WO2020253722A1

WO2020253722A1 - Anti-cd40 antibodies and uses thereof

Info

Publication number: WO2020253722A1
Application number: PCT/CN2020/096574
Authority: WO
Inventors: Yi Yang; Jingshu XIE; Fang Yang; Yuelei SHEN; Jian Ni; Yanan GUO; Yunyun CHEN
Original assignee: Eucure Beijing Biopharma Co Ltd
Current assignee: Eucure Beijing Biopharma Co Ltd
Priority date: 2019-06-17
Filing date: 2020-06-17
Publication date: 2020-12-24
Anticipated expiration: 2021-12-17

Abstract

Provided are anti-CD40 (TNF Receptor Superfamily Member 5) antibodies, antigen -binding fragments, and the uses thereof.

Description

ANTI-CD40 ANTIBODIES AND USES THEREOF

TECHNICAL FIELD

This disclosure relates to anti-CD40 (TNF Receptor Superfamily Member 5) antibodies and uses thereof.

BACKGROUND

Autoimmune diseases are conditions arising from an abnormal immune response to a normal body part. There are at least 80 types of autoimmune diseases. The cause of autoimmune disease is generally not well understood. Some autoimmune diseases such as lupus run in families, and some other autoimmune diseases may be triggered by infections or other environmental factors. Some common autoimmune diseases include e.g., celiac disease, diabetes mellitus type 1, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.

Recent clinical and commercial success of therapeutic antibodies has created great interest in using antibodies to treat various immune-related disorders. There is a need to develop antibodies for use in various antibody-based therapeutics to treat autoimmune diseases.

SUMMARY

This disclosure relates to anti-CD40 antibodies, antigen-binding fragment thereof, and the uses thereof.

In some aspects, the disclosure relates to an antibody or antigen-binding fragment thereof that binds to CD40 (TNF Receptor Superfamily Member 5) comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising

CDRs

1, 2, and 3, wherein 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.

In some embodiments, the selected

VH CDRs

1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are one of the following:

(1) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 2, 3, respectively, and the selected

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 5, 6, respectively;

(2) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 7, 8, 9, respectively, and the selected

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10, 11, 12, respectively;

(3) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 13, 14, 15, respectively, and the selected

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, 18, respectively.

(4) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 19, 20, 21, respectively, and the selected

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 22, 23, 24, respectively.

(5) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 25, 26, 27, respectively, and the selected

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 28, 29, 30, respectively.

(6) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 31, 32, 33, respectively, and the selected

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 34, 35, 36, respectively.

In some embodiments, the antibody or antigen-binding fragment specifically binds to human CD40.

In some embodiments, the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment is a single-chain variable fragment (scFV) .

In some aspects, the disclosure relates to a nucleic acid comprising a polynucleotide encoding a polypeptide comprising:

(1) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) 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, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 79 binds to CD40;

(2) an immunoglobulin light chain or a 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, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 78 binds to CD40;

(3) an immunoglobulin heavy chain or a 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, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 81 binds to CD40;

(4) an immunoglobulin light chain or a 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, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 80 binds to CD40;

(5) an immunoglobulin heavy chain or a 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, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 100, 101, 102, or 83 binds to CD40;

(6) an immunoglobulin light chain or a fragment thereof comprising a

VL comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 16, 17 and 18, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 97, 98, 99, or 82 binds to CD40;

(7) an immunoglobulin heavy chain or a 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: 19, 20, and 21, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 85 binds to CD40;

(8) an immunoglobulin light chain or a fragment thereof comprising a

VL comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 22, 23 and 24, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 84 binds to CD40;

(9) an immunoglobulin heavy chain or a 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: 25, 26, and 27, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 94, 95, 96, or 87 binds to CD40;

(10) an immunoglobulin heavy chain or a 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: 25, 103, and 27, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 94, 95, 96, or 87 binds to CD40;

(11) an immunoglobulin light chain or a fragment thereof comprising a

VL comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 28, 29 and 30, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 90, 91, 92, 93, or 86 binds to CD40;

(12) an immunoglobulin heavy chain or a 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: 31, 32, and 33, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 89 binds to CD40;

(13) an immunoglobulin light chain or a fragment thereof comprising a

VL comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 34, 35 and 36, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 88 binds to CD40;

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 CDRs

1, 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 CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively.

VH comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively.

VL comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively.

VH comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively.

VL comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively.

VH comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively.

VL comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively.

VH comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 25, 26, and 27, respectively.

VL comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively.

VH comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively.

VL comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 34, 35, and 36, respectively.

In some embodiments, the VH when paired with a VL specifically binds to human CD40, or the VL when paired with a VH specifically binds to human CD40.

In some embodiments, the immunoglobulin heavy chain or the fragment thereof is a humanized immunoglobulin heavy chain or a fragment thereof, and the immunoglobulin light chain or the fragment thereof is a humanized immunoglobulin light chain or a fragment thereof. In some embodiments, the nucleic acid encodes a single-chain variable fragment (scFv) . In some embodiments, the nucleic acid is cDNA.

In some aspects, the disclosure relates to a vector comprising one or more of the nucleic acids as described herein. In some aspects, the disclosure relates to a vector comprising two of the nucleic acids as described herein. In some embodiments, the vector encodes the VL region and the VH region that together bind to CD40.

In some aspects, the disclosure relates to a pair of vectors, wherein each vector comprises one of the nucleic acids as described herein. In some embodiments, together the pair of vectors encodes the VL region and the VH region that together bind to CD40.

In some aspects, the disclosure relates to a cell comprising the vector as described herein or the pair of vectors as described herein.

In some embodiments, the cell is a CHO cell.

In some aspects, the disclosure relates to a cell comprising one or more of the nucleic acids as described herein. In some aspects, the disclosure relates to a cell comprising two of the nucleic acids as described herein. In some embodiments, the two nucleic acids together encode the VL region and the VH region that together bind to CD40.

In some aspects, the disclosure relates to a method of producing an antibody or an antigen-binding fragment thereof, the method comprising

(a) culturing the cell as described herein under conditions sufficient for the cell to produce the antibody or the antigen-binding fragment; and

(b) collecting the antibody or the antigen-binding fragment produced by the cell.

In some aspects, 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. 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: 78, and the selected VL sequence is SEQ ID NO: 79;

(2) the selected VH sequence is SEQ ID NO: 80, and the selected VL sequence is SEQ ID NO: 81;

(3) the selected VH sequence is SEQ ID NO: 97, 98, 99, or 82, and the selected VL sequence is SEQ ID NO: 100, 101, 102, or 83;

(4) the selected VH sequence is SEQ ID NO: 84, and the selected VL sequence is SEQ ID NO: 85;

(5) the selected VH sequence is SEQ ID NO: 90, 91, 92, 93, or 86, and the selected VL sequence is SEQ ID NO: 94, 95, 96, or 87;

(6) the selected VH sequence is SEQ ID NO: 88, and the selected VL sequence is SEQ ID NO: 89.

In some embodiments, the VH comprises the sequence of SEQ ID NO: 90 and the VL comprises the sequence of SEQ ID NO: 94. In some embodiments, the VH comprises the sequence of SEQ ID NO: 97 and the VL comprises the sequence of SEQ ID NO: 100.

In some embodiments, the antibody or antigen-binding fragment specifically binds to human CD40. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment is a single-chain variable fragment (scFV) .

In one aspect, provided herein is an antibody or antigen-binding fragment thereof comprising the

VH CDRs

1, 2, 3, and the

VL CDRs

1, 2, 3 of the antibody or antigen-binding fragment thereof as described herein.

In one aspect, provided herein is an antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof as described herein.

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

In some aspects, the disclosure relates to a method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof as described herein, or the antibody-drug conjugate as described herein.

In some embodiments, the subject has a solid tumor. In some embodiments, the cancer is melanoma, pancreatic carcinoma, mesothelioma, or a hematological malignancy. In some embodiments, the cancer is Non-Hodgkin's lymphoma, lymphoma, or chronic lymphocytic leukemia.

In some aspects, the disclosure relates to a method of decreasing the rate of tumor growth, the method comprising contacting a tumor cell with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described herein or the antibody-drug conjugate as described herein.

In some aspects, the disclosure relates to a method of killing a tumor cell, the method comprising contacting a tumor cell with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof as described herein or the antibody-drug conjugate as described herein.

In some aspects, the disclosure relates to a method of inhibiting immune response in a subject, the method comprising administering to the subject an effective amount of a composition comprising the antibody or antigen-binding fragment thereof as described herein or the antibody-drug conjugate as described herein.

In some embodiments, the subject has an autoimmune disease.

In some aspects, the disclosure relates to a method of treating an autoimmune disease, the method comprising administering to the subject an effective amount of a composition comprising the antibody or antigen-binding fragment thereof as described herein or the antibody-drug conjugate as described herein.

In some embodiments, the autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus or lupus nephritis.

In some aspects, the disclosure relates to a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described herein and a pharmaceutically acceptable carrier.

In some aspects, the disclosure relates to a pharmaceutical composition comprising the antibody drug conjugate as described herein, and a pharmaceutically acceptable carrier.

As used herein, the term “cancer” refers to cells having the capacity for autonomous growth. Examples of such cells include cells having an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include cancerous growths, e.g., tumors; oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. Also included are malignancies of the various organ systems, such as respiratory, cardiovascular, renal, reproductive, hematological, neurological, hepatic, gastrointestinal, and endocrine systems; as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, and cancer of the small intestine. Cancer that is “naturally arising” includes any cancer that is not experimentally induced by implantation of cancer cells into a subject, and includes, for example, spontaneously arising cancer, cancer caused by exposure of a patient to a carcinogen (s) , cancer resulting from insertion of a transgenic oncogene or knockout of a tumor suppressor gene, and cancer caused by infections, e.g., viral infections. The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “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 malignant tumors of mesenchymal derivation. The term “hematopoietic neoplastic disorders” includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin. A hematopoietic neoplastic disorder can arise from myeloid, lymphoid or erythroid lineages, or precursor cells thereof.

As used herein, the term “antibody” refers to any antigen-binding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementary determining region (CDR) (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, multi-specific antibodies (e.g., bi-specific 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, e.g., bi-specific antibodies, single- chain antibodies, diabodies, linear antibodies, and multi-specific antibodies formed from antibody fragments.

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

As used herein, the term “human antibody” refers to an antibody that is encoded by an endogenous nucleic acid (e.g., rearranged human immunoglobulin heavy or light chain locus) present in a human. In some embodiments, a human antibody is collected from a human or produced in a human cell culture (e.g., 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 or yeast cell. In some embodiments, a human antibody is produced in a transgenic non-human animal (e.g., a bovine) containing an unrearranged or rearranged human immunoglobulin locus (e.g., heavy or light chain human immunoglobulin locus) .

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

As used herein, the term “humanized antibody” refers to a non-human antibody which contains minimal sequence derived from a non-human (e.g., mouse) immunoglobulin and contains sequences derived from a human immunoglobulin. In non-limiting examples, humanized antibodies are human antibodies (recipient antibody) in which hypervariable (e.g., CDR) region residues of the recipient antibody are replaced by hypervariable (e.g., CDR) region residues from a non-human antibody (e.g., a donor antibody) , e.g., a mouse, rat, or rabbit antibody, having the desired specificity, affinity, and capacity. In some embodiments, the Fv framework residues of the human immunoglobulin are replaced by corresponding non-human (e.g., mouse) immunoglobulin residues. In some embodiments, humanized antibodies may contain residues which 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, the humanized antibody contains substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops (CDRs) correspond to those of a non-human (e.g., mouse) immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin. 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 methods known in the art. Non-limiting examples of methods for generating humanized antibodies are described herein.

As used herein, the term “single-chain antibody” refers to a single polypeptide that contains at least two immunoglobulin variable domains (e.g., a variable domain of a mammalian immunoglobulin heavy chain or light chain) that is capable of specifically binding to an antigen. Non-limiting examples of single-chain antibodies are described herein.

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, the multimeric antibody is able to crosslink 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) .

As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human, to whom treatment according to the methods of the present 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 below the age of 18 years old) . In addition to humans, patients include but are not limited to mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, and primates. Included are, for example, non-human primates (e.g., monkey, chimpanzee, gorilla, and the like) , rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits) , lagomorphs, swine (e.g., pig, miniature pig) , equine, canine, feline, bovine, and other domestic, farm, and zoo animals.

As used herein, when referring to an antibody, the phrases “specifically binding” and “specifically binds” mean that the antibody interacts with its target molecule (e.g., CD40) preferably to other molecules, because the interaction is dependent upon the presence of a particular structure (i.e., the antigenic determinant or epitope) on the target molecule; in other words, the reagent is recognizing and binding to molecules that include a specific structure rather than to all molecules in general. An antibody that specifically binds to the target molecule may be referred to as a target-specific antibody. For example, an antibody that specifically binds to a CD40 molecule may be referred to as a CD40-specific antibody or an anti-CD40 antibody.

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.

As used herein, the terms “polynucleotide, ” “nucleic acid molecule, ” and “nucleic acid sequence” are used interchangeably herein to refer to polymers of nucleotides of any length of at least two nucleotides, and include, without limitation, DNA, RNA, DNA/RNA hybrids, and modifications thereof.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and 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.

Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a flow chart showing the first part of an exemplary protocol of making anti-hCD40 antibodies.

FIG. 2 is a flow chart showing the second part of an exemplary protocol of making anti-hCD40 antibodies.

FIG. 3 is a set of flow cytometry graphs showing that the anti-hCD40 antibodies can bind to cells that express hCD40.

FIG. 4 is a set of graphs showing flow cytometry analysis of the anti-hCD40 antibodies’ cross-reactivity with human-mouse chimeric CD40 (chiCD40) , mouse CD40 (mCD40) , and monkey CD40 (rmCD40) .

FIG. 5 is a graph showing body weight over time of humanized CD40 mice (B-hCD40) with MC-38 tumor cells treated with mouse anti-hCD40 antibodies. PS stands for physiological saline (control) .

FIG. 6 is a graph showing percentage change of body weight over time of humanized CD40 mice (B-hCD40) with MC-38 tumor cells treated with mouse anti-hCD40 antibodies. PS stands for physiological saline (control) .

FIG. 7 is a graph showing tumor size over time in humanized CD40 mice (B-hCD40) with MC-38 tumor cells treated with mouse anti-hCD40 antibodies. PS stands for physiological saline (control) .

FIG. 8 is a graph showing body weight over time of humanized CD40 mice (B-hCD40) with MC-38 tumor cells treated with mouse anti-hCD40 antibodies. PS stands for physiological saline (control) .

FIG. 9 is a graph showing percentage change of body weight over time of humanized CD40 mice (B-hCD40) with MC-38 tumor cells treated with mouse anti-hCD40 antibodies. PS stands for physiological saline (control) .

FIG. 10 is a graph showing tumor size over time in humanized CD40 mice (B-hCD40) with MC-38 tumor cells treated with mouse anti-hCD40 antibodies. PS stands for physiological saline (control) .

FIG. 11 is a graph showing body weight over time of humanized CD40 mice (B-hCD40) with MC-38 tumor cells treated with chimeric anti-hCD40 antibodies. PS stands for physiological saline (control) .

FIG. 12 is a graph showing percentage change of body weight over time of humanized CD40 mice (B-hCD40) with MC-38 tumor cells treated with chimeric anti-hCD40 antibodies. PS stands for physiological saline (control) .

FIG. 13 is a graph showing tumor size over time in humanized CD40 mice (B-hCD40) with MC-38 tumor cells treated with chimeric anti-hCD40 antibodies. PS stands for physiological saline (control) .

FIG. 14 is a diagram showing the experimental protocol for analyzing effects of mouse anti-hCD40 antibodies on immune responses.

FIGS. 15A-15B show ELISA results from analyzing effects of mouse anti-hCD40 antibodies on immune responses at 1: 100 dilution.

FIGS. 16A-16B shows effects of mouse anti-hCD40 antibodies on immune responses after a second immunization as analyzed by ELISA (1: 100) .

FIG. 17 is a diagram showing the experimental protocol for analyzing effects of mouse anti-hCD40 antibodies on immune responses.

FIGS. 18A-18B show effects of mouse anti-hCD40 antibodies on immune responses as analyzed by ELISA (1: 100) .

FIG. 19 is a diagram showing the experimental protocol for analyzing effects of mouse anti-hCD40 antibodies on immune responses.

FIG. 20 shows comparison results of the effects of mouse anti-hCD40 antibodies on immune responses as analyzed by ELISA (1: 100) after the first immunizations.

FIG. 21 shows effects of mouse anti-hCD40 antibodies on immune responses after the second immunization as analyzed by ELISA (1: 100) .

FIG. 22 is a set of flow cytometry graphs showing anti-hCD40 antibodies binding to CHO cells that expressed human CD40 at 70℃.

FIG. 23 shows binding curves of anti-hCD40 antibodies binding to PBMC.

FIG. 24 is a set of flow cytometry graphs showing antibody endocytosis in CHO-K1 cells expressing human CD40.

FIG. 25 is a set of flow cytometry graphs showing antibody endocytosis in Raji cells that expressing endogenous human CD40.

FIG. 26 is a diagram showing the experimental protocol for analyzing effects of anti-hCD40 antibodies on immune responses.

FIGS. 27A-27D show effects of anti-hCD40 antibodies on immune responses as analyzed by ELISA at a dilution ratio of 1: 100, 1: 300, 1: 900 and 1: 2700, respectively.

FIGS. 28A-28B shows effects of anti-hCD40 antibodies on immune responses as analyzed by ELISA at 1: 300 and 1: 900.

FIG. 29 is a set of flow cytometry graphs showing CD20 ⁺/CD19 ⁺ cells isolated from the spleen of B-hCD40 mice that were administered with physiological saline (mouse number 01-07) or 2A7-H1K2-IgG4-FLAA (mouse number 08-15) .

FIG. 30 shows percentages of CD20 ⁺/CD19 ⁺ cells in mouse spleen cells.

FIG. 31A shows curves indicating CD40 receptor occupancy and B cell activation by anti-hCD40 antibody 2A7-H2K2-IgG4-FLAA.

FIG. 31B shows curves indicating CD40 receptor occupancy and B cell activation by lucatumumab.

FIG. 32 lists CDR sequences of mouse anti-hCD40 antibodies (1B10, 9B1, 4D1, 9F5, 2A7, and 6G1) and CDR sequences of humanized anti-hCD40 antibodies thereof as defined by Kabat numbering.

FIG. 33 lists CDR sequences of mouse anti-hCD40 antibodies (1B10, 9B1, 4D1, 9F5, 2A7, and 6G1) and CDR sequences of humanized anti-hCD40 antibodies thereof as defined by Chothia numbering.

FIG. 34 lists amino acid sequences of human CD40 (hCD40) , mouse CD40 (mCD40) , monkey CD40 (rmCD40) , and chimeric CD40 (chiCD40) .

FIG. 35 lists amino acid sequences of heavy chain variable regions and light chain variable regions of humanized anti-hCD40 antibodies based on 2A7.

FIG. 36 lists amino acid sequences of heavy chain variable regions and light chain variable regions of humanized anti-hCD40 antibodies based on 4D1.

FIG. 37 lists the amino acid sequence of the heavy chain variable regions and light chain variable regions of mouse anti-hCD40 antibodies 1B10, 9B1, 4D1, 9F5, 2A7, and 6G1.

DETAILED DESCRIPTION

The present disclosure provides examples of antibodies, antigen-binding fragment thereof, that bind to CD40 (TNF Receptor Superfamily Member 5) .

CD40 and Immune System

The immune system can differentiate between normal cells in the body and those it sees as “foreign, ” which allows the immune system to attack the foreign cells while leaving the normal cells alone. This mechanism sometimes involves proteins called immune checkpoints. Immune checkpoints are molecules in the immune system that either turn up a signal (co-stimulatory molecules) or turn down a signal.

Checkpoint inhibitors can prevent the immune system from attacking normal tissue and thereby preventing autoimmune diseases. Many tumor cells also express checkpoint inhibitors. These tumor cells escape immune surveillance by co-opting certain immune-checkpoint pathways, particularly in T cells that are 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 readily blocked by antibodies against the ligands and/or their receptors.

CD40 (also known as Tumor Necrosis Factor Receptor Superfamily Member 5 or TNFRSF5) is a tumor necrosis factor receptor superfamily member expressed on antigen presenting cells (APC) such as dendritic cells (DC) , macrophages, B cells, and monocytes as well as many non-immune cells and a wide range of tumors. Interaction with its trimeric ligand CD154 (also known as CD40 ligand or CD40L) on activated T helper cells results in APC activation, leading to the induction of adaptive immunity.

Physiologically, signaling via CD40 on APC is thought to represent a major component of T cell help and mediates in large part the capacity of helper T cells to license APC. Ligation of CD40 on DC, for example, induces increased surface expression of costimulatory and MHC molecules, production of proinflammatory cytokines, and enhanced T cell triggering. CD40 ligation on resting B cells increases antigen-presenting function and proliferation.

In pre-clinical models, rat anti-mouse CD40 mAb show remarkable therapeutic activity in the treatment of CD40+ B-cell lymphomas (with 80–100%of mice cured and immune to re-challenge in a CD8 T-cell dependent manner) and are also effective in various CD40-negative tumors. These mAb are able to clear bulk tumors from mice with near terminal disease. CD40 mAb have been investigated in clinical trials and are used for treating melanoma, pancreatic carcinoma, mesothelioma, hematological malignancies, especially Non-Hodgkin's lymphoma, lymphoma, chronic lymphocytic leukemia, and advanced solid tumors.

Therapeutic anti-CD40 antibodies show diverse activities ranging from strong agonism to antagonism. Currently there is no satisfactory explanation for this heterogeneity. The primary mechanistic rationale invoked for agonistic CD40 mAb is to activate host APC in order to induce clinically meaningful anti-tumor T-cell responses in patients. These include T cell-independent but macrophage-dependent triggering of tumor regression. CD40-activated macrophages can become tumoricidal, and least in pancreatic cancer, may also facilitate the depletion of tumor stroma which induces tumor collapse in vivo. Importantly, these mechanisms do not require expression of CD40 by the tumor, which has justified inclusion of patients with a broad range of tumors in many of the clinical trials. Insofar as these strategies aim to activate DC, macrophages, or both, the goal is not necessarily for the CD40 mAb to kill the cell it binds to, for example, via complement mediated cytotoxicity (CMC) or antibody dependent cellular cytoxicity (ADCC) . Thus, by design, the strong agonistic antibody does not mediate CMC or ADCC.

In contrast, other human CD40 mAb can mediate CMC and ADCC against CD40+ tumors, such as nearly all B cell malignancies, a fraction of melanomas, and certain carcinomas. Finally, there is some evidence that ligation of CD40 on tumor cells promotes apoptosis and that this can be accomplished without engaging any immune effector pathway. This has been shown for CD40+ B cell malignancies and certain solid tumors such as CD40+ carcinomas and melanomas.

Because of the centrality of CD40 in generating effective immune responses, CD40 also plays an important role in the pathogenesis of autoimmune disease. 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 negative selection of potentially autoreactive T cells by expressing peripheral tissue-restricted antigens. While the TNFR family member RANK is critically important in embryonic mTEC development, CD40 cooperates with RANK in promoting mTEC development after birth and thus self-tolerance. Disruption of CD40- CD154 interactions in mTECs could potentially contribute to failure of central tolerance. Secondly, CD40 signaling results in the production of pro-inflammatory cytokines, such as IL-6, which can influence T cell differentiation to Th17 cells. CD40 is also upregulated upon antigen presenting cell (APC) activation. Increased levels of CD40, either constitutive or induced, can contribute to increased strength of CD40-CD154 interactions. Another mechanism can be aberrant expression of CD40 in tissues where it is normally undetectable. It has been hypothesized that aberrant expression of MHC class II molecules on endocrine tissues could contribute to the initiation of autoimmune disease. Aberrant CD40 expression on such tissues has also been proposed as a contributing factor to the initiation of autoimmunity in Grave’s disease, and in the production of inflammatory cytokines contributing to the failure of pancreatic islet cell transplants. Finally, CD40 bearing CD4+ T cells play 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 play a role in pathogenesis.

A detailed description of CD40 and its function can be found, e.g., in 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.

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 to treat cancers.

Antibodies and Antigen Binding Fragments

The present disclosure provides anti-CD40 antibodies and antigen-binding fragments thereof. In general, antibodies (also called immunoglobulins) are made up of two classes of polypeptide chains, light chains and heavy chains. A non-limiting antibody of the present disclosure can be an intact, four immunoglobulin chain antibody comprising two heavy chains and two light chains. The heavy chain of the antibody can be of any isotype including IgM, IgG, IgE, IgA, or IgD or sub-isotype including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a kappa light chain or a lambda light chain. An antibody can comprise two identical copies of a light chain and two identical copies of a heavy chain. The heavy chains, which each contain one variable domain (or variable region, V _H) and multiple constant domains (or constant regions) , bind to one another via disulfide bonding within their constant domains to form the “stem” of the antibody. The light chains, which each contain one variable domain (or variable region, V _L) and one constant domain (or constant region) , each bind to one heavy chain via disulfide binding. The variable region of each light chain is aligned with the variable region of the heavy chain to which it is bound. The variable regions of both the light chains and heavy chains contain three hypervariable regions sandwiched between more conserved framework regions (FR) .

These hypervariable regions, known as the complementary determining regions (CDRs) , form loops that comprise the principle antigen binding surface of the antibody. The four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding region.

Methods for identifying the CDR regions of an antibody by analyzing the amino acid sequence of the antibody are well known, and a number of definitions of the CDRs 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 in, e.g., 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, T.T. and Kabat, E.A. (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 herein by reference in its entirety. Unless specifically indicated in the present disclosure, Kabat numbering is used in the present disclosure as a default.

The CDRs are important for recognizing an epitope of an antigen. As used herein, an “epitope” is the smallest portion of a target molecule capable of being specifically bound by the antigen binding domain of an antibody. The minimal size of an epitope may be about three, four, five, six, or seven amino acids, but these amino acids need not be in a consecutive linear sequence of the antigen’s primary structure, as the epitope may depend on an antigen’s three-dimensional configuration based on the antigen’s secondary and tertiary structure.

In some embodiments, the antibody is an intact immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA) . The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, differ in their constant region, particularly in their hinges and upper CH2 domains. The sequences and differences of the IgG subclasses are known in the art, and are described, e.g., 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 herein by reference in its entirety.

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

In some embodiments, the antigen binding fragment can form a part of a chimeric antigen receptor (CAR) . In some embodiments, the chimeric antigen receptor are fusions of single-chain variable fragments (scFv) as described herein, fused to CD3-zeta 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, to increase potency. Thus, in one aspect, the disclosure further provides cells (e.g., T cells) that express the chimeric antigen receptors as described herein.

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

Anti-CD40 Antibodies and Antigen-Binding Fragments

The disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to CD40. The antibodies and antigen-binding fragments described herein are capable of binding to CD40. These antibodies can be agonists or antagonists. In some embodiments, these antibodies can promote CD40 signaling pathway thus increase immune response. In some embodiments, these antibodies can initiate CMC or ADCC.

The disclosure provides e.g., mouse anti-CD40 antibodies 11-1B10 ( “1B10” ) , 13-9B1 ( “9B1” ) , 13-4D1 ( “4D1” ) , 13-9F5 ( “9F5” ) , 20-2A7 ( “2A7” ) , and 11-6G1 ( “6G1” ) , the chimeric antibodies thereof, and the humanized antibodies thereof (e.g., some of the antibodies as shown in Table 1) .

The CDR sequences for 11-1B10, and 11-1B10 derived antibodies (e.g., humanized antibodies) include CDRs of the heavy chain variable domain, SEQ ID NOs: 1-3, and CDRs of the light chain variable domain, SEQ ID NOs: 4-6 as defined by Kabat numbering. The CDRs can also be defined by Chothia system. Under the Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 37-39 and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 40-42.

Similarly, the CDR sequences for 13-9B1, and 13-9B1 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 7-9, and CDRs of the light chain variable domain, SEQ ID NOs: 10-12, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 43-45, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 46-48.

The CDR sequences for 13-4D1, and 13-4D1 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 13-15, and CDRs of the light chain variable domain, SEQ ID NOs: 16-18, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 49-51, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 52-54.

The CDR sequences for 13-9F5, and 13-9F5 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 19-21, and CDRs of the light chain variable domain, SEQ ID NOs: 22-24, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 55-57, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 58-60.

The CDR sequences for 20-2A7, and 20-2A7 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 25-27, and CDRs of the light chain variable domain, SEQ ID NOs: 28-30, as defined by Kabat numbering. In some embodiments, the CDR sequences for 20-2A7, and 20-2A7 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NO: 25, SEQ ID NO: 103 (YINPYNAGTEYNEKFKG) , and SEQ ID NO: 27; and CDRs of the light chain variable domain, SEQ ID NOs: 28-30, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 61-63, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 64-66. In some embodiments, under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NO: 61, SEQ ID NO: 104 (NPYNAG) , and SEQ ID NO: 63; and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 64-66.

The CDR sequences for 11-6G1, and 11-6G1 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 31-33, and CDRs of the light chain variable domain, SEQ ID NOs: 34-36, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 67-69, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 70-72.

The amino acid sequences for heavy chain variable regions and light variable regions of the humanized antibodies are also provided. As there are different ways to humanize a mouse antibody (e.g., a sequence can be modified with different amino acid substitutions) , the heavy chain and the light chain of an antibody can have more than one version of humanized sequences. The amino acid sequences for the heavy chain variable regions of humanized 2A7 antibody are set forth in SEQ ID NOs: 90-93. The amino acid sequences for the light chain variable regions of humanized 2A7 antibody are set forth in SEQ ID NOs: 94-96. Any of these heavy chain variable region sequences (SEQ ID NO: 90-93) can be paired with any of these light chain variable region sequences (SEQ ID NO: 94-96) .

Similarly, the amino acid sequences for the heavy chain variable region of humanized 4D1 antibody are set forth in SEQ ID NOs: 97-99. The amino acid sequences for the light chain variable region of humanized 6A7 antibody are set forth in SEQ ID NOs: 100-102. Any of these heavy chain variable region sequences (SEQ ID NO: 97-99) can be paired with any of these light chain variable region sequences (SEQ ID NO: 100-102) .

Some chimeric and humanized antibodies based on 11-1B10 ( “1B10” ) , 13-9B1 ( “9B1” ) , 13-4D1 ( “4D1” ) , 13-9F5 ( “9F5” ) , 20-2A7 ( “2A7” ) , and 11-6G1 ( “6G1” ) are shown in Table 1.

Table 1

Humanization percentage means the percentage identity of the heavy chain or light chain variable region sequence as compared to human antibody sequences in International Immunogenetics Information System (IMGT) database. The top hit means that the heavy chain or light chain variable region sequence is closer to a particular species than to other species. For example, top hit to human means that the sequence is closer to human than to other species. Top hit to human and Macaca fascicularis means that the sequence has the same percentage identity to the human sequence and the Macaca fascicularis sequence, and these percentages identities are highest as compared to the sequences of other species. In some embodiments, humanization percentage is greater than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%. A detailed description regarding how to determine humanization percentage and how to determine top hits is known in the art, and is described, e.g., in Jones, et al. "The INNs and outs of antibody nonproprietary names. " MAbs. Vol. 8. No. 1. Taylor &Francis, 2016, which is incorporated herein by reference in its entirety. A high humanization percentage often has various advantages, e.g., more safe and more effective in humans, more likely to be tolerated by a human subject, and/or less likely to have side effects.

Furthermore, in some embodiments, the antibodies or antigen-binding fragments thereof described herein can also contain one, two, or three heavy chain variable region CDRs selected from the group of SEQ ID NOs: 1-3, SEQ ID NOs: 7-9, SEQ ID NOs: 13-15, SEQ ID NOs: 19-21, SEQ ID NOs: 25-27, SEQ ID NOs: 25, 103, 27, SEQ ID NOs: 31-33, SEQ ID NOs: 37-39, SEQ ID NOs: 43-45, SEQ ID NOs: 49-51, SEQ ID NOs: 55-57, SEQ ID NOs: 61-63, SEQ ID NOs: 61, 104, 63, and SEQ ID NOs: 67-69; and/or one, two, or three light chain variable region CDRs selected from the group of SEQ ID NOs: 4-6, SEQ ID NOs: 10-12, SEQ ID NOs: 16-18, SEQ ID NOs: 22-24, SEQ ID NOs: 28-30, SEQ ID NOs: 34-36, SEQ ID NOs: 40-42, SEQ ID NOs: 46-48, SEQ ID NOs: 52-54, SEQ ID NOs: 58-60, SEQ ID NOs: 64-66, and SEQ ID NOs: 70-72.

In some embodiments, the antibodies can have a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR3 amino acid sequence, and a light chain variable region (VL) comprising CDRs 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected 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. The selected

VH CDRs

1, 2, 3 amino acid sequences and the selected VL CDRs, 1, 2, 3 amino acid sequences are shown in FIG. 32 (Kabat CDR) and FIG. 33 (Chothia CDR) .

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 1 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 2 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 3 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 7 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 8 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 9 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 13 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 14 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 15 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 19 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 20 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 21 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 25 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 26 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 27 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 25 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 103 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 27 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 31 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 32 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 33 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 37 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 38 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 39 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 43 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 44 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 45 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 49 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 50 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 51 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 55 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 56 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 57 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 61 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 62 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 63 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 61 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 104 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 63 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 67 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 68 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 69 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 4 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 5 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 6 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 10 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 11 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 12 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 16 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 17 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 18 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 22 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 23 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 24 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 28 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 29 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 30 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 34 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 35 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 36 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 40 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 41 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 42 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 46 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 47 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 48 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 52 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 53 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 54 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 58 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 59 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 60 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 64 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 65 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 66 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 70 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 71 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 72 with zero, one or two amino acid insertions, deletions, or substitutions.

The insertions, deletions, and substitutions can be within the CDR sequence, or at one or both terminal ends of the CDR sequence.

The disclosure also provides antibodies or antigen-binding fragments thereof that bind to CD40. The antibodies or antigen-binding fragments thereof contain a heavy chain variable region (VH) comprising or consisting 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) comprising or consisting 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: 78, and the selected VL sequence is SEQ ID NO: 79. In some embodiments, the selected VH sequence is SEQ ID NO: 80 and the selected VL sequence is SEQ ID NO: 81. In some embodiments, the selected VH sequence is SEQ ID NO: 97, 98, 99, or 82, and the selected VL sequence is SEQ ID NO: 100, 101, 102, or 83. In some embodiments, the selected VH sequence is SEQ ID NO: 84 and the selected VL sequence is SEQ ID NO: 85. In some embodiments, the selected VH sequence is SEQ ID NO: 90, 91, 92, 93, or 86, and the selected VL sequence is SEQ ID NO: 94, 95, 96, or 87. In some embodiments, the selected VH sequence is SEQ ID NO: 88 and the selected VL sequence is SEQ ID NO: 89.

The 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.

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes) . The length of a reference sequence aligned for comparison purposes is at least 80%of the length of the reference sequence, and in some embodiments is at least 90%, 95%, or 100%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then 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, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For purposes of the present disclosure, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

The disclosure also provides 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 comprises CDRs as shown in FIG. 32 or FIG. 33, or have sequences as shown in FIGS. 35-37. When the polypeptides are paired with corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region) , the paired polypeptides bind to CD40 (e.g., human CD40) .

The anti-CD40 antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments and multi-specific (e.g., bi-specific) antibodies or antibody fragments. Additional antibodies provided herein are polyclonal, monoclonal, multi-specific (multimeric, e.g., bi-specific) , human antibodies, chimeric antibodies (e.g., human-mouse chimera) , single-chain antibodies, intracellularly-made 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 antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof.

Fragments of antibodies are suitable for use in the methods provided so long as they retain the desired affinity and specificity of the full-length antibody. Thus, a fragment of an antibody that binds to CD40 will retain an ability to bind to CD40. An Fv fragment is an antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight association, which can be covalent in nature, for example in scFv. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, 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 comprising only three CDRs specific for an antigen) can have the ability to recognize and bind antigen, although usually at a lower affinity than the entire binding site.

Single-chain Fv or (scFv) antibody fragments comprise the VH and VL domains (or regions) of 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. In some embodiments, the linker connecting scFv VH and VL domains is GGGGSGGGGSGGGGS (SEQ ID NO: 77) .

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

Diabodies are small antibody fragments with two antigen-binding sites, which fragments comprise a VH connected to a VL in the same polypeptide chain (VH and VL) . By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

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

Antibodies and antibody fragments of the present disclosure can be modified in the Fc region to provide desired effector functions or serum half-life.

Multimerization of antibodies may be accomplished through natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art. For example, some percentage of purified antibody preparations (e.g., purified IgG ₁ molecules) spontaneously form protein aggregates containing antibody homodimers and other higher-order antibody multimers.

Alternatively, antibody homodimers may be formed through chemical linkage techniques known in the art. For example, heterobifunctional crosslinking agents including, but not limited to SMCC (succinimidyl 4- (maleimidomethyl) cyclohexane-1-carboxylate) and SATA (N-succinimidyl S-acethylthio-acetate) can be used to form antibody multimers. An exemplary protocol for the formation of antibody homodimers is described in Ghetie et al. (Proc. Natl. Acad. Sci. U.S.A. 94: 7509-7514, 1997) . Antibody homodimers can be converted to Fab’ ₂ homodimers through digestion with pepsin. Another way to form antibody homodimers is through the use of the autophilic T15 peptide described in Zhao et al. (J. Immunol. 25: 396-404, 2002) .

In some embodiments, the multi-specific antibody is a bi-specific antibody. Bi-specific antibodies can be made by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers that are recovered from recombinant cell culture. For example, the interface can contain at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan) . Compensatory “cavities” of identical or similar size to the large side chain (s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine) . This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. This method is described, e.g., in WO 96/27011, which is incorporated by reference in its entirety.

Bi-specific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin. Heteroconjugate antibodies can also be made using any convenient cross-linking methods. Suitable cross-linking agents and cross-linking techniques are well known in the art and are disclosed in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.

Methods for generating bi-specific antibodies from antibody fragments are also known in the art. For example, bi-specific antibodies can be prepared using chemical linkage. Brennan et al. (Science 229: 81, 1985) describes a procedure where 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 Fab’ fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab’ TNB derivatives is then reconverted to the Fab’ thiol by reduction with mercaptoethylamine, and is mixed with an equimolar amount of another Fab’ TNB derivative to form the bi-specific antibody.

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

In some embodiments, the antibodies or antigen-binding fragments described herein can be conjugated to a therapeutic agent. The antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof can covalently or non-covalently bind 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, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin, 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) .

In some embodiments, the antibody or antigen-binding fragment thereof described herein recognizes a recombinant CD40. In some embodiments, the antibody or antigen-binding fragment thereof described herein recognizes an endogenous CD40, e.g., on the cell surface of PBMC. In some embodiments, EC ₅₀ (half maximal effective concentration) of the antibody or antigen-binding fragment thereof described herein are determined by flow cytometry. In some embodiments, the determined EC ₅₀ is at least or about 50 ng/ml, 100 ng/ml, 120 ng/ml, 140 ng/ml, 160 ng/ml, 180 ng/ml, 200 ng/ml, 220 ng/ml, 240 ng/ml, 260 ng/ml, 280 ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml, 600 ng/ml, 700 ng/ml, 800 ng/ml, 900 ng/ml, or 1000 ng/ml.

In some embodiments, the antibody or antigen-binding fragment thereof described herein can enter a cell (e.g., CHO cells) expressing a recombinant CD40. In some embodiments, the antibody or antigen-binding fragment thereof described herein can enter a cell (e.g., Raji cells) expressing an endogenous CD40. In some embodiments, the antibody or antigen-binding fragment thereof described herein can enter a cell through endocytosis. In some embodiments, the antibody or antigen-binding fragments described herein enters 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 the cells (e.g., CD40-expressing cells) through endocytosis.

In some embodiments, the antibody or antigen-binding fragment thereof described herein exhibits immune-stimulating effects. In some embodiments, the antibody or antigen-binding fragment thereof described herein exhibits immune-suppressing effects. In some embodiments, the antibody or antigen-binding fragment thereof described herein suppresses one or more immune functions (e.g., antigen-induced antibody production) to less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%as compared to the same immune function when the antibody or antigen-binding fragment thereof is not administered.

In some embodiments, the immune-suppressing effects of the antibody or antigen-binding fragment thereof described herein is reversible. In some embodiments, the immune-suppressing effects of the antibody or antigen-binding fragment thereof described herein is irreversible. In some embodiments, immune functions (e.g., T-cell dependent humoral immune function) of a subject (e.g., a mouse) are recovered after at least 10 days, at least 15 days, at least 20 days, at least 25 days, at least 30 days, at least 32 days, at least 35 days, at least 40 days, at least 45 days, or at least 60 days after the subject is administered with the antibody or antigen-binding fragment thereof. In some embodiments, immune functions (e.g., T-cell dependent humoral immune function) of a subject (e.g., a mouse) are recovered to at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more as compared to the same immune function before the subject is administered with the antibody or antigen-binding fragment thereof.

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

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

In some embodiments, the antibody or antigen-binding fragment thereof described herein decreases CD154 binding to CD40 to less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. In some embodiments, the percentage of 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 decreases 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%as compared to the percentage of activated B cells when the antibody or antigen-binding fragment thereof is not administered.

Antibody Characteristics

The antibodies or antigen-binding fragments thereof described herein can block the binding between CD40 and CD40 ligands (e.g., CD154) .

The antibodies or antigen-binding fragments thereof as described herein can be CD40 agonist or antagonist. In some embodiments, by binding to CD40, the antibody can inhibit CD40 signaling pathway. In some embodiments, the antibody can upregulate immune response or downregulate immune response.

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

In some implementations, the antibody (or antigen-binding fragments thereof) specifically binds to CD40 (e.g., human CD40, monkey CD40 (e.g., rhesus macaques, Macaca fascicularis) , 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.

In some embodiments, kinetic association rates (kon) is greater than 1 x 10 ²/Ms, greater than 1 x 10 ³/Ms, greater than 1 x 10 ⁴/Ms, greater than 1 x 10 ⁵/Ms, or greater than 1 x 10 ⁶/Ms. In some embodiments, kinetic association rates (kon) is less than 1 x 10 ⁵/Ms, less than 1 x 10 ⁶/Ms, or less than 1 x 10 ⁷/Ms.

Affinities can be deduced from the quotient of the kinetic rate constants (KD=koff/kon) . In some embodiments, KD is less than 1 x 10 ^-6 M, less than 1 x 10 ^-7 M, less than 1 x 10 ^-8 M, less than 1 x 10 ^-9 M, or less than 1 x 10 ^-10 M. In some embodiments, the KD is less than 50nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, KD is greater than 1 x 10 ^-7 M, greater than 1 x 10 ^-8 M, greater than 1 x 10 ^-9 M, greater than 1 x 10 ^-10 M, greater than 1 x 10 ^-11 M, or greater than 1 x 10 ^-12 M. In some embodiments, the antibody binds to human CD40 with KD less than or equal to about 6 nM.

General techniques for measuring the affinity of an antibody for an antigen include, e.g., ELISA, RIA, and surface plasmon resonance (SPR) . In some embodiments, the antibody binds to human CD40 (SEQ ID NO: 73) , monkey CD40 (e.g., rhesus macaque CD40, SEQ ID NO: 75) , chimeric CD40 (SEQ ID NO: 76) , and/or mouse CD40 (SEQ ID NO: 74) . In some embodiments, the antibody does not bind to human CD40 (SEQ ID NO: 73) , monkey CD40 (e.g., rhesus macaque CD40, SEQ ID NO: 75; cynomolgus CD40) , chimeric CD40 (SEQ ID NO: 76) , and/or mouse CD40 (SEQ ID NO: 74) .

In some embodiments, thermal stabilities are determined. The antibodies or antigen binding fragments as described herein can have a Tm 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 ℃. In some embodiments, 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 ℃.

In some embodiments, the antibody has a tumor growth inhibition percentage (TGI%) that is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. In some embodiments, the antibody has a tumor growth inhibition percentage that is less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. The TGI%can be determined, e.g., at 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 treatment starts, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the treatment starts. As used herein, the tumor growth inhibition percentage (TGI%) is calculated using the following formula:

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

Ti is the average tumor volume in the treatment group on day i. T0 is the average tumor volume in the treatment group on day zero. Vi is the average tumor volume in the control group on day i. V0 is the average tumor volume in the control group on day zero.

In some embodiments, the antibodies or antigen-binding fragments thereof as described herein are CD40 antagonist. In some embodiments, the antibodies or antigen binding fragments decrease CD40 signal transduction in a target cell that expresses CD40.

In some embodiments, the antibodies or antigen binding fragments can enhance APC (e.g., DC cell) function, for example, inducing surface expression of costimulatory and MHC molecules, inducing production of proinflammatory cytokines, and/or enhancing T cell triggering function.

In some embodiments, the antibodies or antigen binding fragments can bind to tumor cells that express CD40. In some embodiments, the antibodies or antigen binding fragments can induce complement mediated cytotoxicity (CMC) and/or antibody dependent cellular cytoxicity (ADCC) , and kill the tumor cell.

In some embodiments, the antibodies or antigen binding fragments have a functional Fc region. In some embodiments, effector function of a functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC) . In some embodiments, effector function of a functional Fc region is phagocytosis. In some embodiments, effector function of a functional Fc region is ADCC and phagocytosis.

In some embodiments, the antibodies or antigen binding fragments can induce complement mediated cytotoxicity (CMC) .

In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4.

In some embodiments, the antibodies or antigen binding fragments do not have a functional Fc region. For example, the antibodies or antigen binding fragments are Fab, Fab’, F (ab’) 2, and Fv fragments. In some embodiments, the Fc region has LALA mutations (L234A and L235A mutations in EU numbering) , or LALA-PG mutations (L234A, L235A, P329G mutations in EU numbering) . In some embodiments, the antibodies or antigen binding fragments have a FLAA mutation (F234A and L235A mutations in EU numbering) .

Methods of Making Anti-CD40 Antibodies

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 an 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 to be immunized. Animals can be injected with the antigenic peptide or protein more than one time (e.g., twice, three times, or four times) .

The full-length polypeptide or protein can be used or, alternatively, antigenic peptide fragments thereof can be used as immunogens. The antigenic peptide of a protein comprises 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 an antibody raised against the peptide forms a specific immune complex with the protein. As described above, the full length sequence of human CD40 is known in the art (SEQ ID NO: 73) .

An immunogen typically is used to prepare antibodies by immunizing a suitable subject (e.g., human or transgenic animal expressing at least one human immunoglobulin locus) . An appropriate immunogenic preparation can contain, for example, a recombinantly-expressed or a 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 immunostimulatory agent.

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

Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding a human, humanized, or chimeric antibody, or antigen-binding fragment thereof described herein, or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acids sequences that make-up the antigen-binding site of the antibody or an antigen-binding domain. In a population of such variants, some antibodies or antigen-binding fragments will have increased affinity for the target protein, e.g., CD40. Any combination of deletions, insertions, and/or combinations can be made to arrive at an antibody or antigen-binding fragment thereof that has increased binding affinity for the target. The amino acid changes introduced into the antibody or antigen-binding fragment can also alter or introduce new post-translational modifications into the antibody or antigen-binding fragment, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence such that a different sugar is attached by enzymes present in a cell) , or introducing new glycosylation sites.

Antibodies disclosed herein can be derived from any species of animal, including mammals. Non-limiting examples of native antibodies include antibodies derived from humans, primates, e.g., monkeys and apes, cows, pigs, horses, sheep, camelids (e.g., camels and llamas) , chicken, goats, and rodents (e.g., rats, mice, hamsters and rabbits) , including transgenic rodents genetically engineered to produce human antibodies.

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

A humanized antibody, typically has a human framework (FR) grafted with non-human CDRs. Thus, a humanized antibody has one or more amino acid sequence introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed by e.g., substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. These methods are described in e.g., 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 by reference herein in its entirety. Accordingly, “humanized” antibodies are chimeric antibodies wherein 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 mouse antibodies in which some CDR residues and some FR residues are substituted by residues from analogous sites in human antibodies.

The choice of human VH and VL domains to be used in making the humanized antibodies is very important for reducing 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 which is closest to that of the mouse is then accepted as the human FR for the humanized antibody (Sims et al., J. Immunol., 151: 2296 (1993) ; Chothia et al., J. Mol. Biol., 196: 901 (1987) ) .

It is further important that antibodies be humanized with retention of high specificity and affinity for the antigen and 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 which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen (s) , is achieved.

Ordinarily, amino acid sequence variants of the human, humanized, or chimeric anti-CD40 antibody will contain an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%percent identity with a sequence present in the light or heavy chain of the original antibody.

Identity or homology with respect to an original sequence is usually the percentage of amino acid residues present within the candidate sequence that are identical with a sequence present within the 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, and not considering any conservative substitutions as part of the sequence identity.

Additional modifications to the anti-CD40 antibodies or antigen-binding fragments can be made. For example, a cysteine residue (s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have any increased half-life in vitro and/or in vivo. Homodimeric antibodies with increased half-life in vitro and/or in vivo can also be prepared using heterobifunctional cross-linkers as described, for example, in Wolff et al. (Cancer Res. 53: 2560-2565, 1993) . Alternatively, an antibody can be engineered which has dual Fc regions (see, for example, Stevenson et al., Anti-Cancer Drug Design 3: 219-230, 1989) .

In some embodiments, a covalent modification can be made to the anti-CD40 antibody or antigen-binding fragment thereof. These covalent modifications can be made by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of the antibody or antibody fragment are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with an organic derivatization agent that is capable of reacting with selected side chains or the N-or C-terminal residues.

In some embodiments, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1%to 80%, from 1%to 65%, from 5%to 65%or from 20%to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues; or position 314 in Kabat numbering) ; however, Asn297 may also be located about ±3 amino acids upstream or downstream of 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, to reduce glycan heterogeneity, the Fc region of the antibody can be further engineered to replace the Asparagine at position 297 with Alanine (N297A) .

In some embodiments, to facilitate production efficiency by avoiding Fab-arm exchange, the Fc region of the antibodies was further engineered to replace the serine at position 228 (EU numbering) of IgG4 with proline (S228P) . A detailed description regarding S228 mutation is described, e.g., 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.

Recombinant Vectors

The present disclosure also provides recombinant vectors (e.g., an expression vectors) that include an isolated polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein) , host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or a vector comprising the polynucleotide) , and the production of recombinant antibody polypeptides or fragments thereof by recombinant techniques.

As used herein, a “vector” is any construct capable of delivering one or more polynucleotide (s) of interest to a host cell when the vector is introduced to the host cell. An “expression vector” is capable of delivering and expressing the one or more polynucleotide (s) of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the polynucleotide of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, and/or a poly-A tail, either within the vector or in the genome of the host cell at or near or flanking the integration site of the polynucleotide of interest such that the polynucleotide of interest will be translated in the host cell introduced with the expression vector.

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

In some implementations, a polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein) is introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus) , which may involve the use of a non-pathogenic (defective) , replication competent virus, or may use a replication defective virus. In the latter case, viral propagation generally will occur only in complementing virus packaging cells. Suitable systems are disclosed, for example, in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86: 317-321; Flexner et al., 1989, Ann. N.Y. Acad Sci. 569: 86-103; Flexner et al., 1990, Vaccine, 8: 17-21; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner-Biotechniques, 6: 616-627, 1988; Rosenfeld et al., 1991, Science, 252: 431-434; 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 of ordinary skill in the art. The DNA may also be “naked, ” as described, for example, in Ulmer et al., 1993, Science, 259: 1745-1749, and Cohen, 1993, Science, 259: 1691-1692. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads that are efficiently transported into the cells.

For expression, the DNA insert comprising an antibody-encoding or polypeptide-encoding polynucleotide disclosed herein can be operatively linked to an appropriate 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 LTRs, to name a few. Other suitable promoters are known to the skilled artisan. The expression constructs can further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs may include a translation initiating at the beginning and a termination codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated.

As indicated, the expression vectors 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 culturing in E. coli and other bacteria. Representative examples of appropriate 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 S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells; and plant cells. Appropriate culture mediums and conditions for the host cells described herein are known in the art.

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 the skilled artisan.

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, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus (RSV) , and metallothionein promoters, such as the mouse metallothionein-I promoter.

In the yeast Saccharomyces cerevisiae, a number of 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, N. Y, and Grant et al., Methods Enzymol., 153: 516-544 (1997) .

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

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

For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. The signals may be endogenous to the polypeptide or they may be heterologous signals.

The polypeptide (e.g., antibody) can be expressed in a modified form, such as a fusion protein (e.g., a GST-fusion) or with a histidine-tag, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to the polypeptide to facilitate purification. Such regions can be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.

Methods of Treatment

The antibodies or antigen-binding fragments thereof of the present disclosure can be used for various therapeutic purposes.

In one aspect, the disclosure provides methods for treating a cancer in a subject, methods of reducing the rate of the increase of volume of a tumor in a subject over time, methods of reducing the risk of developing a metastasis, or methods of reducing the risk of developing an additional metastasis in a subject. In some embodiments, the treatment can halt, slow, retard, or inhibit progression of a cancer. In some embodiments, the treatment can result in the reduction of in the number, severity, and/or duration of one or more symptoms of the cancer in a subject.

In one aspect, the disclosure features methods that include 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, a 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 hematologic malignancy. In some embodiments, the cancer is unresectable melanoma or metastatic melanoma, non-small cell lung carcinoma (NSCLC) , small cell lung cancer (SCLC) , bladder cancer, or metastatic hormone-refractory prostate cancer. In some embodiments, the subject has a solid tumor. In some embodiments, the cancer is squamous cell carcinoma of the head and neck (SCCHN) , renal cell carcinoma (RCC) , triple-negative breast cancer (TNBC) , or colorectal carcinoma. In some embodiments, the subject has Hodgkin's lymphoma. In some embodiments, the subject has triple-negative breast cancer (TNBC) , gastric cancer, urothelial cancer, Merkel-cell carcinoma, or head and neck cancer. In some embodiments, the cancer is melanoma, pancreatic carcinoma, mesothelioma, hematological malignancies, especially Non-Hodgkin's lymphoma, lymphoma, chronic lymphocytic leukemia, or advanced solid tumors.

In some embodiments, the compositions and methods disclosed herein can be used for treatment of patients at risk for a cancer. Patients with cancer can be identified with various methods known in the art.

In one aspect, the disclosure provides methods for treating, preventing, or reducing the risk of developing disorders associated with an abnormal or unwanted immune response, e.g., an autoimmune disorder, e.g., by affecting the functional properties of the APC cells (e.g., by blocking the interaction between CD40 and CD40L) . These autoimmune disorders include, but are not limited to, 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, Behcet's disease, pernicious anemia, bullous pemphigoid, polyarthritis nodosa, cardiomyopathy, polychondritis, celiac sprue-dermatitis, polyglandular syndromes, chronic fatigue syndrome (CFIDS) , polymyalgia rheumatica, chronic inflammatory demyelinating, polymyositis and dermatomyositis, chronic inflammatory polyneuropathy, primary agammaglobulinemia, Churg-Strauss syndrome, primary biliary cirrhosis, cicatricial pemphigoid, psoriasis, CREST syndrome, Raynaud's phenomenon, cold agglutinin disease, Reiter's syndrome, Crohn's disease, Rheumatic fever, discoid lupus, rheumatoid arthritis, Cryoglobulinemia sarcoidosis, fibromyalgia, scleroderma, Grave's disease,

syndrome, Guillain-Barre, stiff-man syndrome, Hashimoto's thyroiditis, Takayasu arteritis, idiopathic pulmonary fibrosis, temporal arteritis/giant cell arteritis, idiopathic thrombocytopenia purpura (ITP) , ulcerative colitis, IgA nephropathy, uveitis, diabetes (e.g., Type I) , vasculitis, lichen planus, and vitiligo. The 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 an abnormal or unwanted immune response associated with cell, tissue or organ transplantation, e.g., renal, hepatic, and cardiac transplantation, e.g., graft versus host disease (GVHD) , or to prevent allograft rejection. In some embodiments, the subject has Crohn's disease, ulcerative colitis or type 1 diabetes. In some embodiments, the subject has autoimmune thyroid disease, Grave’s disease, multiple sclerosis, psoriasis, inflammatory bowel disease (e.g., Crohn’s Disease (CD) and ulcerative colitis) , rheumatoid arthritis,

syndrome, autoimmune nephritis, or systemic lupus erythematosus. A relationship of CD40 and various autoimmune diseases are described e.g., 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) ; 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 are incorporated herein by reference in the entirety.

As used herein, by an “effective amount” is meant an amount or dosage sufficient to effect beneficial or desired results including halting, slowing, retarding, or inhibiting progression of a disease, e.g., an autoimmune disease or a cancer. An effective amount will vary depending upon, e.g., an age and a body weight of a subject to which the antibody, antigen binding fragment, antibody-encoding polynucleotide, vector comprising the polynucleotide, and/or compositions thereof is to be administered, a severity of symptoms and a route of administration, and thus administration can be determined on an individual basis.

An effective amount can be administered in one or more administrations. By way of example, an effective amount of an antibody or an antigen binding fragment is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow and/or delay progression of an autoimmune disease or a cancer in a patient or is an amount sufficient to ameliorate, stop, stabilize, reverse, slow and/or delay proliferation of a cell (e.g., a biopsied cell, any of the cancer cells described herein, or 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, inter alia, patient history as well as other factors such as the type (and/or dosage) of antibody used.

Effective amounts and schedules for administering the antibodies, antibody-encoding polynucleotides, and/or compositions disclosed herein may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage that must be administered will vary depending on, for example, the mammal that will receive the antibodies, antibody-encoding polynucleotides, and/or compositions disclosed herein, the route of administration, the particular type of antibodies, antibody-encoding polynucleotides, antigen binding fragments, and/or compositions disclosed herein used and other drugs being administered to the mammal. Guidance in selecting appropriate doses for antibody or antigen binding fragment can be found in the literature on therapeutic uses of antibodies and antigen binding fragments, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., 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.

A typical daily dosage of an effective amount of an antibody is 0.01 mg/kg to 100 mg/kg. In some embodiments, the dosage 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 dosage 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 dosage is about 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.9 mg/kg, 0.8 mg/kg, 0.7 mg/kg, 0.6 mg/kg, 0.5 mg/kg, 0.4 mg/kg, 0.3 mg/kg, 0.2 mg/kg, or 0.1 mg/kg.

In any of the methods described herein, the 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 the 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, the at least one antibody or antigen-binding fragment and the 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, the 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.

In some embodiments, the one or more additional therapeutic agents can be administered to the subject prior to, or after administering 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) . 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 bioactive period of the one or more additional therapeutic agents and the at least one antibody or antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein) in the subject.

In some embodiments, the subject can be administered 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) over an extended period of time (e.g., over a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years) . A skilled medical professional may determine the length of the treatment period using any of the methods described herein for diagnosing or following the effectiveness of treatment (e.g., the observation of at least one symptom of cancer) . As described herein, a skilled medical professional can also change the identity and number (e.g., increase or decrease) of antibodies or antigen-binding antibody fragments (and/or one or more additional therapeutic agents) administered to the subject and can also 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 the subject based on an assessment of the effectiveness of the treatment (e.g., using any of the methods described herein and known in the art) .

In some embodiments, one or more additional therapeutic agents can be administered to the subject. The additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of B-Raf, an EGFR inhibitor, an inhibitor of a MEK, an inhibitor of ERK, an inhibitor of K-Ras, an inhibitor of c-Met, an inhibitor of anaplastic lymphoma kinase (ALK) , an inhibitor of a phosphatidylinositol 3-kinase (PI3K) , an inhibitor of an Akt, an inhibitor of mTOR, a dual PI3K/mTOR inhibitor, an inhibitor of Bruton's tyrosine kinase (BTK) , and an inhibitor 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) .

In some embodiments, the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of HER3, an inhibitor of LSD1, an inhibitor of MDM2, an inhibitor of BCL2, an inhibitor of CHK1, an inhibitor of activated hedgehog signaling pathway, and an agent that selectively degrades the estrogen receptor.

In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of Trabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, Pazopanib, IMA-901, AGS-003, cabozantinib, Vinflunine, an Hsp90 inhibitor, Ad-GM-CSF, Temazolomide, IL-2, IFNa, vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomid, amrubicine, carfilzomib, pralatrexate, and enzastaurin.

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, tumor necrosis factor (TNF) alpha, 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 treatment targeting CX3CL1, a treatment targeting CXCL9, a treatment targeting CXCL10, a treatment targeting CCL5, an LFA-1 agonist, an ICAM1 agonist, and a Selectin agonist.

In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI are administered to the subject.

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, or an anti-GITR antibody.

Pharmaceutical Compositions and Routes of Administration

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

Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) . The compositions can include a sterile diluent (e.g., sterile water or saline) , a fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvents, antibacterial or antifungal agents, such as benzyl alcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, antioxidants, such as ascorbic acid or sodium bisulfite, chelating agents, such as ethylenediaminetetraacetic acid, buffers, such as acetates, citrates, or phosphates, and isotonic agents, such as sugars (e.g., dextrose) , polyalcohols (e.g., mannitol or sorbitol) , or salts (e.g., sodium chloride) , or any combination thereof. Liposomal suspensions can also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Patent No. 4,522,811) . Preparations of the compositions can be formulated and enclosed in ampules, disposable syringes, or multiple dose vials. Where required (as in, for example, injectable formulations) , proper fluidity can be maintained by, for example, 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, controlled 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. ) .

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

Toxicity and therapeutic efficacy of compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys) . One can, for example, determine the LD50 (the dose lethal to 50%of the population) and the ED50 (the dose therapeutically effective in 50%of the population) : the therapeutic index being the ratio of LD50: ED50. Agents that exhibit high therapeutic indices are preferred. Where an agent exhibits an undesirable side effect, care should be taken to minimize potential damage (i.e., reduce unwanted side effects) . Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

Data obtained from cell culture assays and animal studies can be used in formulating an appropriate dosage of any given agent for use in a subject (e.g., a human) . A therapeutically effective amount of the 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) will be an amount that treats the disease in a subject (e.g., kills cancer cells ) in a subject (e.g., a human subject identified as having cancer) , or a subject identified as being at risk of developing the disease (e.g., a subject who has previously developed cancer but now has been cured) , decreases the severity, frequency, and/or duration of one or more symptoms of a disease in a subject (e.g., a human) . The effectiveness and dosing of any of the antibodies or antigen-binding fragments described herein can be determined by a health care professional or veterinary professional using methods known in the art, as well as by the observation of one or more symptoms of disease in a subject (e.g., a human) . Certain factors may influence 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) .

Exemplary doses include milligram or microgram amounts of any of the antibodies or antigen-binding fragments described herein per kilogram of the subject’s 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 broad range, one of ordinary skill in the art will understand that therapeutic agents, including antibodies and antigen-binding fragments thereof, vary in their potency, and effective amounts can be determined by methods known in the art. Typically, relatively low doses are administered at first, and the attending health care professional or veterinary professional (in the case of therapeutic application) or a researcher (when still working at the development stage) can subsequently and gradually increase the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and the half-life of the antibody or antibody fragment in vivo.

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

EXAMPLES

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

Example 1. Generating Mouse Anti-hCD40 Antibodies

To generate mouse antibodies against human CD40 (hCD40; SEQ ID NO: 73) , 6-8 weeks old female BALB/c mice were immunized with human CD40. Anti-hCD40 antibodies were collected by the methods as described below and shown in FIG. 1 and FIG. 2.

Immunization of mice

6-8 weeks old female BALB/c mice were immunized with His-tagged human CD40 proteins at 20 μg/mouse at a concentration of 100 μg/ml. The His-tagged human CD40 proteins were emulsified with adjuvant and injected at four positions on the back of the mice. For the first subcutaneous (s. c. ) injection, the diluted antigen was emulsified with Complete Freund’s Adjuvant (CFA) in equal volume. In the following subcutaneous injections, the protein was emulsified with Incomplete Freund’s Adjuvant (IFA) in equal volume. Three days after the third injection or the booster immunization, blood (serum) was collected and analyzed for antibody titer using ELISA.

In another experiment, 6-8 weeks old female BALB/c mice were immunized by injecting the expression plasmid encoding human CD40 into the mice. The plasmids encoding the antigen were injected into the tibialis anterior muscle (intramuscular injection; i.m. injection) of the mice by using gene guns at the concentration of 1000 μg/ul at 60 μg per mouse. At least four injections were performed with at least 14 days between two injections. Blood (serum) was collected seven days after the last immunization and the serum was tested for antibody titer by ELISA.

Procedures to enhance immunization were also performed at least fourteen days after the previous immunization (either by injecting the plasmid or by injecting the proteins) . CHO cells that express CD40 antigen on the surface were intravenously injected into the mice through tail veins. Spleen was then collected four days after the injection.

Fusion of SP2/0 cells and spleen cells

Spleen tissues were grinded. Spleen cells were first selected by CD3ε Microbeads and Anti-Mouse IgM Microbeads, and then fused with SP2/0 cells. The cells were then plated in 96-well plates with hypoxanthine-aminopterin-thymidine (HAT) medium.

Primary screening of hybridoma

Primary screening of the hybridoma supernatant in the 96-well plates was performed using Fluorescence-Activated Cell Sorting (FACS) pursuant to standard procedures. Chinese hamster ovary (CHO) cells were added to 96-well plates (2 × 10 ⁴ cells per well) before the screening. 50 μl of supernatant was used. The antibodies that were used in experiments were

(1) Fluorescein (FITC) -conjugated AffiniPure F (ab) ₂ Fragment Goat Anti-Mouse IgG, Fcγ Fragment Specific, and

(2) Alexa

647-conjugated AffiniPure F (ab) ₂ Fragment Goat Anti-Human IgG, Fcγ Fragment Specific.

Sub-cloning

Sub-cloning was performed using ClonePix2. In short, the positive wells identified during the primary screening were transferred to semisolid medium, and IgG positive clones were identified and tested. FITC anti-mouse IgG Fc antibody was used.

Ascites fluid antibodies

1 × 10 ⁶ positive hybridoma cells were injected intraperitoneally (i.p. ) to B-NDG ^TM mice (Beijing Biocytogen, Beijing, China; Cat#B-CM-002) . Monoclonal antibodies were produced by growing hybridoma cells within the peritoneal cavity of the mouse. The hybridoma cells multiplied and produced ascites fluid in the abdomens of the mice. The fluid contained a high concentration of antibody which can be harvested for later use.

Purification of antibodies

Antibodies in ascites fluid were purified using GE AKTA protein chromatography (GE Healthcare, Chicago, Illinois, United States) . 11-1B10 ( “1B10” ) , 13-9B1 ( “9B1” ) , 13-4D1 ( “4D1” ) , 13-9F5 ( “9D7” ) , 20-2A7 ( “2A7” ) , 11-6G1 ( “6G1” ) , 13-8C6 ( “8C6” ) , 11-5B2 ( “5B2” ) , 11-10A5 ( “10A5” ) , and 06-9B9 ( “9B9” ) were among the mouse antibodies produced by the methods described above.

The VH, VL and CDR regions of the antibodies were determined. The heavy chain CDR1, CDR2, CDR3, and light chain CDR1, CDR2, and CDR3 amino acid sequences of 1B10 are shown in SEQ ID NOs: 1-6 (Kabat numbering) or SEQ ID NOs: 37-42 (Chothia numbering) .

The heavy chain CDR1, CDR2, CDR3, and light chain CDR1, CDR2, and CDR3 amino acid sequences of 9B1 are shown in SEQ ID NOs: 7-12 (Kabat numbering) or SEQ ID NOs: 43-48 (Chothia numbering) .

The heavy chain CDR1, CDR2, CDR3, and light chain CDR1, CDR2, and CDR3 amino acid sequences of 4D1 are shown in SEQ ID NOs: 13-18 (Kabat numbering) or SEQ ID NOs: 49-54 (Chothia numbering) .

The heavy chain CDR1, CDR2, CDR3, and light chain CDR1, CDR2, and CDR3 amino acid sequences of 9F5 are shown in SEQ ID NOs: 19-24 (Kabat numbering) or SEQ ID NOs: 55-60 (Chothia numbering) .

The heavy chain CDR1, CDR2, CDR3, and light chain CDR1, CDR2, and CDR3 amino acid sequences of 2A7 are shown in SEQ ID NOs: 25-30 (Kabat numbering) or SEQ ID NOs: 61-66 (Chothia numbering) .

The heavy chain CDR1, CDR2, CDR3, and light chain CDR1, CDR2, and CDR3 amino acid sequences of 6G1 are shown in SEQ ID NOs: 31-36 (Kabat numbering) or SEQ ID NOs: 67-72 (Chothia numbering) .

Example 2. Humanization of mouse antibodies

The starting point for humanization was the mouse antibodies (e.g., 2A7, and 4D1) . The amino acid sequences for the heavy chain variable region and the light chain variable region of these mouse antibodies were determined.

Four humanized heavy chain variable region variants (SEQ ID NOs: 90-93) and three humanized light chain variable region variants (SEQ ID NOs: 94-96) for 2A7 were constructed, containing different modifications or substitutions.

Three humanized heavy chain variable region variants (SEQ ID NOs: 97-99) and three humanized light chain variable region variants (SEQ ID NOs: 100-102) for 4D1 were constructed, containing different modifications or substitutions.

These humanized heavy chain variable region variants can be combined with any of the humanized light chain variable region variants based on the same mouse antibody. For example, 2A7-H4 (SEQ ID NO: 93) can be combined with any humanized light chain variable region variant based on the same mouse antibody 2A7 (e.g., 2A7-K2 (SEQ ID NO: 95) ) , and the antibody is labeled accordingly (e.g., 2A7-H4K2) .

Expression levels of combinations of the humanized heavy chain variable region variants (H1, H2, H3, and H4) paired with the humanized light chain variable region variants (K1, K2, and K3) based on the antibody 2A7 were determined using Biacore (Biacore, INC, Piscataway N. J. ) T200 biosensor. The experiments were repeated once. The results are summarized in the table below.

Table 2

According to the results, expression levels of humanized anti-hCD40 antibodies with the H1 or H2 variable regions were relatively high. In contrast, expression levels of the humanized antibodies with the H4 variable region were significantly lower than antibodies comprising the other heavy chain variable region variants. No significant variance was observed between antibodies with different light chain variable region variants, which had little effect on antibody expression. Overall, the expression levels of H1K1, H1K2, H2K1, and H2K2 were relatively high.

Example 3. In vitro testing of the mouse anti-hCD40 antibodies

The anti-hCD40 antibodies were collected from mouse ascites fluid and purified by chromatography. 25 μl CHO cells transiently transfected with human CD40 were added to each well in a plate. The purified antibodies were titrated to final concentrations of 10, 1, 0.1, 0.01, and 0.001 μg/ml. The titrated antibodies were added to each well at 25 μl per well at 4 ℃ and incubated for 30 minutes.

After being washed with phosphate-buffered saline (PBS) twice (1200 rpm; 5 min) , 50 μl of FITC labeled anti-mouse IgG Fc antibody (anti-mIgG Fc-FITC) at 1: 100 dilution was added into each well, and incubated for 30 minutes at 4 ℃, followed by PBS wash. The signals for FITC was determined by flow cytometry.

As shown in FIG. 3, when the concentration of the mouse anti-hCD40 antibodies 13-4D1 and 13-9F5 increased, the signal for cells binding to the mouse anti-hCD40 antibodies increased, suggesting that the anti-hCD40 antibodies can bind to cells express human CD40.

Example 4. Cross-reactivity of anti-hCD40 antibodies against monkey, mouse, and human-mouse chimeric CD40

In each experiment, the CHO cells were transfected with mouse CD40 (mCD40, SEQ ID NO: 74) , monkey (rhesus macaque) CD40 (rmCD40, SEQ ID NO: 75) , or chimeric (mouse and human) CD40 (chiCD40, SEQ ID NO: 76) .

25 μl CHO cells were added to each well. 25 μl purified anti-hCD40 antibodies (1 μg/ml) (4D1 or 9F5) were added to each well and were incubated at 4 ℃ for 30 minutes.

After being washed with PBS (1200 rpm, 5 min) twice, 50 μl of FITC labeled anti-mouse IgG Fc antibody (anti-mIgG Fc-FITC) was added into each well at 1: 100 dilution, followed by incubating at 4 ℃ for 30 minutes, and then PBS wash (1200 rpm, 5 min) . The signals for FITC were detected by flow cytometry.

As shown in FIG. 4, 4D1 and 9F5 did not cross react with mouse CD40, but had strong cross reactivity with rmCD40 and chimeric CD40. In FIG. 4, NC stands for negative control. The tables below summarize the results from the cross-reactivity studies.

Table 3

The cross-reactivity with monkey, mouse, and human-mouse chimeric CD40 were also tested for several chimeric antibodies. The chimeric antibodies have the heavy chain variable domain and the light chain variable domain from the corresponding mouse anti-hCD40 antibodies, with the constant domains from human antibody (including, e.g., the CL, CH1, CH2, and CH3 domains) . The term mHvKv indicates mouse heavy chain variable region and mouse light chain variable region. As shown in Table 4, 13-9B1-mHvKv-IgG2, 11-1B10-mHvKv-IgG2, 20-2A7-mHvKv-IgG2, and 11-6G1-mHvKv-IgG2 can bind to rmCD40 and chiCD40. However, they cannot bind to mCD40.

Table 4

Example 5. Binding affinity of anti-hCD40 antibodies

Determination of binding affinity to human CD40

The binding affinity of the anti-hCD40 antibodies were measured using surface plasmon resonance (SPR) using Biacore (Biacore, INC, Piscataway N.J. ) T200 biosensor equipped with pre-immobilized Protein A sensor chips.

Anti-hCD40 antibodies were collected by transfecting CHO-Scells and then purified. The antibodies (1 μg/mL) were injected into Biacore T200 biosensor at 10 μL/min for about 33 seconds to achieve a desired protein density (e.g., about 55.8 response units (RU) ) . Histidine-tagged human CD40 proteins (hCD40-His) at the concentration of 200, 50, 12.5, 3.125, or 0.78125 nM were then injected at 30 μL/min for 60 seconds. Dissociation was monitored for 120 seconds. The chip was regenerated after the last injection of each titration with Glycine (pH 2.0, 30 μL/min for 12 seconds) .

Kinetic association rates (kon) and dissociation rates (koff) were obtained simultaneously by fitting the data globally to a 1: 1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. Methods Enzymology 6.99-110) using Biacore T200 Evaluation Software 3.0. Affinities were deduced from the quotient of the kinetic rate constants (KD=koff/kon) .

As a person of ordinary skill in the art would understand, the same method with appropriate adjustments for parameters (e.g., antibody concentration) was performed for each tested antibody. The results for the tested antibodies are summarized in the table below.

Table 5

The antibodies tested, including, 6G1-mHvKv-IgG2, 1B10-mHvKv-IgG2, and 9B1-mHvKv-IgG2, and 2A7-mHvKv-IgG2 are chimeric anti-hCD40 antibodies; 2A7-H1K1-IgG2, 2A7-H1K2-IgG2, 2A7-H1K3-IgG2, 2A7-H2K1-IgG2, 2A7-H2K2-IgG2, 2A7-H2K3-IgG2, 2A7-H3K1-IgG2, 2A7-H3K2-IgG2, 2A7-H3K3-IgG2, 2A7-H4K1-IgG2, 2A7-H4K2-IgG2, and 2A7-H4K3-IgG2 are humanized anti-hCD40 antibodies. The name and the sequences of the chimeric anti-CD40 antibodies are summarized in Table 1. Lucatumumab is a human monoclonal antibody against CD40. It was included in the experiment form comparison purpose. The results show that these chimeric and humanized antibodies have very high binding affinity with human CD40.

Determination of binding affinity to human and monkey CD40

Anti-hCD40 antibodies were collected by transfecting CHO-Scells and then culture supernatants comprising the antibodies were collected. The supernatants were diluted 25-50 folds and then injected into Biacore T200 biosensor at 10 μL/min for about 30 seconds to achieve a desired protein density. Histidine-tagged human CD40 proteins (hCD40-His) or monkey (Rhesus macaque) CD40 protein (rmCD40-His) at the concentrations of 200 nM to 0.1953125 nM were then injected at 30 μL/min for 180 seconds. Dissociation was monitored for 400 seconds. The chip was regenerated after the last injection of each titration with Glycine (pH 2.0, 30 μL/min for 20 seconds) .

Kinetic association rates (kon) and dissociation rates (koff) were obtained simultaneously by fitting the data globally to a 1: 1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. Methods Enzymology 6.99-110) . Affinities were deduced from the quotient of the kinetic rate constants (KD=koff/kon) .

Table 6

The antibodies tested, including, 20-2A7-mHvKv-IgG2, 20-2A7-mHvKv-IgG2, 13-9F5-mHvKv-IgG1, 13-9F5-mHvKv-IgG1, 13-4D1-mHvKv-IgG1, 13-4D1-mHvKv-IgG1, 11-1B10-mHvKv-IgG2, and 11-1B10-mHvKv-IgG2 are chimeric anti-hCD40 antibodies. They have mouse heavy chain and light chain variable domains, and human IgG1 or IgG2 constant domains. The results showed that after binding with rmCD40, 20-2A7-mHvKv-IgG2 had a high dissociation rate, which reduced its overall binding affinity. 13-9F5-mHvKv-IgG1 exhibited relatively high binding affinities with both hCD40 and rmCD40. 13-4D1-mHvKv-IgG1 exhibited relatively low binding affinities with both hCD40 and rmCD40, with a high dissociation rate. 11-1B10-mHvKv-IgG2 had a relatively low binding affinity to rmCD40.

Example 6. In vivo testing of mouse and chimeric anti-hCD40 antibodies

In order to test the anti-hCD40 antibodies in vivo and to predict the effects of these antibodies in human, a humanized CD40 mouse model was generated. The humanized CD40 mouse model was engineered to express a chimeric CD40 protein (SEQ ID NO: 76) wherein a part of the extracellular region of the mouse CD40 protein was replaced with the corresponding human CD40 extracellular region. The amino acid residues 20-192 of mouse CD40 (SEQ ID NO: 74) were replaced by amino acid residues 20-192 of human CD40 (SEQ ID NO: 73) . The humanized mouse model (B-hCD40 mice) provides a new tool for testing new therapeutic treatments in a clinical setting by significantly decreasing the difference between clinical outcome in human and in ordinary mice expressing mouse CD40. A detailed description regarding humanized CD40 mouse model can be found in PCT/CN2018/091845, which is incorporated herein by reference in its entirety.

The anti-hCD40 antibodies were tested for their effect on tumor growth in vivo in a model of colon carcinoma. MC-38 cancer tumor cells (colon adenocarcinoma cell) were injected subcutaneously in B-hCD40 mice. When the tumors in the mice reached a volume of 150±50 mm ³, the mice were randomly placed into different groups based on the volume of the tumor (five mice in each group) (day 0) .

The mice were then injected with physiological saline (PS) and anti-hCD40 antibodies by intraperitoneal administration (i.p. ) . The antibody was given on the second day and the fifth day of each week for 3 weeks (6 injections in total) .

The injected amount was calculated based on the weight of the mouse at 3 mg/kg. The length of the long axis and the short axis of the tumor were measured and the volume of the tumor was calculated as 0.5 × (long axis) × (short axis) ². The weight of the mice was also measured before the injection, when the mice were placed into different groups (before the first antibody injection) , twice a week during the antibody injection period, and before euthanization.

The tumor growth inhibition percentage (TGI%) was calculated using the following formula: TGI (%) = [1- (Ti-T0) / (Vi-V0) ] ×100. Ti is the average tumor volume in the treatment group on day i. T0 is the average tumor volume in the treatment group on day zero. Vi is the average tumor volume in the control group on day i. V0 is the average tumor volume in the control group on day zero.

T-test was performed for statistical analysis. A TGI%higher than 60%indicates significant suppression of tumor growth. P < 0.05 is a threshold to indicate significant difference.

In vivo results for mouse anti-hCD40 antibodies

To analyze the first set of mouse anti-hCD40 antibodies, , B-hCD40 mice in each group were injected with physiological saline (PS) as a negative control (G1) , the mouse anti-hCD40 antibody 13-4D1 (G2; 3 mg/kg) , the mouse anti-hCD40 antibody 13-8C6 (G3; 3 mg/kg) , the mouse anti-hCD40 antibody 11-1B10 (G4; 3 mg/kg) , the mouse anti-hCD40 antibody 13-9B1 (G5; 3 mg/kg) , the mouse anti-hCD40 antibody 13-9F5 (G6; 3 mg/kg) , or the mouse anti-hCD40 antibody 20-2A7 (G7; 3 mg/kg) , or Selicrelumab as a positive control (G8; 3 mg/kg) .

The weight of the mice was monitored during the entire treatment period (FIG. 5, and FIG. 6) . Not much difference in weight was observed among these groups. The results showed that the anti-hCD40 antibodies were well tolerated and not toxic to the mice.

The tumor size in groups treated with 13-8C6 and Selicrelumab increased to a lesser extent compared to the control group (FIG. 7) and other antibody treatment groups. The data further suggest that other antibodies can suppress immune response.

The TGI%at day 19 (28 days after injection of MC-38 cells) was also calculated as shown in the table below.

Table 7

A second set of mouse anti-hCD40 antibodies were also tested. B-hCD40 mice in each group were injected with physiological saline (PS) as a negative control (G1) , the mouse anti-hCD40 antibody 11-5B2 (G2; 3 mg/kg) , the mouse anti-hCD40 antibody 11-10A5 (G3; 3 mg/kg) , the mouse anti-hCD40 antibody 11-1B10 (G4; 3 mg/kg) , the mouse anti-hCD40 antibody 11-6G1 (G5; 3 mg/kg) , or the mouse anti-hCD40 antibody 13-9B1 (G6; 3 mg/kg) .

The weight of the mice was monitored during the entire treatment period (FIG. 8, and FIG. 9) . The body weight of mice in G5 slightly decreased at day 21. Not much difference in weight was observed among the remaining groups. The results showed that most anti-hCD40 antibodies were tolerated and not toxic to the mice.

The tumor size in groups treated with 11-6G1 and 11-10A5 increased to a lesser extent compared to the control group (FIG. 10) and other antibody treatment groups. The data further suggest that 13-9B1 and 11-1B10 can suppress immune response.

The TGI%at day 20 (27 days after injection of MC-38 cells) was also calculated as shown in the table below.

Table 8

In vivo results for chimeric anti-hCD40 antibodies

Chimeric anti-hCD40 antibodies 11-5B2-mHvKv-IgG2 (G3) , 06-6A7-mHvKv-IgG2 (G4) , and 11-6G1-mHvKv-IgG2 (G5) were administered into B-hCD40 mice (humanized CD40 mice) by intraperitoneal administration. Physiological saline was injected as a negative control (Group 1, G1) , and selicrelumab was used as a positive control (Group 2, G2)

The injected amount of the antibodies was calculated based on the weight of the mouse at 3 mg/kg. The antibodies were given on the first day and the fourth day of each week (6 injections in total) .

The weight of the mice was monitored during the entire treatment period. The weight of mice in different groups all increased (FIG. 11, and FIG. 12) . No clear difference in weight was observed among the different groups. The results showed that the anti-hCD40 antibodies were well tolerated and not toxic to the mice.

The tumor size showed significant difference in groups treated with certain chimeric antibodies compared to the control group (FIG. 13) .

The TGI%at day 23 (31 days after grouping) for each treatment group was calculated as shown in the table below.

Table 9

The results showed that chimeric antibodies 11-6G1-mHvKv-IgG2, 06-6A7-mHvKv-IgG2 and 11-5B2-mHvKv-IgG2 significantly inhibited tumor growth.

Example 7: Immune-suppressing effects of mouse anti-hCD40 antibodies

Experiments were performed to analyze the effects of the mouse anti-hCD40 antibodies on immune responses. Ovalbumin (OVA) was used as an antigen to stimulate immune responses in the humanized CD40 mouse model (B-hCD40 mice) . Briefly B-hCD40 mice (6-8 weeks old) were divided into 6 groups (Groups 1-6; 5 mice per group) , with Group 1 receiving physiological saline ( “PS” ) as a negative control, and Groups 2-6 receiving mouse anti-hCD40 antibodies 06-6A7, 13-4D1, 13-9F5, 20-2A7, 06-9B9 at 3mg/kg body weight, respectively. The physiological saline or mouse anti-hCD40 antibodies were administered intraperitoneally every first and fourth day of the week for four weeks (8 times total) . In all six groups, one dose of OVA at 100 μg/animal (0.1 ml of 1000 μg/mL, BioSS, Beijing, Cat#bs-0283P) and an immunologic adjuvant (QuickAntibody ^TM ( “QA” ) ; Cat#KX0210041; Beijing Biodragon Immunotechnologies, Inc. ) were administered intramuscularly along with intraperitoneal administration of the third dose of PS or anti-hCD40 antibodies. Following the ninth dose of PS or mouse anti-hCD40 antibody administration serum was collected from each animal and subjected to ELISA analysis. A second round of immunization with the OVA and QA was carried out on the first day of week 6 and serum was collected for ELISA analysis 14 days after OVA administration at week 8. FIG. 14 shows the experiment protocol. OVA was embedded on the ELISA plate. Goat Anti-Mouse IgG H&L (HRP) (ab97040) was used for ELISA analysis.

As shown in FIGS. 15A-15B, during the first round OVA administration, anti-hCD40 antibodies 20-2A7, 13-4D1, and 13-9F5 reduced immune response in B-hCD40 mice as compared to control, whereas 06-6A7 increased immune response. FIGS. 16A-16B show the ELISA results for the serum collected at week 8 after the second round of OVA administration. The results were similar to the first round of OVA administration. 20-2A7, 13-4D1, and 13-9F5 reduced the immune response in the subjects.

The table below shows a summary of the anti-tumor activity and immune-suppressing effects of the antibodies 06-6A7, 13-4D1, 13-9F5, 20-2A7, and 06-9B9. Consistent with the pharmacodynamics and pharmacokinetics studies, mouse anti-hCD40 antibodies that induced tumor growth can reduce immune response, and those that inhibited tumor growth can increase immune response.

Table 10

Effects of frequency of administration

A second set of experiments was carried out where B-hCD40 mice (6-8 weeks old) were divided into 10 groups, with Group 1 receiving physiological saline (PS) as a negative control, and Groups 2-10 receiving mouse anti-hCD40 antibodies 06-6A7, 06-9B9, 13-4D1, 13-9F5, 20-2A7, 13-8C6, 11-1B10, 13-9B1, and 20-2C12 at 3mg/kg body weight, respectively. The table below shows a summary of the experiment setup. FIG. 17 shows the experiment protocol, where OVA and adjuvant (e.g., incomplete Freund's adjuvant (IFA) (Freund’s Adjuvant, Incomplete; Cat#F5506; Sigma-Aldrich China, Inc. ) or complete Freund's adjuvant (CFA) ) (Freund’s Adjuvant, Complete; Cat#F5881; Sigma-Aldrich China, Inc. ) were administered twice subcutaneously.

Table 11

OVA was embedded on the ELISA plate. Goat Anti-Mouse IgG H&L (HRP) (Cat#ab97040) was used for ELISA analysis. FIGS. 18A-18B show ELISA results of each tested antibody. At 1: 100 dilutions, mouse anti-hCD40 antibodies 13-9F5, 13-4D1, 20-2A7-1B1, 11-1B10, and 13-9B1 significantly reduced immune response as compared to the control.

Testing effects of different adjuvants

B-hCD40 mice (6-8 weeks old) were divided into 6 groups, with Groups 1-3 receiving OVA and Freund's adjuvant (FA) , and Groups 4-6 receiving OVA and the excipient QA (QuickAntibody ^TM) . The table below shows a summary of the experimental setup. OVA/FA was administered twice at 100 μg/animal subcutaneously, and OVA/QA was administered twice at 100 μg/animal intramuscularly. FIG. 19 shows the experimental protocol.

Table 12

As shown in FIG. 20, both 20-2A7 and 13-9F5 can suppress immune response. At week 5 (six days after the second OVA administration) , serum was collected for ELISA analysis. As shown in FIG. 21, the immune response increased after the second OVA administration in the control group, suggesting that the second antigen administration increased immune response. The result shows both 20-2A7 and 13-9F5 can suppress immune response after the second OVA administration and the effects were not affected by the frequency of administration or the adjuvants that were used.

Example 8: Thermal stability measurement of anti-hCD40 antibodies

Measuring Thermal Stability by Kits

Thermal stability of humanized anti-hCD40 antibodies 2A7-H1K2-IgG4-FLAA, 2A7-H2K2-IgG4-FLAA, 2A7-mHvKv-IgG2, and lucatumumab were measured by a Protein Thermal Shift ^TM Dye Kit using QuantStudio ^TM 5 Real Time PCR Systems. In a 96-well plate, each reaction system (20 μl) was built according to the table below.

Table 13

Reaction Component

Volume (μl)

Protein Thermo Shift Buffer	5
Antibody	4
Water	10.5
Diluted Protein Thermal Shift Dye	2.5

Reactions were performed continuously in two steps. Specifically, the first step was carried out at 1.6℃ per second at 25℃ for 2 minutes and the second step was carried out at 0.05℃ per second at 99℃ for 2 minutes. Melting temperature (Tm) of each anti-hCD40 antibody was determined, as shown in the table below.

Table 14

Protein	Tm (℃)
2A7-H1K2-IgG4-FLAA	71.21
2A7-H2K2-IgG4-FLAA	71.46
2A7-mHvKv-IgG2	65.53
Lucatumumab	69.94

The results showed that Tm of 2A7-H1K2-IgG4-FLAA and 2A7-H2K2-IgG4-FLAA were higher than the Tm of Lucatumumab. 2A7-mHvKv-IgG2 had the lowest Tm of the four antibodies.

Measuring Thermal Stability by FACS

Fluorescence-Activated Cell Sorting (FACS) was applied for measuring thermal stability of the humanized anti-hCD40 antibodies. Culture supernatant (day 3) from transiently transfected cells expressing the humanized antibodies were collected. The measurement was performed as follows. 25 μl CHO cells transiently transfected with human CD40 were added to each well in a plate. The supernatants were titrated to final concentrations of 1, 0.1, and 0.01 μg/ml. The titrated supernatants were added to each well at 25 μl per well at 4℃ (control) or 70℃, then incubated for 30 minutes.

After being washed with phosphate-buffered saline (PBS) twice (1200 rpm; 5 min) , 50 μl of Alexa Fluor 647 labeled anti-human IgG Fc antibody (Alexa

647 AffiniPure F (ab') ₂ Fragment Goat Anti-Human IgG, Fcγ fragment specific; Jackson ImmunoResearch Inc., Catalog number 109-606-170) at 1: 500 dilution was added into each well, and incubated for 30 minutes at 4 ℃, followed by PBS wash. The signals for FITC was determined by flow cytometry.

As shown in FIG. 22, 2A7-H1K2-IgG4-FLAA and 2A7-H2K2-IgG4-FLAA maintained high binding activities (e.g., at 1 μg/ml) when heated at 70℃ for 5 minutes. In contrast, lucatumumab lost its binding activity (e.g., at 1 μg/ml) at the same temperature.

Example 9: EC ₅₀ determination of anti-hCD40 antibodies binding to PBMC

EC ₅₀ values of anti-hCD40 antibodies were determined by detecting the binding of anti-hCD40 antibodies to the endogenous CD40 on the cell surface of human PBMC (peripheral blood mononuclear cell) . The determined EC ₅₀ indicates the binding ability of anti-hCD40 antibodies to endogenous CD40, and provides support for subsequent drug development. B cells in human PBMC has a high level of endogenous CD40 expression, and were used to detect the binding activity of anti-hCD40 antibodies (2A7) . More specifically, anti-hCD40 antibodies 2A7-H1K2-IgG4-FLAA, 2A7-H2K2-IgG4-FLAA and hIgG4 (Human IgG4 kappa Isotype Control; Crown Bioscience Inc., Catalog number C0004) were respectively labelled with Alexa Fluor 647 fluorescent dye (Alexa Fluor ^TM 647 NHS Ester (Succinimidyl Ester) ; Thermo Fisher Scientific, Catalog number A20106-1MG) , and then titrated to final concentrations of 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, and 1.95 ng/ml. The titrated antibodies were added to each well to incubate with PBMC at 4℃ for 30 minutes. Flow cytometry was used to detect the binding level of different concentrations of antibodies to PBMC endogenous CD40. Results are shown in FIG. 23 and the table below. The determined EC ₅₀ value of 2A7-H1K2-IgG4-FLAA and 2A7-H2K2-IgG4-FLAA were 236.6 and 140.3 ng/ml, respectively.

Table 15

Antibody Name	EC ₅₀ (ng/ml)
2A7‐H1K2‐IgG4‐FLAA	236.6
2A7‐H2K2‐IgG4‐FLAA	140.3
Isotype control	/

Example 10: Endocytosis of anti-hCD40 antibodies

In one experiment, anti-hCD40 antibodies 2A7-H1K2-IgG4-FLAA, 2A7-H2K2-IgG4-FLAA and hIgG4 (Human IgG4 kappa Isotype Control; Crown Bioscience Inc., Catalog number C0004) were respectively labelled using an antibody labelling kit (pHAb Amine Reactive Dye; Promega, Catalog number G9845) . 5 × 10 ⁵ CHO-K1-hCD40 cells (CHO-K1 cells transiently transfected with human CD40) were seeded to each well in a 6-well plate and incubated at 37℃ overnight. When cell confluence reached about 60%, the labelled antibodies were added to the wells (at a final concentration of 5 nM) and incubated with the CHO-K1-hCD40 cells at 4℃ for 30 minutes. After the incubation, the cells were washed with PBS three times. 2 ml of CHO cell medium was added to each well, followed by an incubation at 37℃ for 3, 6, or 24 hours, or at 4℃ for 0 hour (as a negative control) . Then, the cells were digested and subjected for FACS analysis. Results are shown in FIG. 24.

In another experiment, anti-hCD40 antibodies 2A7-H1K2-IgG4-FLAA, 2A7-H2K2-IgG4-FLAA and hIgG4 (Human IgG4 kappa Isotype Control; Crown Bioscience Inc., Catalog number C0004) were respectively labelled using the antibody labelling kit as described herein. 2.5 × 10 ⁶ Raji cells were seeded in a 10 mm dish and incubated at 37℃ overnight. When the cells were in good conditions (e.g., even distribution) , they were collected by centrifugation. Then, the labelled antibodies were diluted to a final concentration of 5 nM in 5 ml cell culture medium, which was then used to resuspend the centrifuged cells. The resuspended cells were incubated at 4℃ for 30 minutes, then diluted with 5 ml PBS in a 15 ml centrifuge tube, followed by centrifugation at 130 g for 10 minutes. The cells were then resuspended by 10 ml cell culture medium, and added to a 6-well plate at 2 ml per well. The cells were incubated at 37℃ for 3, 6, or 24 hours, or at 4℃ for 0 hour (as a negative control) . Then, the cells were collected, washed by PBS, and subjected for FACS analysis. Results are shown in FIG. 25.

In summary, both 20-2A7-H1K2-IgG4-FLAA and 20-2A7-H2K2-IgG4-FLAA can undergo endocytosis after binding to human CD40 on cell surface with similar endocytosis rates. The anti-hCD40 antibodies had basically completed endocytosis at 6 hours, and the endocytosis signal decreased at 24 hours. The transfected CHO cells and the Raji cells (expressing endogenous CD40) exhibited comparable rates of CD40 antibody endocytosis, but the endocytosis signal representation was quite different. In addition, the isotype control did not exhibit any endocytosis signals by the CHO cells or the Raji cells, eliminating interferences of the labelling dyes on endocytosis.

Example 11: Immune-suppressing effects of humanized anti-hCD40 antibodies

Experiments were performed to analyze the effects of the humanized anti-hCD40 antibodies on immune responses. Ovalbumin (OVA) was used as an antigen to stimulate immune responses in the humanized CD40 mouse model (B-hCD40 mice) . Briefly B-hCD40 mice (6-8 weeks old) were divided into 4 groups (Groups 1-4; 10 mice per group) , with Group 1 receiving physiological saline ( “PS” ) as a negative control, Group 2 receiving mouse anti-hCD40 antibody 20-2A7, and Groups 3-4 receiving humanized anti-hCD40 antibodies 2A7-H1K2-IgG4 and 2A7-H2K2-IgG4 at 3mg/kg body weight, respectively. The physiological saline or mouse anti-hCD40 antibodies were administered intraperitoneally on the fourth day of the first week, and every first and fourth day of the following four weeks (9 times total) . In all four groups, one dose of OVA at 100 μg/animal (0.1 ml of 1000 μg/mL, BioSS, Beijing, Cat#bs-0283P) and an immunologic adjuvant (QuickAntibody ^TM ( “QA” ) ; Cat#KX0210041; Beijing Biodragon Immunotechnologies, Inc. ) were administered intramuscularly along with intraperitoneal administration of the third dose of PS or anti-hCD40 antibodies. A second round of immunization with the OVA and QA was carried out on the fourth day of week 4 and serum was collected for ELISA analysis 7 days after OVA administration at week 5. FIG. 26 shows the experiment protocol. OVA was embedded on the ELISA plate. Goat Anti-Mouse IgG H&L (HRP) (ab97040) was used for ELISA analysis.

FIGS. 27A-27D showed the ELISA results for the serum collected at week 5 after the second round of OVA administration. The results indicated that after the second round of OVA administration, 2A7-H1K2-IgG4 and 2A7-H2K2-IgG4 exhibited good humoral immuno-suppressive effects.

Example 12: In vivo immune system recovery after anti-hCD40 antibody administration

32 days after the PS or anti-hCD40 antibody administration, the immunized B-hCD40 mice in Example 11 were used for re-immunization after the anti-hCD40 antibodies were cleared from the mouse body. Experiments were performed to investigate whether humoral immunity of the B-hCD40 mice can return to normal after the anti-hCD40 administration was stopped, and whether specific antibodies can be produced in these mice.

Briefly, the Group 1 mice (10 mice) in Example 11 were re-grouped into two groups (Group 1 and Group 2; 5 mice per group) , and the Group 2 mice (10 mice) in Example 11 were re-grouped into three groups (4 mice in

Group

3, 3 mice in

Group

4 and 3 mice in Group 5) . The physiological saline (PS) or mouse anti-hCD40 antibody 20-2A7 (at 3 mg/kg) were administered intraperitoneally every first and fourth day of the week for 2 weeks (4 times total) in Group 2 and Group 5. In Groups 1-3 and 5, one dose of OVA at 100 μg/animal (BioSS, Beijing, Cat#bs-0283P) emulsified with Incomplete Freund’s Adjuvant (IFA) in equal volume were administered intramuscularly on the fourth day of the first week. On the first day of the first week and the fourth day of the second week, serum was collected from each animal and subjected to ELISA analysis. The table below shows the experiment protocol.

Table 16

As shown in FIGS. 28A-28B, the comparison between Group 5 and Group 3 shows that after the anti-hCD40 antibody administration was stopped for 32 days, the immune function of the B-hCD40 mice continued to be suppressed when the mice were re-immunized and re-administered with the anti-hCD40 antibodies. In addition, the comparison between Group 4 and Group 5 shows that after the anti-hCD40 antibody administration was stopped for 32 days, and clearance of the anti-hCD40 antibodies from the mouse body, the CD40/CD40L pathway and T-cell-dependent humoral immune function were recovered. Either re-immunizing by OVA, or relying on previous immunizations, activated the immune system and the mice produced specific antibodies. Further, the results demonstrated that the T cell-dependent humoral immunosuppressive effect of the mouse anti-hCD40 antibody 20-2A7 was reversible. Therefore, the immune function of the B-hCD40 mice can return to normal after the anti-hCD40 administration was stopped.

Example 13: In vivo toxicity testing of humanized hCD-40 antibodies

B-hCD40 mice were administered with physiological saline (PS) or humanized anti-hCD40 antibody 2A7-H1K2-IgG4-FLAA. After 3 days, mouse spleens were collected to analyze the percentage of CD20 ⁺/CD19 ⁺ cells by flow cytometry. The table below shows the experiment protocol and the results are shown in FIG. 29.

Table 17

The percentages of CD20 ⁺/CD19 ⁺ cells in mouse spleen cells are summarized in FIG. 30. The results indicate that the percentage of CD20 ⁺/CD19 ⁺ cells in mice administered with 2A7-H1K2-IgG4-FLAA did not decrease, indicating that the antibody did not exhibit any B cell clearance or B-cell depletion effects.

Example 14: CD40 receptor occupancy and B cell activation

CD40 receptor occupancy and B cell activation of anti-hCD40 antibodies were analyzed by FACS. Mean fluorescence intensity (MFI) was used to calculate percentage of CD40 receptor occupancy (RO%) and percentage of activated B cells (CD69+ B cells%) . Specifically, fresh PBMCs were added to a 96-well plate at 1 × 10 ⁶ cells per well. Anti-hCD40 antibodies 2A7-H2K2-IgG4-FLAA or lucatumumab-IgG1 were diluted and added to corresponding wells at final concentrations of 200 μg/ml, 40 μg/ml, 8 μg/ml, 1.6 μg/ml, 0.32 μg/ml, 0.064 μg/ml, 0.0128 μg/ml, or 0.00256 μg/ml. Recombinant CD154 (Soluble CD40L (TRAP) Recombinant Human Protein (rCD154) ; Thermo Fisher Scientific, Catalog number PHP0024) was then added to corresponding wells at a final concentration of 100 ng/ml. The plate was incubated at 37℃, 5%CO ₂ for 18 hours. The table below shows the experiment protocol.

Table 18

CD40 receptor occupancy analysis

CD40 receptor occupancy of 2A7-H2K2-IgG4-FLAA and lucatumumab-IgG1 were analyzed as follows. First, an Fc receptor blocking solution (Human TruStrain FcX; BioLegend, Catalog number 422302) and a viability test dye (Zombie NIR ^TM Fixable Viability Kit (DMSO) ; BioLegend, Catalog number 423106) were added to each well. The plate was incubated at room temperature in dark. Next, PE anti-Human IgG, Fc (eBioscience, Catalog number 12-4998-82) and APC anti-human CD20 (BioLegend, Catalog number 302310) were used to stain the cells. The stained cells were resuspended in 250 μl PBS and subjected for FACS analysis. Percentage of CD40 receptor occupancy (RO%) was calculated as follows:

RO%= ( (CD20+PE+ MFI) ₂ - (CD20+PE+ MFI) ₁) / ( (CD4+PE+ MFI) ₃- (CD4+PE+ MFI) ₁) ; wherein (CD20+PE+ MFI) ₁ is the background MFI value from wells without adding any anti-hCD40 antibodies or rCD154; (CD20+PE+ MFI) ₂ is the MFI value from wells with 200 μg/ml-0.00256 μg/ml anti-hCD40 antibodies and 100 ng/ml rCD154; and (CD20+PE+ MFI) ₃ is the MFI value from wells with 200 μg/ml anti-hCD40 antibodies and 100 ng/ml rCD154. As shown in FIG. 31A (the C40-2A7-H2K2-IgG4-FLAA-RO curve) , at a concentration of 0.32 μg/ml of anti-hCD40 antibody 2A7-H2K2-IgG4-FLAA, the CD40 receptor occupancy reached saturation (RO%= 100%) . In contrast, as shown in FIG. 31B (the C40-lucatumumab-IgG1-RO curve) , lucatumumab did not saturate the CD40 receptor at 200 μg/ml.

B cell activation analysis

B cell activation of 2A7-H2K2-IgG4-FLAA and lucatumumab-IgG1 were analyzed as follows. First, cells were stained using APC anti-human CD20 (BioLegend, Catalog number 302310) and PE/Cy7 anti-human CD69 (BioLegend, Catalog number 310912) . Then, the stained cells were resuspended in 250 μl PBS and subjected for FACS analysis. Percentage of activated B cells (CD69+ B cells%) was calculated as follows, using the MFI values of PE/Cy7 in B cells:

CD69 ⁺ B cells%= (E –B) / (M –B) ;

wherein B is the background MFI value from wells without adding any anti-hCD40 antibodies or rCD154; E is the MFI value from wells with 200 μg/ml-0.00256 μg/ml anti-hCD40 antibodies and 100 ng/ml rCD154; and M is the MFI value from wells with only 100 ng/ml rCD154 added. Thus, wells with only rCD154 added had a CD69+ B cells%of 100%. As shown in FIG. 31A (the C40-2A7-H2K2-IgG4-FLAA-CD69 curve) , as the antibody concentration increased, percentage of activated B cells decreased continuously. In FIG. 31B (the lucatumumab-CD69 curve) , lucatumumab exhibited maximum effect of inhibiting B cell activation at a concentration of 1.6 μg/ml. As there was a deep curve in the lucatumumab-CD69 curve, this might be undesirable as it indicates that the window between the toxicity level and therapeutic effective dose for lucatumumab may be small.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

An antibody or antigen-binding fragment thereof that binds to CD40 (TNF Receptor Superfamily Member 5) comprising:

a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR3 amino acid sequence; and

a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein 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,

wherein the selected VH CDRs 1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are one of the following:

(1) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 2, 3, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 5, 6, respectively;

(2) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 7, 8, 9, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10, 11, 12, respectively;

(3) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 13, 14, 15, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, 18, respectively.

(4) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 19, 20, 21, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 22, 23, 24, respectively.

(5) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 25, 26, 27, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 28, 29, 30, respectively.

(6) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 31, 32, 33, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 34, 35, 36, respectively.
The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3 respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively.
The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively.
The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively.
The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively.
The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 25, 26 and 27, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively.
The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 34, 35, and 36, respectively.
The antibody or antigen-binding fragment thereof of any one of claims 1-7, wherein the antibody or antigen-binding fragment specifically binds to human CD40.
The antibody or antigen-binding fragment thereof of any one of claims 1-8, wherein the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof.
The antibody or antigen-binding fragment thereof of any one of claims 1-9, wherein the antibody or antigen-binding fragment is a single-chain variable fragment (scFV) .
A nucleic acid comprising a polynucleotide encoding a polypeptide comprising:

(1) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) 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, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 79 binds to CD40;

(2) an immunoglobulin light chain or a 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, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 78 binds to CD40;

(3) an immunoglobulin heavy chain or a 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, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 81 binds to CD40;

(4) an immunoglobulin light chain or a 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, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 80 binds to CD40;

(5) an immunoglobulin heavy chain or a 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, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 100, 101, 102, or 83 binds to CD40;

(6) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 16, 17 and 18, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 97, 98, 99, or 82 binds to CD40;

(7) an immunoglobulin heavy chain or a 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: 19, 20, and 21, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 85 binds to CD40;

(8) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 22, 23 and 24, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 84 binds to CD40;

(9) an immunoglobulin heavy chain or a 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: 25, 26, and 27, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 94, 95, 96, or 87 binds to CD40;

(10) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 28, 29 and 30, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 90, 91, 92, 93, or 86 binds to CD40;

(11) an immunoglobulin heavy chain or a 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: 31, 32, and 33, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 89 binds to CD40;

(12) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 34, 35 and 36, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 88 binds to CD40;
The nucleic acid of claim 11, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively.
The nucleic acid of claim 11, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively.
The nucleic acid of claim 11, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively.
The nucleic acid of claim 11, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively.
The nucleic acid of claim 11, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively.
The nucleic acid of claim 11, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively.
The nucleic acid of claim 11, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively.
The nucleic acid of claim 11, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively.
The nucleic acid of claim 11, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 25, 26, and 27, respectively.
The nucleic acid of claim 11, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively.
The nucleic acid of claim 11, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively.
The nucleic acid of claim 11, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 34, 35, and 36, respectively.
The nucleic acid of any one of claims 11-23, wherein the VH when paired with a VL specifically binds to human CD40, or the VL when paired with a VH specifically binds to human CD40.
The nucleic acid of any one of claims 11-24, wherein the immunoglobulin heavy chain or the fragment thereof is a humanized immunoglobulin heavy chain or a fragment thereof, and the immunoglobulin light chain or the fragment thereof is a humanized immunoglobulin light chain or a fragment thereof.
The nucleic acid of any one of claims 11-25, wherein the nucleic acid encodes a single-chain variable fragment (scFv) .
The nucleic acid of any one of claims 11-26, wherein the nucleic acid is cDNA.
A vector comprising one or more of the nucleic acids of any one of claims 11-27.
A vector comprising two of the nucleic acids of any one of claims 11-28, wherein the vector encodes the VL region and the VH region that together bind to CD40.
A pair of vectors, wherein each vector comprises one of the nucleic acids of any one of claims 11-27, wherein together the pair of vectors encodes the VL region and the VH region that together bind to CD40.
A cell comprising the vector of claim 28 or 29, or the pair of vectors of claim 30.
The cell of claim 31, wherein the cell is a CHO cell.
A cell comprising one or more of the nucleic acids of any one of claims 11-27.
A cell comprising two of the nucleic acids of any one of claims 11-27.
The cell of claim 34, wherein the two nucleic acids together encode the VL region and the VH region that together bind to CD40.
A method of producing an antibody or an antigen-binding fragment thereof, the method comprising

(a) culturing the cell of any one of claims 31-35 under conditions sufficient for the cell to produce the antibody or the antigen-binding fragment; and

(b) collecting the antibody or the antigen-binding fragment produced by the cell.
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 the selected VH sequence and the selected VL sequence are one of the following:

(1) the selected VH sequence is SEQ ID NO: 78, and the selected VL sequence is SEQ ID NO: 79;

(2) the selected VH sequence is SEQ ID NO: 80, and the selected VL sequence is SEQ ID NO: 81;

(3) the selected VH sequence is SEQ ID NO: 97, 98, 99, or 82, and the selected VL sequence is SEQ ID NO: 100, 101, 102, or 83;

(4) the selected VH sequence is SEQ ID NO: 84, and the selected VL sequence is SEQ ID NO: 85;

(5) the selected VH sequence is SEQ ID NO: 90, 91, 92, 93, or 86, and the selected VL sequence is SEQ ID NO: 94, 95, 96, or 87;

(6) the selected VH sequence is SEQ ID NO: 88, and the selected VL sequence is SEQ ID NO: 89.
The antibody or antigen-binding fragment thereof of claim 37, wherein the VH comprises the sequence of SEQ ID NO: 90 and the VL comprises the sequence of SEQ ID NO: 94.
The antibody or antigen-binding fragment thereof of claim 28, wherein the VH comprises the sequence of SEQ ID NO: 97 and the VL comprises the sequence of SEQ ID NO: 100.
The antibody or antigen-binding fragment thereof of any one of claims 37-39, wherein the antibody or antigen-binding fragment specifically binds to human CD40.
The antibody or antigen-binding fragment thereof of any one of claims 37-40, wherein the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof.
The antibody or antigen-binding fragment thereof of any one of claims 37-41, wherein the antibody or antigen-binding fragment is a single-chain variable fragment (scFV) .
An antibody or antigen-binding fragment thereof comprising the VH CDRs 1, 2, 3, and the VL CDRs 1, 2, 3 of the antibody or antigen-binding fragment thereof of any one of claims 1-10 and 37-42.
An antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof of any one of claims 1-10 and 37-43.
An antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-10 and 37-44 covalently bound to a therapeutic agent.
The antibody drug conjugate of claim 45, wherein the therapeutic agent is a cytotoxic or cytostatic agent.
A method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-10 and 37-44, or the antibody-drug conjugate of claims 45 or 46, to the subject.
The method of claim 47, wherein the subject has a solid tumor.
The method of claim 47, wherein the cancer is melanoma, pancreatic carcinoma, mesothelioma, or a hematological malignancy.
The method of claim 47, wherein the cancer is Non-Hodgkin's lymphoma, lymphoma, or chronic lymphocytic leukemia.
A method of decreasing the rate of tumor growth, the method comprising

contacting a tumor cell with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof of any one of claims 1-10 and 37-44, or the antibody-drug conjugate of claims 45 or 46.
A method of killing a tumor cell, the method comprising

contacting a tumor cell with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-10 and 37-44, or the antibody-drug conjugate of claims 45 or 46.
A method of inhibiting immune response in a subject, the method comprising

administering to the subject an effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-10 and 37-44, or the antibody-drug conjugate of claims 45 or 46.
The method of claim 53, wherein the subject has an autoimmune disease.
A method of treating an autoimmune disease, the method comprising

administering to the subject an effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-10 and 37-44, or the antibody-drug conjugate of claims 45 or 46.
The method of claim 55, wherein the autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus or lupus nephritis.
A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-10 and 37-44, and a pharmaceutically acceptable carrier.
A pharmaceutical composition comprising the antibody drug conjugate of claim 45 or 46, and a pharmaceutically acceptable carrier.