fragments described herein comprise 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.

[0133] In some embodiments, the antibodies or antigen-binding fragments described herein can inhibit PMBC proliferation by less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, or less than 30%, for example, compared to an isotype control antibody (e.g., hIgG1).

[0134] In some embodiments, the antibodies or antigen-binding fragments described herein can inhibit B cell proliferation and / or B cell activation. For example, upon treatment with an antibody or antigen-binding fragment described herein, the percentage of activated B cell subsets in PMBC cells is 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% compared to that upon treatment with an isotype-controlled antibody (e.g., hIgG1).

[0135] Method for producing anti-CD40 antibody An isolated fragment of human CD40 can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies can be raised in animals by multiple injections (e.g., subcutaneous or intraperitoneal injections) of the antigenic peptide or protein. In some embodiments, the antigenic peptide or protein is injected with at least one adjuvant. In some embodiments, the antigenic peptide or protein can be conjugated to an agent that is immunogenic in the species being immunized. Animals can be injected with the antigenic peptide or protein two or more times (e.g., two, three, or four times).

[0136] A full-length polypeptide or protein can be used, or an antigenic peptide fragment thereof can be used as the immunogen. An antigenic peptide of a protein contains at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acid sequence of CD40 and encompasses an epitope of the protein such that antibodies generated against the peptide form specific immune complexes with the protein. As mentioned above, the full-length sequence of human CD40 (SEQ ID NO: 42) is well known in the art. In some embodiments, Fc-tagged or His-tagged CD40 protein is used as the immunogen.

[0137] An immunogen is typically used for the preparation of antibodies by immunizing a suitable subject (e.g., a human or a transgenic animal expressing at least one human immunoglobulin locus). An appropriate immunogenic preparation can contain, for example, a recombinantly expressed or chemically synthesized polypeptide (e.g., a fragment of human CD40). The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulant.

[0138] 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 enzyme-linked immunosorbent assay (ELISA) using immobilized CD40 polypeptide or peptide. If desired, the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein G or protein A chromatography to obtain an IgG fraction. At an appropriate time after immunization, e.g., when the titer of specific antibodies is at its 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 256:495-497, 1975), the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4:72, 1983), and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96, 1985), or the trioma technique. Techniques for producing hybridomas are well known (see generally Current Protocols in Immunology, 1994, Coligan et al. (Eds.), John Wiley & Sons, Inc., New York, NY)). For example, hybridoma cells producing monoclonal antibodies are detected by screening hybridoma culture supernatants for antibodies that bind the polypeptide or epitope of interest using a standard ELISA assay.

[0139] 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 antibodies or antigen-binding fragments thereof described herein, or by peptide synthesis. Such variants include, for example, deletion, insertion, or substitution of residues within the amino acids of the sequence that make up the antigen-binding site or domain of the antibody. In such a population of variants, some antibodies or antigen-binding fragments have increased affinity for the target protein, e.g., CD40. Any combination of deletions, insertions, and / or combinations can be achieved in antibodies or antigen-binding fragments thereof with increased binding affinity for the target. Antibodies or antigen-binding fragments can be altered or new post-translational modifications can be introduced into the antibodies or antigen-binding fragments by changing the number (e.g., increasing or decreasing) of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence so that a different sugar is attached by an enzyme present in the cell), or introducing new glycosylation sites.

[0140] The antibodies disclosed herein can be derived from any species of animal, including mammals. Non-limiting examples of natural antibodies include antibodies derived from humans, primates, such as monkeys and apes, cows, pigs, horses, sheep, camelids (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits), including transgenic rodents that have been genetically engineered to produce human antibodies.

[0141] Human and humanized antibodies include antibodies having variable and constant regions derived from (or having the same amino acid sequences derived from) human germline immunoglobulin sequences. Human antibodies can include, for example, amino acid residues within the CDRs that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0142] A humanized antibody typically has a human framework (FR) into which non-human CDRs have been grafted. Thus, a humanized antibody has one or more amino acid sequences introduced into a human from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can essentially be performed, for example, by substituting rodent CDRs or CDR sequences with the corresponding sequences of a human antibody. These methods are described, for example, in Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988), each of which is incorporated herein by reference in its entirety. Thus, a "humanized" antibody is a chimeric antibody in which substantially less than an intact human V domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically murine antibodies in which some CDR residues and some FR residues have been substituted by residues from analogous sites in human antibodies.

[0143] The selection of human VH and VL domains used in the production of humanized antibodies is very important to reduce immunogenicity. According to the so-called "best-fit" method, the sequence of the V domain of a mouse antibody is screened against the entire library of known human domain sequences. The human sequence that is closest to the mouse sequence is then accepted as the human FR for the humanized antibody (Sims et al., J.Immunol., 151:2296 (1993) and Chothia et al., J.Mol.Biol., 196:901 (1987)).

[0144] Furthermore, it is important that antibodies be humanized while retaining high specificity and affinity for the antigen, as well as other favorable biological properties. To achieve this goal, humanized antibodies can be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that can illustrate and display possible three-dimensional conformations of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the possible role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences to achieve the desired antibody characteristic, e.g., increased affinity for the target antigen.

[0145] Typically, an amino acid sequence variant of a human, humanized, or chimeric anti-CD40 antibody contains an amino acid sequence that has a percent identity of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% with a sequence present in the light or heavy chain of the original antibody.

[0146] In some embodiments, a mouse having a humanized heavy chain immunoglobulin locus and a humanized kappa chain immunoglobulin locus (e.g., RenMab TMThe antibody is produced using a human immunoglobulin locus (HbA1, HbA2, HbA3, HbA4, HbA5, HbA6, HbA7, HbA8, HbA9, HbB10, HbB11, HbB20, HbB12, HbB13, HbB21, HbB14, HbB15, HbB22, HbB16, HbB17, HbB23, HbB18, HbB19, HbB24, HbB19, HbB25, HbB18, HbB19, HbB26, HbB19, HbB18, HbB19, HbB27, HbB19 ... TM A detailed description of the mice can be found in PCT / CN2020 / 075698, which is incorporated by reference in its entirety.

[0147] In some embodiments, a mouse having a humanized heavy chain immunoglobulin locus and a humanized kappa chain immunoglobulin locus (e.g., RenLite TM The RenLite immunoglobulin locus is a region on a chromosome that contains the genes for the heavy chain of an antibody. The locus can include, for example, the human IGHV (variable) gene, the human IGHD (diversity) gene, the human IGHJ (joining) gene, and the mouse heavy chain constant domain gene. The kappa chain immunoglobulin locus is a region on a chromosome that contains the genes encoding the common light chain. The kappa chain immunoglobulin locus can include, for example, the human IGKV (variable) gene, the human IGKJ (joining) gene, and the mouse light chain constant domain gene. RenLite TM A detailed description of the mice can be found in PCT / CN2021 / 097652, which is incorporated by reference in its entirety.

[0148] The mouse-generated antibody has a fully human VH, a fully human VL, and a mouse constant region. In some embodiments, the human VH and human VL bind to a human IgG constant region (e.g., IgG1, IgG2, IgG3, and IgG4). In some embodiments, the constant region has a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 52, 53, 54, 55, 56, 57, 58, or 59.

[0149] Identity or homology to a source sequence is usually the percentage of amino acid residues present in a candidate sequence that are identical to a sequence present in a human, humanized, or chimeric anti-CD40 antibody or fragment, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering conservative substitutions as part of the sequence identity.

[0150] Further modifications can be made to the anti-CD40 antibody or antigen-binding fragment. For example, cysteine ​​residues can be introduced into the Fc region to allow interchain disulfide bond formation in this region. The homodimeric antibodies thus generated may have some increased in vitro and / or in vivo half-life. Heterobifunctional cross-linkers, as described, for example, in Wolff et al. (Cancer Res. 53:2560-2565, 1993), can also be used to prepare homodimeric antibodies with increased in vitro and / or in vivo half-lives. Alternatively, antibodies with dual Fc regions can be engineered (see, for example, Stevenson et al., Anti-Cancer Drug Design 3:219-230, 1989).

[0151] In some embodiments, covalent modifications can be made to anti-CD40 antibodies or antigen-binding fragments thereof. These covalent modifications can be made by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of antibodies or antibody fragments are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with organic derivatizing agents capable of reacting with selected side chains, or with the N- or C-terminal residues.

[0152] In some embodiments, antibody variants are provided that have carbohydrate structures that lack fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies may be between 1% and 80%, between 1% and 65%, between 5% and 65%, or between 20% and 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycan structures (e.g., complex, hybrid, and high mannose structures) attached to Asn297, as measured, for example, by MALDI-TOF mass spectrometry as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at position 297 (position 314 in the Eu numbering of Fc region residues, or Kabat numbering) in the Fc region, however, Asn297 may also be located about ±3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. In some embodiments, the Fc region of the antibody is further engineered to replace the asparagine at position 297 with alanine (N297A) to reduce glycan heterogeneity.

[0153] In some embodiments, the Fc region of the antibody is further engineered to replace the serine at position 228 (EU numbering) of IgG4 with a proline (S228P) to promote production efficiency by avoiding Fab-arm exchange. Details regarding the S228 mutation are described, for example, in 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 by reference in its entirety.

[0154] Recombinant Vectors 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 the recombinant vectors have been introduced (i.e., such that the host cell contains the polynucleotides and / or vectors containing the polynucleotides), and the production of recombinant antibody polypeptides or fragments thereof by recombinant techniques.

[0155] As used herein, a "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. An "expression vector" is capable of delivering and expressing one or more polynucleotides of interest as encoded polypeptides in a host cell into which the expression vector is introduced. Thus, in an expression vector, a polynucleotide of interest is positioned for expression in the vector by being operably linked to control elements, such as a promoter, enhancer, and / or polyA tail, either in the vector or in the genome of the host cell at, near, or adjacent to the integration site of the polynucleotide of interest, such that the polynucleotide of interest is translated in the host cell into which it is introduced with the expression vector.

[0156] Vectors can be introduced into host cells by methods well known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., by recombinant viruses). Thus, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0157] 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., variola or other poxvirus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication-competent virus, or may use a replication-incompetent virus. In the latter case, viral propagation generally occurs only in complementary viral packaging cells. For example, Fisher-Hoch et al.,1989,Proc.Natl.Acad.Sci.USA 86:317~321, Flexner et al.,1989,Ann.NYAcad Sci.569:86~103, Flexner et al. al., 1990, Vaccine, 8:17~21, U.S. Pat. 91 / 02805, Berkner-Biotechniques, 6:616~627, 1988; Rosenfeld et al. Suitable systems are disclosed in: Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91: 215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90: 11498-11502; Guzman et al., 1993, Circulation, 88: 2838-2848; and Guzman et al., 1993, Cir. Res., 73: 1202-1207. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. The DNA can also be "naked," as described, for example, in Ulmer et al., 1993, Science, 259:1745-1749, and Cohen, 1993, Science, 259:1691-1692. Uptake of naked DNA can be increased by coating the DNA onto biodegradable beads that are efficiently transported into cells.

[0158] For expression, a DNA insert comprising an antibody-encoding or polypeptide-encoding polynucleotide disclosed herein can be operably linked to a suitable promoter (e.g., a heterologous promoter), such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters, and promoters of retroviral-type LTRs, to name a few. Other suitable promoters are known to those of skill in the art. The expression construct can further contain sites for transcription initiation and termination, and, within the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct can include a translation initiation at the beginning and a termination codon (UAA, UGA or UAG) positioned approximately at the end of the polypeptide to be translated.

[0159] As indicated, the expression vector can include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for E. coli and other bacterial culture. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as E. coli, Streptomyces, and Salmonella typhimurium cells, fungal cells such as yeast cells, insect cells such as Drosophila melanogaster S2 and Spodoptera litura Sf9 cells, animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells, and plant cells. Appropriate culture media and conditions for the host cells described herein are well known in the art.

[0160] Non-limiting vectors for use in bacteria include pQE70, pQE60 and pQE-9 available from Qiagen, pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A available from Stratagene, and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG available from Stratagene, and pSVK3, pBPV, pMSG, and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to those of skill in the art.

[0161] Non-limiting bacterial promoters suitable for use include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters, and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, promoters of retroviral-type LTRs such as those of Rous sarcoma virus (RSV), and metallothionein promoters such as the mouse metallothionein-I promoter.

[0162] In the yeast Saccharomyces cerevisiae, several vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, may be used (for reviews, see Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, and Grant et al., Methods Enzymol., 153:516-544 (1997)).

[0163] Introduction of the construct into the host cell can be accomplished by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, or other methods, which are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), which are incorporated herein by reference in their entirety.

[0164] Transcription of DNA encoding the antibody of the present disclosure in more eukaryotic organisms can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10-300 bp, that serve to increase the transcriptional activity of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer, located at base pairs 100-270 on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0165] To allow secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals can be incorporated into the expressed polypeptide. The signals can be endogenous to the polypeptide or the signals can be heterologous signals.

[0166] Polypeptides (e.g., antibodies) can be expressed in modified forms, such as fusion proteins (e.g., GST fusions) or with histidine tags, and can contain not only secretion signals, 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 a polypeptide to improve stability and durability in the host cell during purification or during subsequent handling and storage. Peptide moieties can also be added to the polypeptide to facilitate purification. Such regions can be removed before final preparation of the polypeptide. It is well known and routine in the art to add peptide moieties to polypeptides to, among other things, effect secretion or excretion, improve stability, and facilitate purification.

[0167] Treatment method The antibodies or antigen-binding fragments thereof of the present disclosure can be used for a variety of therapeutic purposes.

[0168] In one aspect, the present disclosure provides a method of treating cancer in a subject, reducing the rate of increase in tumor volume in a subject over time, reducing the risk of developing metastases, or reducing the risk of developing further metastases in a subject. In some embodiments, the treatment can halt, slow, prevent, or inhibit the progression of cancer. In some embodiments, the treatment can result in a reduction in the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0169] In one aspect, the disclosure relates to a method comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein to a subject in need thereof (e.g., a subject having, or identified or diagnosed as having, cancer, e.g., breast cancer (e.g., triple-negative breast cancer), carcinoid cancer, 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, renal cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or a malignant hematological disorder). In some embodiments, the cancer is unresectable 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 is afflicted with 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 colon cancer. In some embodiments, the subject has 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, hematological malignancies, particularly non-Hodgkin's lymphoma, lymphoma, chronic lymphocytic leukemia, or advanced solid tumors.

[0170] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk for cancer. Patients suffering from cancer can be identified in a variety of ways well known in the art.

[0171] In one aspect, the disclosure provides methods for treating, preventing, or reducing the risk of developing an aberrant or unwanted immune response, e.g., a disorder associated with an autoimmune disease, e.g., by affecting the functional properties of an APC cell (e.g., by blocking the interaction of CD40 and CD40L). These autoimmune diseases include alopecia areata, lupus, ankylosing spondylitis, Meniere's disease, antiphospholipid syndrome, mixed connective tissue disease, autoimmune Addison's disease, multiple sclerosis, autoimmune hemolytic anemia, myasthenia gravis, autoimmune hepatitis, pemphigus vulgaris, Behçet's disease, pernicious anemia, bullous pemphigoid, nodular polyarthritis, cardiomyopathy, polychondritis, celiac sprue dermatitis, polyglandular syndrome, chronic fatigue syndrome (CFIDS), polymyalgia rheumatica, chronic inflammatory demyelination, polymyositis and dermatomyositis, chronic inflammatory polyneuropathy, primary agammaglobulinemia, Churg-Strauss syndrome, primary biliary tract infections, and chronic inflammatory polyneuropathy. These conditions include, but are not limited to, idiopathic cirrhosis, cicatricial pemphigoid, psoriasis, CREST syndrome, Raynaud's phenomenon, cold agglutinin disease, Reiter's syndrome, Crohn's disease, rheumatic fever, discoid lupus, rheumatoid arthritis, cryoglobulinemic sarcoidosis, fibromyalgia, scleroderma, Graves' disease, Sjogren's syndrome, Guillain-Barre, stiff-man syndrome, Hashimoto's thyroiditis, Takayasu's arteritis, idiopathic alveolar fibrosis, temporal arteritis / giant cell arteritis, idiopathic thrombocytopenic purpura (ITP), ulcerative colitis, IgA nephropathy, uveitis, diabetes mellitus (e.g., type I), 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 disorders associated with aberrant or unwanted immune responses associated with cell, tissue, or organ transplants, such as kidney, liver, and heart transplants, such as graft-versus-host disease (GVHD), or to prevent allograft rejection. In some embodiments, the subject suffers from skin disease, liver disease, (e.g., cirrhosis), hidradenitis, experimental autoimmune encephalomyelitis. In some embodiments, the subject has renal disease, lupus, Sjogren's syndrome, ulcerative colitis, psoriasis, allergic dermatitis, atopic dermatitis, hidradenitis suppurativa, 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 diabetes mellitus type 1. 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, Sjogren's syndrome, autoimmune nephritis, or systemic lupus erythematosus. The relationship between CD40 and various autoimmune diseases is described, for example, in 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: Humoral immunity and beyond." Advanced drug delivery reviews (2018), and Albach, et al., "Safety, pharmacokinetics and pharmacodynamics of single rising doses of BI 655064, an antagonistic anti-CD40 antibody in healthy subjects: a potential novel treatment for autoimmune diseases." European journal of clinical pharmacology 74.2 (2018): 161-169, each of which is incorporated by reference herein in its entirety.

[0172] As used herein, an "effective amount" refers to an amount or dosage sufficient to bring about a beneficial or desired result, including halting, slowing, preventing, 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, polynucleotide encoding the antibody, vector comprising the polynucleotide, and / or composition is administered, the severity of the symptoms, and the route of administration, and thus dosing can be determined on an individual basis.

[0173] An effective amount can be administered in one or more administrations. For example, an effective amount of an antibody or antigen-binding fragment is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow, and / or delay the progression of an autoimmune disease or cancer in a patient, or an amount sufficient to ameliorate, stop, stabilize, reverse, slow, and / or delay the proliferation of a cell (e.g., a biopsy cell, any of the cancer cells described herein, or a cell line (e.g., a cancer cell line)) in vitro. As is understood in the art, an effective amount of an antibody or antigen-binding fragment may vary depending on other factors, such as, inter alia, the medical history of the patient, as well as the type (and / or dose) of antibody used.

[0174] Effective amounts and schedules for administering the antibodies, antibody-encoding polynucleotides, and / or compositions disclosed herein can be determined empirically, and making such determinations is within the skill of one in the art. One of skill in the art will understand that the dosage required to be administered will vary depending on, for example, the mammal receiving the antibodies, antibody-encoding polynucleotides, and / or compositions disclosed herein, the route of administration, the particular type of antibody, antibody-encoding polynucleotide, antigen-binding fragment, and / or composition disclosed herein that is used, and other agents administered to the mammal. Guidance for selecting appropriate doses for antibodies or antigen-binding fragments can be found in the literature on the therapeutic use of antibodies and antigen-binding fragments, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, NJ, 1985, ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York, 1977, pp. 365-389.

[0175] A typical daily dose of an effective amount of antibody is 0.01 mg / kg to 100 mg / kg. In some embodiments, the dose can 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 dose can 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 dose 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.

[0176] In any of the methods described herein, at least one antibody, antigen-binding fragment thereof, 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 can 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 in 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 in 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 in two different compositions (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 as a pill, tablet, or capsule. In some embodiments, the at least one additional therapeutic agent is administered in a sustained release oral formulation.

[0177] In some embodiments, one or more additional therapeutic agents can be administered to the 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, the one or more additional therapeutic agents and the 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 the subject such that there is an overlap in the subject between the period of biological activity of the one or more additional therapeutic agents and the period of biological activity of the at least one antibody or antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein).

[0178] 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) can 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 can determine the length of time for treatment 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 can also vary (e.g., increase or decrease) the identity and number of antibodies or antigen-binding antibody fragments (and / or one or more additional therapeutic agents) administered to a subject, and can adjust (e.g., increase or decrease) the dosage or frequency of administration of at least one antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) to a subject based on an evaluation of the effectiveness of the treatment (e.g., using any of the methods described herein and well known in the art).

[0179] In some embodiments, one or more additional therapeutic agents can be administered to the subject. The additional therapeutic agents can include one or more inhibitors selected from the group consisting of inhibitors of B-Raf, EGFR inhibitors, inhibitors of MEK, inhibitors of ERK, inhibitors of K-Ras, inhibitors of c-Met, inhibitors of anaplastic lymphoma kinase (ALK), inhibitors of phosphatidylinositol 3-kinase (PI3K), inhibitors of Akt, inhibitors of mTOR, dual PI3K / mTOR inhibitors, inhibitors of Bruton's tyrosine kinase (BTK), and inhibitors of isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2). In some embodiments, the additional therapeutic agent is an inhibitor of indoleamine 2,3-dioxygenase-1 (IDO1) (e.g., epacadostat).

[0180] In some embodiments, the additional therapeutic agent can include one or more inhibitors selected from the group consisting of inhibitors of HER3, inhibitors of LSD1, inhibitors of MDM2, inhibitors of BCL2, inhibitors of CHK1, inhibitors of the activated hedgehog signaling pathway, and agents that selectively degrade the estrogen receptor.

[0181] In some embodiments, the additional therapeutic agent is trabectedin, nab-paclitaxel, trebananib, pazopanib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, leolysin, Alimta, Dicaida, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, pazopanib, IMA-901, AGS-00 3, cabozantinib, vinflunine, Hsp90 inhibitors, Ad-GM-CSF, temozolomide, IL-2, IFNa, vinblastine, thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacitidine, lenalidomide, bortezomib, amrubicin, carfilzomib, pralatrexate, and enzastaurin.

[0182] In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, a tumor necrosis factor (TNF), 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.

[0183] In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI is administered to the subject.

[0184] 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-OX40 antibody, an anti-4-1BB antibody, and / or an anti-GITR antibody. In some embodiments, an anti-CD40 antibody or antigen-binding fragment thereof described herein can be administered with an anti-PD-1 antibody and an anti-TIGIT antibody.

[0185] Pharmaceutical Compositions and Routes of Administration Any one or more (e.g., 1, 2, 3, or 4) of any of the antibodies or antigen-binding fragments described herein can be present in a pharmaceutical composition in any combination. Any two or more (e.g., 2, 3, or 4) of any of the antibodies or antigen-binding fragments described herein can be present in a pharmaceutical composition in any combination. The pharmaceutical compositions can be formulated in any manner known in the art.

[0186] Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). The compositions may contain a sterile diluent (e.g., sterile water or saline), fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents, antibacterial or antifungal agents (e.g., benzyl alcohol or methylparaben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.), antioxidants (e.g., ascorbic acid or sodium bisulfite), chelating agents (e.g., ethylenediaminetetraacetic acid), buffers (e.g., acetates, citrates, or phosphates), and isotonic agents (e.g., sugars (e.g., dextrose), polyalcohols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride)), or any combination thereof. Liposomal suspensions may also be used as pharma- ceutically acceptable carriers (see, e.g., U.S. Pat. No. 4,522,811). The preparation of the composition can be formulated and enclosed in ampoules, disposable syringes, or multiple dose vials. Where necessary (e.g., in injectable formulations), proper fluidity can be maintained, for example, by the use of a coating such as lecithin, or a surfactant. Absorption of the antibody or antigen-binding fragment thereof can be prolonged by including an agent that delays absorption (e.g., aluminum monostearate and gelatin). Alternatively, sustained release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.).

[0187] 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 unit dosage form (i.e., physically discrete units containing a predetermined amount of active compound for ease of administration and uniformity of dosage).

[0188] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic, and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions may be provided in unit dosage form (i.e., a dose for a single administration). Pharmaceutical compositions may be formulated using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the chosen route of administration. For injection, the antibody may be formulated in an aqueous solution, preferably in a physiologically compatible buffer, to reduce discomfort at the injection site. The solution may contain formulating agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the antibody may be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0189] The toxicity and therapeutic efficacy of the composition can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys). For example, the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population) can be determined, and the therapeutic index is the ratio of LD50:ED50. Drugs that exhibit a high therapeutic index are preferred. If a drug exhibits undesirable side effects, care should be taken to minimize the potential for harm (i.e., to reduce the undesirable side effects). The toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

[0190] Data obtained from cell culture assays and animal studies can be used in formulating an appropriate dose of any given agent for use in a subject (e.g., a human). A therapeutically effective amount of one or more (e.g., one, two, three, or four) antibodies or antigen-binding fragments thereof (e.g., any of the antibodies or antibody fragments described herein) is an amount that treats the disease in a subject (e.g., a human subject identified as having cancer) or at risk of developing the disease (e.g., a subject who previously developed cancer but has now been cured), (e.g., an amount that reduces the severity, frequency, and / or duration of one or more symptoms of the disease in the subject (e.g., a human). The efficacy and administration of any of the antibodies or antigen-binding fragments described herein can be determined by a health care professional or veterinary professional using methods well known in the art, as well as by observing one or more symptoms of the disease in the subject (e.g., a human). Certain factors can affect the dosage and timing required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and the presence of other diseases).

[0191] Exemplary doses include amounts (milligrams or micrograms) 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). While these doses cover a wide range, one of skill in the art will appreciate that therapeutic agents including antibodies and antigen-binding fragments thereof can be determined for their efficacy and effective amounts by methods well known in the art. Typically, a relatively low dose is administered initially, and the dose can be subsequently and gradually increased by the attending health care professional or veterinary professional (for therapeutic applications) or by a researcher (if still working in the development phase) until an appropriate response is obtained. In addition, it is 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 subject's age, weight, general health, sex, and diet, time of administration, route of administration, rate of excretion, and half-life of the antibody or antibody fragment in vivo.

[0192] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.The present disclosure also provides methods of making the antibodies, or antigen-binding fragments thereof, for the various uses described herein.

[0193] Working Example The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0194] Example 1. Generation of human anti-CD40 antibodies To generate antibodies against human CD40, RenMab TM Mouse or RenLite TM Mice were immunized with human CD40, and anti-CD40 antibodies were produced as described below.

[0195] RenMabTM The mouse has both 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 genes for the heavy chain of an antibody. The locus includes the 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 that code for the light chain (kappa chain) of an antibody. The kappa chain immunoglobulin locus includes the IGKV (variable), IGKJ (joining), and light chain constant domain genes. RenMab TM A detailed description of the mice can be found in PCT / CN2020 / 075698, which is incorporated by reference in its entirety.

[0196] RenLite TM The RenLite mouse can be used as a genetically engineered model in which the heavy chain variable regions are fully humanized while maintaining a common light chain that has been strategically engineered into the antibody genes. TM Details of the mice can be found, for example, in PCT / CN2021 / 097652, which is incorporated by reference in its entirety.

[0197] Immunization of mice RenMab TM Mouse or RenLite TMMice were immunized with Fc-tagged human CD40 protein (hCD40-Fc, Beijing ACROBiosystems Co.Ltd., Catalog No.: CD0-H5253) or canine CD40 (dCD40-Fc, Sino Biological Inc., Catalog No.: 70105-D02H). Specifically, hCD40-Fc or dCD40-Fc was emulsified with adjuvant and injected into the heel, neck, or base of the tail of mice. Mice were immunized a total of three 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 2 weeks, and the interval between the second and third immunizations was 2 weeks. One week after the third immunization, orbital bleeds were collected and the antibody titers were analyzed by ELISA. Two weeks later, mice with high titers were selected and injected with hCD40-His (human CD40 protein with His-Tag, Beijing ACROBiosystems Co.Ltd., Catalog No.: CD0-H5228) or dCD40-His (CD40 protein, dog, recombinant (His tag), Sino Biological Inc., Catalog No.: 70105-D08H) through the tail vein.

[0198] In a separate experiment, RenMab was developed by injecting expression plasmids encoding human CD40 and / or canine CD40 into the tibialis anterior muscle of mice (by intramuscular (im) injection). TM Mouse or RenLite TM Mice were immunized. Mice were injected at least four times with at least 14 days between each injection. Blood was collected 7 days after the last immunization and serum was tested for antibody titers by ELISA.

[0199] At least 7 days after immunization, a boosting procedure (either by injecting an expression plasmid or by injecting a protein) was performed. For pulse immunization, mice were also 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 canine CD40 protein). Immune system organs (e.g. bone marrow, lymph nodes, and spleen) were then harvested 5 days after injection.

[0200] Antigen-specific B cells were also isolated directly from immunized mice without fusion with myeloma cells. Antibody light and heavy chain variable region sequences were obtained directly from antigen-specific B cells. For example, single cell technology (e.g., Beacon® Optofluidic System, Berkeley Lights Inc.) was used to screen plasma cells secreting antigen-specific monoclonal antibodies. 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 antibodies and CD40. Exemplary anti-CD40 antibodies obtained by this method include 2F8, 6A4, 15B4, 16D5, and 12B5.

[0201] Antibodies were named according to the following rules. For example, when the heavy chain variable region (VH) and light chain variable region (VL) of 2F8 were combined with the constant region of human IgG1, the antibody was named 2F8-IgG1. Similarly, 2F8-IgG2 and 2F8-IgG4 were generated, which contain the same VH and VL sequences as 2F8, but the constant regions are from human IgG2 and IgG4 subtypes, respectively.

[0202] The constant region can also contain 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) were introduced into the Fc region of 2F8-IgG1 to generate 2F8-IgG1-LALA, 2F8-IgG1-N297A, 2F8-IgG1-YTE, 2F8-IgG1-FLAA, and 2F8-IgG1-SI, respectively. The YTE mutation was also introduced into the Fc region of 2F8-IgG1-LALA or 2F8-IgG1-N297A to generate 2F8-IgG1-LALA-YTE or 2F8-IgG1-N297A-YTE, respectively. Antibodies with different constant region sequences were also generated against 6A4, 15B4, 16D5, or 12B5.

[0203] The amino acid sequences of the heavy chain CDR1, 2, and 3 and the light chain CDR1, 2, and 3 for 2F8 are shown in SEQ ID NOs: 1 to 6 (Kabat numbering) or SEQ ID NOs: 19 to 24 (Chothia numbering), respectively. The human heavy chain variable region and human light chain variable region for 2F8 are shown in SEQ ID NOs: 65 and 37, respectively.

[0204] The amino acid sequences of the heavy chain CDR1, 2, and 3 and the light chain CDR1, 2, and 3 for 6A4 are shown in SEQ ID NOs: 7 to 12 (Kabat numbering) or SEQ ID NOs: 25 to 30 (Chothia numbering), respectively. The human heavy chain variable region and human light chain variable region for 6A4 are shown in SEQ ID NOs: 38 and 39, respectively.

[0205] The amino acid sequences of the heavy chain CDR1, 2, and 3 and the light chain CDR1, 2, and 3 for 12B5 are shown in SEQ ID NOs: 13 to 18 (Kabat numbering) or SEQ ID NOs: 31 to 36 (Chothia numbering), respectively. The human heavy chain variable region and human light chain variable region for 12B5 are shown in SEQ ID NOs: 40 and 41, respectively.

[0206] Preparation of antibodies The positive antibody sequences from the sequence verification stage were subjected to plasmid extraction and transfected into 100 mL systems. After 10-13 days of cell culture, the expression supernatants were collected and subjected to affinity chromatography. The obtained antibody samples were used in in vitro testing and screening.

[0207] Example 2. Binding affinity of anti-CD40 antibodies 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 that targets CD40 and has been approved for the treatment of lupus.

[0208] The binding affinity of the anti-CD40 antibodies 2F8-IgG4-FLAA, 6A4-IgG4-FLAA, 12B5-IgG4-FLAA, and bleselumab analogues to human CD40 (hCD40), monkey (cynomolgus) CD40 (fasCD40), or dog CD40 (dCD40) was assayed using a Biacore™ ELISA kit equipped with a pre-immobilized Protein A sensor chip. TM Measurements were performed by surface plasmon resonance (SPR) using an 8K biosensor (Biacore, Inc, Piscataway NJ).

[0209] Specifically, hCD40-His, fasCD40-His (cynomolgus monkey CD40 / TNFRSF5 protein, His tag, Beijing ACRO Biosystems Co. Ltd., Catalog number: CD0-C52H6), or dCD40-His was diluted with 1×HBS-EP+ buffer (pH 7.4) to 200 nM, 100 nM, 50 nM, 25 nM, 6.25 nM, 3.125 nM, or 0 nM, and then subjected to a Biacore ELISA at 10 μL / min for about 50 seconds. TM The 8K biosensor was injected with 1 μg / mL of purified anti-CD40 antibody in 1×HBS-EP+ buffer (pH 7.4) at 1 μL / min for 50 s. Dissociation was monitored for 400 s. After the last injection of each titration, the chip was regenerated with glycine solution (pH 2.0) at 30 μL / min for 30 s.

[0210] Kinetic association rates (k) and dissociation rates (k) were obtained simultaneously by fitting the entire data to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. Methods Enzymology 6.99-110) using the Biacore™ 8K Evaluation software 3.0. Affinity was estimated from the quotient of the kinetic rate constants (K = k / k).

[0211] The same method was performed for each antibody tested, with appropriate adjustments to parameters (e.g., antibody concentration), as would be understood by one of skill in the art. The results for the antibodies tested are summarized in the table below.

[0212] The results showed 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.

[0213] [Table 1]

[0214] Example 3. Jurkat-luc-hCD40 receptor cell activation assay 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, which was developed by Novartis and XOMA for the treatment of Sjogren's syndrome.

[0215] 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, which was developed by Boehringer Ingelheim for the treatment of diseases such as rheumatoid arthritis (RA) and lupus nephritis.

[0216] Experiments were performed to test whether the anti-CD40 antibodies 2F8-IgG4-FLAA, 6A4-IgG4-FLAA, 15B4-IgG4-FLAA, 12B5-IgG4-FLAA, BI655064 analogs, iscalimab analogs, and bleselumab analogs could block the CD40 signaling pathway.

[0217] CHO-K1-FcγRIIB cells (Promega, Catalog No.: CS1979A09) were seeded in 96-well plates (cell density 4×104 cells / well) and incubated overnight at 37°C. Test antibodies were serially diluted (3-fold) with a maximum concentration of 6 μg / mL. Human CD40 ligand CD40L-His (Human CD40 Ligand / TNFSF5 Protein, His, Flag Tag (Active Trimer) (MALS Validated), Beijing ACROBiosystems Co.Ltd., Catalog No.: CDL-H52Db) was serially diluted with a maximum concentration of 3 μg / mL. Jurkat-Luc-hCD40 cells (transfected Jurkat-Luc cells expressing human CD40 protein) were seeded in a 96-well plate (cell density 5×105 cells / well), and then 25 μL of serially diluted antibody solutions and / or human CD40 ligand were added to each well. The working concentrations of the antibodies were 0.5 μg / mL, 1 μg / mL, and 2 μg / mL. The 96-well plate was incubated in a 37° C. incubator for 6 hours. After incubation, the plate was removed and 75 μL of Bio-lite TMLuciferase assay reagent (Vazyme Biotech Co., Ltd., Catalog No.: DD1201-02-AB) was added to each well. After incubating the plate at room temperature for 5-10 min, a luminescence detector was placed to detect the fluorescent signal.

[0218] When the concentration of all anti-CD40 antibodies was decreased, the fluorescent signal (indicating cell binding to CD40 ligand) increased, suggesting that the binding of human CD40 to human CD40 ligand was blocked by the anti-CD40 antibodies.

[0219] Example 4: Immunosuppressive effect of anti-CD40 antibodies in vivo Immunosuppressive effect of anti-CD40 antibody in hCD40 mice A CD40 genetic humanized mouse model was generated expressing a chimeric CD40 protein (SEQ ID NO: 49) in which a portion of the extracellular domain of the mouse CD40 protein was replaced with the corresponding human CD40 extracellular domain. Specifically, amino acids 20-192 of mouse CD40 (SEQ ID NO: 50) were replaced with amino acids 20-192 of human CD40 (SEQ ID NO: 42). Humanized mouse models (e.g., hCD40 mice) provide a tool for testing novel therapeutic treatments in a clinical setting by significantly reducing the difference in clinical outcomes in laboratory mice expressing human and mouse CD40. A detailed description of the CD40 genetic humanized mouse model can be found in PCT / CN2018 / 091845, which is incorporated by reference herein in its entirety.

[0220] Experiments were performed to analyze the effect of human anti-CD40 antibodies on immune responses. Ovalbumin (OVA) was used as an antigen to stimulate immune responses in hCD40 mice. Briefly, hCD40 mice (6-8 weeks old) were placed into one control group and six treatment groups (five mice per group). Treatment group mice were randomly selected for intraperitoneal (ip) administration of anti-CD40 antibodies bleselumab analog (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, respectively. Control group mice (G1) were injected with an equal volume of phosphate-buffered saline (PBS). Two doses were administered in total, on day 0 (the day of grouping) and day 4 (four days after grouping), respectively. Ovalbumin (OVA) was diluted to 1 mg / mL with PBS and mixed with CFA at a volume ratio of 1:1, with an immunization volume of 200 μL / mouse. On day 1 (one day after grouping), mice were intraperitoneally administered with the OVA / CFA mixture. Serum was collected from each animal on days 10 and 17 and passed through ELISA analysis. Figure 1 shows the experimental scheme. OVA was precoated on the ELISA plate. Goat anti-mouse IgG H&L (HRP) (Abcam, Cat. No.: ab97265) was used for ELISA analysis. The details of the administration scheme are shown in the table below.

[0221] [Table 2]

[0222] As shown in Figure 2, ELISA results for sera collected on day 10 indicate that the anti-CD40 antibodies bleselumab analog (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 in hCD40 mice compared to PBS (G1). Figure 3 shows the ELISA results for sera collected on day 17. The results on day 17 were similar to those obtained on day 10. Specifically, bleselumab analogs (G2), 12B5-IgG4-FLAA (G3), 2F8-IgG4-FLAA (G5), and 6A4-IgG4-FLAA (G6) reduced immune responses with lower OD than 16D5-IgG4-FLAA (G4) and 15B4-IgG4-FLAA (G7). It is likely that more antibodies showed reduced immune responses on day 10 due to increased antibody degradation on day 17.

[0223] Immunosuppressive effect of anti-CD40 antibody in hCD40 / hFcRn mice A CD40 / FcRn bi-gene humanized mouse model was also generated by crossing hCD40 mice with FcRn gene humanized mice (hFcRn mice). The FcRn gene humanized mice were engineered to express human FcRn protein (SEQ ID NO: 51). A detailed description of the FcRn gene humanized mice and the CD40 / FcRn bi-gene humanized mice (hCD40 / hFcRn mice) can be found in PCT / CN2022 / 075057, which is incorporated by reference in its entirety.

[0224] Similar to the above experiment, the pharmacokinetics (PK) and in vivo efficacy of the anti-CD40 antibody bleselumab 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 administered 1-4 mg / kg (i.e., 1 mg / kg, 2 mg / kg, or 4 mg / kg) of the anti-CD40 antibody bleselumab analogs, 12B5-IgG1-LALA-YTE and 2F8-IgG1-LALA-YTE, respectively, by intraperitoneal (ip) administration on day 0 (day of grouping). Control group mice (G1) were injected with an equal volume of PBS. On day 1 (one day after grouping), mice were administered an OVA / CFA mixture intraperitoneally. 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 run through PK analysis. Serum was collected from each animal on days 10, 17, and 21 and also run through ELISA analysis. The details of the dosing scheme are shown in the table below.

[0225] [Table 3]

[0226] The pharmacokinetic results show that after injection of different antibodies, the antibody concentration in the serum of hCD40 / hFcRn mice decreased over time, which is consistent with the pharmacokinetic characteristics. The ELISA results of the serum collected on days 10, 17, and 21 are shown in the table below and in Figures 6-8, respectively. In general, the anti-OVA antibodies of all mice in the control and treatment groups showed a continuous increasing trend throughout the experiment. In addition, the mice in the treatment group produced less anti-OVA antibodies than the mice in the control group. The results also showed a dose correlation, i.e., the higher the dose level of the antibody, the less the anti-OVA antibody production, and the better the immunosuppressive effect was achieved. In general, the immunosuppressive effect of 2F8-IgG1-LALA-YTE was better than that of 12B5-IgG1-LALA-YTE, which was better than that of the bleselumab analogue.

[0227] [Table 4-1] [Table 4-2]

[0228] CV: Coefficient of variation Example 5. Pharmacokinetic Analysis PK analysis in hCD40 and C57BL / 6 mice The pharmacokinetic clearance rate of anti-CD40 antibodies was measured in hCD40 mice. Specifically, mice were placed into four groups (three mice per group) and administered 3 mg / kg of bleselumab analog (G1), 12B5-IgG4-FLAA (G2), 6A4-IgG4-FLAA (G3), and 2F8-IgG4-FLAA (G4) by intravenous injection, respectively. Blood samples were collected 15 min, 1 day, 3 days, 7 days, 10 days, and 14 days after administration, and 4 days before administration.

[0229] Serum levels of human antibodies were measured by sandwich ELISA. Briefly, goat anti-human IgG (H+L) (Jackson ImmunoResearch Inc., Catalog No.: 109-005-088) was diluted to a final concentration of 2000 ng / mL and added to a 96-well plate (ELISA plate) at 100 μL / well, then incubated overnight at 4° C. After incubation, the plate was resuspended in PBS-T buffer (Tween®) and diluted with PBS-T buffer. TM The plate was washed four times with PBS-T buffer (supplemented with 20% BSA). The antibody-free areas were blocked with 2% BSA (bovine serum albumin) for 2 hours at 37°C. The plate was then washed four times with PBS-T buffer. After washing, 100 μL of serum (1 / 20 dilution (MRD) in 1% BSA) was added to each well. The wells were sealed and incubated for 1 hour at 37°C. After washing the plate with a plate washer, Peroxidase AffiniPure F(ab')2 Fragment Goat Anti-Human IgG, Fcγ Fragment Specific (Jackson ImmunoResearch Inc., Catalog No.: 109-036-098) was added to each well of the plate at 100 μL / well and incubated for 1 hour at 37°C. After washing the plate, tetramethylbenzidine (TMB) solution was added as a substrate at 100 μL / well to the 96-well plate. After incubation at room temperature in the dark, 100 μL of stop solution (Beyotime, Catalog No.: P0215) was added to each well. The absorbance value of each well was read at wavelengths of 450 nm and 630 nm using a microplate reader. Analysis software Gen5 TM The data were analyzed using the Phoenix Quantitative Analysis System. The absorbance values ​​of the calibration samples prepared with each test product and the corresponding concentrations were used to construct a standard curve with four parameters (i.e., T1 / 2, Cmax, AUC0-14 days, and CL). The antibody concentration of each serum sample was calculated using the standard curve. Drug concentration-time curves were constructed using the calculated sample concentrations at each time point. TM Pharmacokinetic parameters were calculated using WinNolin 8.3 and the results are shown in the table below.

[0230] Similar to the above experiments, the pharmacokinetic clearance rates of the anti-CD40 antibody bleselumab analogs 12B5-IgG4-FLAA, 6A4-IgG4-FLAA, and 2F8-IgG4-FLAA were also measured in C57BL / 6 mice. The details of the dosing scheme are shown in the table below.

[0231] [Table 5]

[0232] Four days before antibody administration, antibody concentrations were detected as 0 μg / mL (results not shown). As shown in Figure 4 and the table below, the results indicate that the concentration of antibodies in the serum of hCD40 and C57BL / 6 mice decreased over time after injection of different antibodies, consistent with typical pharmacokinetic properties. The half-life of anti-CD40 antibodies in C57BL / 6 (B6) mice ranged from 12.37 to 17.70 days, whereas the half-life of anti-CD40 antibodies in hCD40 mice ranged from 1.87 to 4.84 days. The clearance rate (CL) of anti-CD40 antibodies in C57BL / 6 mice ranged from 4.65 to 6.35 mL / day / kg, whereas the CL of anti-CD40 antibodies in hCD40 mice ranged from 12.92 to 14.69 mL / day / kg. The results showed that the antibody concentrations in wild-type C57BL / 6 mice were all similar. In contrast, in hCD40 mice, all antibody concentrations declined more rapidly, which can be explained by TMDD (target-mediated drug disposition).

[0233] [Table 6-1] [Table 6-2]

[0234] T1 / 2: Terminal half-life Cmax: maximum concentration AUC0~14 days: Blood concentration-time curve area 0~14 days CL: Clearance PK analysis in hFcRn mice Similar to the above experiment, the pharmacokinetic clearance rate of anti-CD40 antibody 12B5 was measured in hFcRn mice, which were placed into four groups (five mice per group). 2 mg / kg of 12B5-IgG1-LALA-YTE (G1), 12B5-IgG1-N297A-YTE (G2), 12B5-IgG4-YTE (G3), or 12B5-IgG4-FLAA (G4) was administered by intravenous injection. Blood samples were collected 2 hours, 1 day, 3 days, 7 days, 10 days, 14 days, 21 days, 28 days, and 35 days after administration, as well as 4 days before administration. The details of the administration scheme are shown in the table below.

[0235] [Table 7]

[0236] Four days before antibody administration, the antibody concentration was detected as 0 μg / mL (results not shown). As shown in FIG. 5 and the table below, the results show that the concentration of the antibody in the serum of hFcRn mice decreased over time after injection of the different antibodies, consistent with typical pharmacokinetic properties. The half-lives (T1 / 2) of 12B5-IgG1-LALA-YTE (G1) and 12B5-IgG4-YTE (G3) in mice 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).

[0237] [Table 8]

[0238] T1 / 2: terminal half-life, Cmax: maximum concentration, AUC0~35 days: Blood concentration-time curve area 0~35 days CL: Clearance Example 6. Binding affinity of anti-CD40 antibodies to human FcRn The binding affinity of the anti-CD40 antibodies 12B5-IgG1-LALA-YTE, 12B5-IgG1-N297A-YTE, 12B5-IgG4-YTE, and 12B5-IgG4 to human FcRn was measured by surface plasmon resonance (SPR) using a Biacore™ (Biacore, Inc, Piscataway NJ) 8K or T200 biosensor equipped with a pre-immobilized Protein A sensor chip.

[0239] hFcRn-His (FCGRT&B2M heterodimer protein, His Tag&Strep II tag (SPR&BLI&MALS verified, Beijing ACROBiosystems Co.Ltd., Cat. No.: FCM-H5286)) was diluted to 1μg / mL with 1×HBS-EP+ buffer (pH 7.4), and then incubated at 10μL / min for approximately 50 seconds with Biacore. TM 8K or T200 biosensor to achieve the required capture level (e.g., about 200 response units (RU)). Purified anti-CD40 antibodies with 1×HBS-EP+buffer (pH 7.4) at concentrations of 1250 nM, 625 nM, 312.5 nM, 156.25 nM, 78.125 nM, or 0 nM were then injected at 30 μL / min for 50 s. Dissociation was monitored for 200 s. After the last injection of each titration, the chip was regenerated with glycine solution (pH 1.7) at 30 μL / min for 30 s. Binding curves were obtained. Data analysis was performed by the analysis software, and the association and dissociation curves were fitted using a steady-state affinity binding model to obtain affinity kinetic data.

[0240] The above experiment was repeated, except that the 1×HBS-EP+ buffer (pH 7.4) was replaced with 1×HBS-EP+ buffer (pH 6.0).

[0241] The same method was performed for each antibody tested, with appropriate adjustments to parameters (e.g., antibody concentration), as would be understood by one of skill in the art. The results for the antibodies tested are summarized in the table below.

[0242] The results are shown in the table below. At a pH value of 7.4, 12B5-IgG4-FLAA did not bind to hFcRn, while the other antibodies bound to hFcRn. At pH 6.0, all of the antibodies showed high binding affinity to hFcRn. The results show that under acidic conditions, all of the antibodies had increased affinity to hFcRn. In addition, 12B5-IgG1-LALA-YTE and 12B5-IgG4-YTE showed higher binding affinity to hFcRn than 12B5-IgG1-N297A-YTE.

[0243] [Table 9]

[0244] Example 7. Measurement of antibody-dependent cellular cytotoxicity (ADCC) Rituximab (VH SEQ ID NO: 67, VL SEQ ID NO: 68) is a chimeric monoclonal antibody directed against CD20 and was first launched in 1997 as an intravenous treatment for relapsed or refractory low-grade or follicular, CD20-positive B-cell non-Hodgkin's lymphoma (NHL).

[0245] In this experiment, FcR-TANK cells (ImmuneOnco Biopharmaceuticals (Shanghai) Inc.) 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 or 4 hours, and the ADCC activity was measured.

[0246] The results are shown in Figure 12. 2F8-IgG1 and the rituximab analogue have ADCC activity, whereas 2F8-IgG1-LALA-YTE showed no ADCC effect.

[0247] Example 8. Measurement of complement-dependent cytotoxicity (CDC) Experiments were performed to evaluate the CDC effect of anti-CD40 antibodies. In the experiment, Raji cells (cell density 5x104 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 for 30 min at 37°C, followed by the addition of diluted normal human serum complement (QIDEL, Cat. No.: A113). After 1.5 h of incubation at 37°C and 5% CO2, PrestoBlue TM Cell viability reagent (Invitrogen, Catalog No: 2413466) was added to each well and the plates were incubated for 2 hours at 37° C., 5% CO 2 .

[0248] The results are shown in Figure 13. 2F8-IgG1-LALA-YTE did not show a CDC effect, whereas the positive control, a rituximab analogue, showed a strong CDC effect.

[0249] Example 9. Inhibitory effect of anti-CD40 antibodies on PBMC proliferation Peripheral blood mononuclear cells (PBMC) cells were seeded in 96-well plates (5×104 cells / well). Serially diluted antibodies 2F8-IgG1-LALA-YTE or hIgG1 (100ng / mL, 10ng / mL, 8ng / mL, 5ng / mL, 4ng / mL, 2ng / mL, 1ng / mL, 0.5ng / mL, or 0.25ng / mL) were added to each well and incubated for 1 hour at 37°C. Then, 10 μL of anti-FLAG antibody (Sigma, Cat. No.: F1804) conjugated shCD154 (human CD40 ligand / TNFSF5 protein, His, Flag tag (active trimer MALS verified), ACRO, Cat. No.: CDL-H52Db) was added to each well and the plate was incubated for 64 hours at 37°C. Fluorescent signals were measured using Vazyme Biotech Cell Counting-Lite® 2.0 Luminescent Cell Viability Assay (Vazyme, Catalog Number: DD1101-02) with a microplate reader.

[0250] The results are shown in Figure 14 and demonstrated that 2F8-IgG1-LALA-YTE effectively inhibited PBMC proliferation.

[0251] Example 10. Inhibitory effect of anti-CD40 antibodies on B cell proliferation In peripheral blood, B cell subsets can be distinguished corresponding to various stages of differentiation, maturation and activation, which 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 special surface markers for activated B cells.

[0252] In this experiment, human PBMC cells were plated in 96-well plates at a density of 1x105 cells / well. Anti-CD40 antibodies 2F8-IgG1-LALA-YTE and hIgG1 were serially diluted (3-fold) with a maximum concentration of 1000ng / mL. 10μL of antibodies were added to the 96-well plate. After 1 hour of incubation at 37°C, 10μL of anti-FLAG® M2 antibody (Sigma, Cat. No.: F1804) conjugated shCD154 was added and incubated for 18 hours at 37°C. The cells were then stained with PE anti-human CD20 (BioLegend, Cat. No.: 302306), PerCP / Cyanine5.5 anti-human CD69 antibody (BioLegend, Cat. No.: 310926), Brilliant Violet 421 TM Anti-human CD86 antibody (BioLegend, Catalog No.: 305426), FITC anti-human CD80 antibody (BioLegend, Catalog No.: 375406), APC / Cy7 anti-human CD23 (BioLegend, Catalog No.: 338502), human CD40 ligand / TNFSF5 protein, His, Flag tag (active trimer) (MALS verified) (ACRO, Catalog No.: CDL-H52Db-100μg), and monoclonal anti-FLAG® M2 antibody (Sigma, Catalog No.: F1804-1MG) were incubated in the dark at 4°C for 30 minutes, followed by flow cytometry analysis. The results are shown in Figures 15A-15B.

[0253] With increasing concentrations of 2F8-IgG1-LALA-YTE, the percentages of all activated B cell subsets decreased, indicating that 2F8-IgG1-LALA-YTE efficiently inhibited B cell proliferation and activation.

[0254] Example 11. Evaluation of the efficacy of anti-CD40 antibodies using the hCD40 / hFcRn mouse EAE model Experimental autoimmune encephalomyelitis (EAE) is the most commonly used experimental model for multiple sclerosis (MS), a human inflammatory demyelinating disease. In this experiment, an EAE model was established in hCD40 / hFcRn mice, and the effect of human anti-CD40 antibodies was analyzed. Specifically, hCD40 / hFcRn mice were placed into a control group (G1) and two treatment groups (G2 and G3). Then, on day 1 (one day after grouping), hCD40 / hFcRn mice were immunized by injecting 100 μL of MOG (myelin oligodendrocyte glycoprotein (35-55)) (at a dose level of 300 μg / mouse) at two sites on the posterior ribs, and 250 μL of PTX (pertussis toxin) was injected intraperitoneally 2 and 48 h after immunization to obtain the EAE model. The control group (G1) mice were injected with an equal volume of PBS.

[0255] Mice in groups G2 and G3 were administered 10 mg / kg hIgG1 or 2F8-IgG1-LALA-YTE by intraperitoneal injection on day 0 (day of grouping), day 4 (4 days after grouping), and day 8 (8 days after grouping), respectively. Control mice (G1) were not injected. Mice were weighed and examined daily for neurological symptoms of EAE and scored according to the following scale: 0=no symptoms, 1=droopy tail, 2=weakness of hind limbs, 3=paralysis of hind limbs, 4=paralysis of front limbs, 5=moribund or dead mouse, and a grading of 0.5 represents an intermediate score. At the end of the experiment (30 days after grouping), spinal cords were collected for histopathological analysis. The details of the administration scheme are shown in the table below.

[0256] [Table 10]

[0257] As shown in Figures 16 to 19, 2F8-IgG1-LALA-YTE (G3) can prevent weight loss caused by animal modeling (Figure 16) and reduce clinical scores (Figure 17). In EAE mice administered 2F8-IgG1-LALA-YTE, there was also a certain improvement effect on meningitis cell infiltration (Figure 18) and spinal cord demyelination (Figure 19).

[0258] Example 12. Evaluation of the efficacy of anti-CD40 antibodies using the hCD40 mouse CIA model Collagen-induced arthritis (CIA) model is a commonly used model because it shares immunological and pathological similarities with human rheumatoid arthritis (RA). We established a CIA model in hCD40 mice and analyzed the effects of human anti-CD40 antibodies.

[0259] Specifically, hCD40 mice were placed into a control group (G1) and two model groups (G2 and G3). Then, 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 Cat. No.: C9301)) into two sites at the base of the tail on day 0 (the first day of immunization). A second immunization was performed with equal doses on day 21 (21 days after the first immunization) to obtain the CIA model. The control group (G1) was injected with an equal volume of PBS.

[0260] 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 (A) the interphalangeal joints of the fingers, (B) the metacarpophalangeal joints, and (C) the wrists of the forepaws and the metatarsophalangeal and ankle joints of the hind paws. The severity of clinical arthritis in individual paws was scored on a scale of 0 to 4 as follows: 0 = Normal. No significant difference in appearance from a healthy mouse. 1 = erythema and swelling of one type of joint (A, B, or C); 2 = erythema and swelling of two types of joints (A, B, or C); 3 = Erythema and swelling of all three joints (A, B, or C) and 4 = Erythema and swelling over the entire leg.

[0261] The animals were scored on a scale of 0 to 16, based on the sum of the scores for all four limbs. At the end of the experiment (56 days after the first immunization), limbs were collected for histopathological analysis. The details of the dosing scheme are shown in the table below.

[0262] [Table 11]

[0263] As shown in Figures 20-23, 12B5-IgG1-LALA-YTE (G3) can prevent weight loss caused by animal modeling (Figure 20) and significantly reduce clinical severity (Figures 21-22), as evidenced by lower clinical scores and incidence rates compared to the control group G2. As shown in Figure 23, 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 versus the control.

[0264] Example 13. In vivo effects of anti-CD40 antibodies Allergic dermatitis (AD) is an inflammatory condition of the skin characterized by erythema, pruritus, scaling, lichenification, and vesicular papules.

[0265] In this study, the in vivo efficacy of anti-CD40 antibody 2F8-IgG1-LALA-YTE in dogs diagnosed with AD was evaluated. Prior to the study, the dog had recurrent AD symptoms for over a year and had been receiving repeated glucocorticoid treatment. Specifically, the antibody was administered by intravenous (iv) injection. The injection volume was calculated based on the dog's weight. The administration frequency was once every two weeks (a total of six administrations). After two injections, it was observed that erythema was significantly reduced on the dog's skin. Peripheral blood was also collected to test blood biochemistry and complete blood count (CBC). The results showed that the tested antibody, 2F8-IgG1-LALA-YTE, was well tolerated and had no toxicity in dogs. No rejection reactions were observed.

[0266] Other embodiments Although the present invention has been described in conjunction with its detailed description, it should be understood that the above description is for illustrative purposes only and is not intended to limit the scope of the invention as defined by 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), A heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region includes an amino acid sequence that is at least 80% identical to the selected VH CDR1 amino acid sequence, the VH CDR2 region includes an amino acid sequence that is at least 80% identical to the selected VH CDR2 amino acid sequence, and the VH CDR3 region includes an amino acid sequence that is at least 80% identical to the selected VH CDR3 amino acid sequence, and A light chain variable region (VL) comprising CDR1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VL CDR3 amino acid sequence, and the light chain variable region comprises the VL CDR1 region comprising an amino acid sequence that is at least 80% identical to the selected VL CDR3 amino acid sequence, The selected VH CDR1, 2, and 3 amino acid sequences, and the selected VL CDR1, 2, and 3 amino acid sequences are as follows: (1) (1-a) The selected VH CDR 1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 1, 2, and 3, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 4, 5, and 6, respectively; or (1-b) The selected VH CDR 1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 19, 20, and 21, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 22, 23, and 24, respectively; (2) (2-a) The selected VH CDR 1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 7, 8, and 9, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 10, 11, and 12, respectively; or (2-b) The selected VH CDR 1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 25, 26, and 27, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 28, 29, and 30, respectively; (3) (3-a) The selected VH CDR 1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 13, 14, and 15, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 16, 17, and 18, respectively; or (3-b) The selected VH CDR 1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 31, 32, and 33, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are described in SEQ ID NOs: 34, 35, and 36, respectively; One of these is an antibody or its antigen-binding fragment.

2. (1) (1-a) The VH comprises CDR1, 2, and 3 having the amino acid sequences described in SEQ ID NOs: 1, 2, and 3, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences described in SEQ ID NOs: 4, 5, and 6, respectively; or (1-b) The VH comprises CDR1, 2, and 3 having the amino acid sequences described in SEQ ID NOs: 19, 20, and 21, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences described in SEQ ID NOs: 22, 23, and 24, respectively; (2) (2-a) The VH comprises CDR1, 2, and 3 having the amino acid sequences described in SEQ ID NOs: 7, 8, and 9, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences described in SEQ ID NOs: 10, 11, and 12, respectively; or (2-b) The VH comprises CDR1, 2, and 3 having the amino acid sequences described in SEQ ID NOs: 25, 26, and 27, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences described in SEQ ID NOs: 28, 29, and 30, respectively; or (3) (3-a) The VH comprises CDR1, 2, and 3 having the amino acid sequences described in SEQ ID NOs: 13, 14, and 15, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences described in SEQ ID NOs: 16, 17, and 18, respectively; or (3-b) The VH comprises CDR1, 2, and 3 having the amino acid sequences described in SEQ ID NOs: 31, 32, and 33, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences described in SEQ ID NOs: 34, 35, and 36, respectively; The antibody or antigen-binding fragment thereof according to claim 1.

3. The following: (i) The antibody or its antigen-binding fragment specifically binds to CD40 in humans, monkeys, or dogs; (ii) The antibody or its antigen-binding fragment is a human antibody or its antigen-binding fragment, a single-strand variable fragment (scFv), and / or a multispecific antibody (e.g., a bispecific antibody); (iii) The antibody or its antigen-binding fragment is a human IgG1 antibody or its antigen-binding fragment (optionally having the YTE and / or LALA mutation), or a human IgG4 antibody or its antigen-binding fragment (optionally having the YTE); An antibody or antigen-binding fragment thereof according to claim 1, having one or more features selected from the above.

4. A nucleic acid comprising a polynucleotide encoding a polypeptide, The aforementioned polypeptide is (1) An immunoglobulin heavy chain or fragment thereof, each comprising a heavy chain variable region (VH) containing complementarity-determining regions (CDRs) 1, 2, and 3, each containing the amino acid sequence described in SEQ ID NOs: 1, 2, and 3; or SEQ ID NOs: 19, 20, and 21, wherein the VH, when paired with a light chain variable region (VL) containing the amino acid sequence described in SEQ ID NO: 37, binds to CD40. (2) An immunoglobulin light chain or fragment thereof, each comprising a VL containing complementarity-determining regions (CDRs) 1, 2, and 3, each comprising the amino acid sequence described in SEQ ID NOs. 4, 5, and 6; or SEQ ID NOs. 22, 23, and 24, wherein the VL, when paired with a VH containing the amino acid sequence described in SEQ ID NOs. 65, binds to CD40. (3) An immunoglobulin heavy chain or fragment thereof, each comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences described in SEQ ID NOs. 7, 8, and 9; or SEQ ID NOs. 25, 26, and 27, wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence described in SEQ ID NO. 39, binds to CD40. (4) An immunoglobulin light chain or fragment thereof, each comprising a VL containing complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which include the amino acid sequences described in SEQ ID NOs. 10, 11, and 12; or SEQ ID NOs. 28, 29, and 30, wherein the VL, when paired with a VH containing the amino acid sequence described in SEQ ID NOs. 38, binds to CD40. (5) An immunoglobulin heavy chain or fragment thereof comprising a VH comprising complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences described in SEQ ID NOs. 13, 14, and 15; or SEQ ID NOs. 31, 32, and 33, wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence described in SEQ ID NOs. 41, binds to CD40. (6) An immunoglobulin light chain or fragment thereof comprising a VL containing CDR1, 2, and 3, each containing the amino acid sequence described in SEQ ID NOs. 16, 17, and 18; or SEQ ID NOs. 34, 35, and 36, wherein the VL, when paired with a VH containing the amino acid sequence described in SEQ ID NOs. 40, binds to the CD40. Nucleic acids, including

5. The following: (i) When VH is paired with VL, it specifically binds to the CD40 of humans, monkeys, or dogs, or when VL is paired with VH, it specifically binds to the CD40 of humans, monkeys, or dogs; (ii) The immunoglobulin heavy chain or fragment thereof is a human immunoglobulin heavy chain or fragment thereof (for example, a human IgG1 heavy chain or fragment thereof, optionally having the YTE and / or LALA mutation; or a human IgG4 heavy chain or fragment thereof, optionally having the YTE), and the immunoglobulin light chain or fragment thereof is a human immunoglobulin light chain or fragment thereof; (iii) The nucleic acid encodes a single-stranded variable fragment (scFv) or a multispecific antibody (e.g., a bispecific antibody); (iv) The nucleic acid is cDNA; The nucleic acid according to claim 4, having one or more features selected from the above.

6. A vector comprising one or more nucleic acids according to claim 4 or 5, wherein the vector optionally comprises two of the nucleic acids, and the vectors both encode the VH region and the VL region that bind to CD40.

7. A pair of vectors, each vector comprising one of the nucleic acids described in claim 4 or 5, wherein the pair of vectors together encode the VL region and the VH region that both bind to CD40.

8. A cell comprising (i) one or more nucleic acids according to claim 4 or 5, (ii) a vector comprising one or more of the nucleic acids, or (iii) a pair of vectors, wherein each vector comprises one of the nucleic acids, and the pair of vectors together encode the VL region and the VH region that both bind to CD40; Selectively, the cell comprises two of the nucleic acids, and selectively, the two nucleic acids both encode the VL region and the VH region that bind to CD40; The cells are, optionally, CHO cells.

9. A method for producing an antibody or its antigen-binding fragment, (a) Culturing the cells according to claim 8 under conditions sufficient for the cells to produce the antibody or the antigen-binding fragment, (b) A method comprising collecting the antibody or antigen-binding fragment produced by the cells.

10. An antibody that binds to CD40 or an antigen-binding fragment thereof, The selected heavy chain variable region (VH) sequence comprises a VH containing an amino acid sequence that is at least 90% identical to the selected heavy chain variable region (VL) sequence, and a VL containing an amino acid sequence that is at least 90% identical to the selected light chain variable region (VL) sequence, wherein the selected VH sequence and the selected VL sequence are as follows: (1) The selected VH sequence is sequence number 65, and the selected VL sequence is sequence number 37; (2) The selected VH sequence is sequence number 38, and the selected VL sequence is sequence number 39; and (3) The selected VH sequence is sequence number 40, and the selected VL sequence is sequence number 41; One of these is an antibody or its antigen-binding fragment.

11. (i) VH comprises the sequence of sequence number 65, and VL comprises the sequence of sequence number 37; (ii) The VH includes the sequence of sequence number 38, and the VL includes the sequence of sequence number 39; or (iii) The VH includes the sequence of sequence number 40, and the VL includes the sequence of sequence number 41; The antibody or antigen-binding fragment thereof according to claim 10.

12. The following: (i) The antibody or its antigen-binding fragment specifically binds to CD40 in humans, monkeys, or dogs; (ii) The antibody or its antigen-binding fragment is a human antibody or its antigen-binding fragment, a single-strand variable fragment (scFv), and / or a multispecific antibody (e.g., a bispecific antibody); (iii) The antibody or antigen-binding fragment is a human IgG1 antibody or its antigen-binding fragment (optionally having the YTE and / or LALA mutation), or a human IgG4 antibody or its antigen-binding fragment (optionally having the YTE); An antibody or antigen-binding fragment thereof according to claim 10, having one or more features selected from the above.

13. An antibody or antigen-binding fragment thereof, comprising VH CDR1, 2, 3 and VL CDR1, 2, 3, according to any one of claims 1 to 3 and 10 to 12.

14. An antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 and 10 to 12, covalently bound to a therapeutic agent, The therapeutic agent is optionally a cytotoxic agent or a cell proliferation inhibitor, wherein the therapeutic agent is an antibody-drug conjugate.

15. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 and 10 to 12, or an antibody-drug conjugate, for the treatment of a subject having cancer, reduction of tumor growth rate, and / or killing tumor cells, wherein the antibody-drug conjugate comprises the antibody or antigen-binding fragment thereof covalently bound to a therapeutic agent.

16. The pharmaceutical composition according to claim 15, wherein the subject suffers from a solid tumor; and / or the cancer is melanoma, pancreatic cancer, mesothelioma, or hematological malignancy; or the cancer is non-Hodgkin lymphoma, lymphoma, or chronic lymphocytic leukemia.

17. A pharmaceutical composition for inhibiting an immune response in a target and / or for treating an autoimmune disease, comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3 and 10 to 12, or an antibody-drug conjugate, wherein the drug-antibody conjugate comprises the antibody or an antigen-binding fragment thereof covalently bound to a therapeutic agent.

18. The subject is suffering from an autoimmune disease, and / or the autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, allergic dermatitis, or multiple sclerosis. The pharmaceutical composition according to claim 17.

19. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof, or an antibody-drug conjugate, according to any one of claims 1 to 3 and 10 to 12, and a pharmaceutically acceptable carrier, wherein the antibody-drug conjugate comprises the antibody or an antigen-binding fragment thereof covalently bound to a therapeutic agent.

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