Anti-TNFR2 antibodies and their uses

Abstract

The present disclosure provides anti-TNFR2 (tumor necrosis factor receptor 2) antibodies, antigen-binding fragments thereof, and uses thereof.

Description

[0001] Priority claim

[0002] This application claims the benefit of International Application No. PCT / CN2021 / 140487, filed on December 22, 2021. The entire contents of the foregoing are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to anti-TNFR2 (tumor necrosis factor receptor 2) antibodies and uses thereof. Background Art

[0004] Cancer is one of the diseases with the highest mortality rates among human beings. According to statistics from the World Health Organization, in 2012, global cancer cases and deaths reached 14 million and 8.2 million, respectively. In China, there were 3.07 million newly diagnosed cancer cases and 2.2 million deaths.

[0005] The recent clinical and commercial success of anti-cancer antibodies has generated significant interest in antibody-based therapeutics. There is a need to develop antibodies for use in various antibody-based therapies to treat cancer or autoimmune diseases. Summary of the Invention

[0006] The present disclosure relates to anti-TNFR2 antibodies, antigen-binding fragments thereof, and uses thereof.

[0007] In one aspect, the disclosure relates to an antibody or antigen-binding fragment thereof that binds to TNFR2, 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 at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence 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 at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence at least 80% identical to a selected VL CDR3 amino acid sequence, wherein the selected VH CDR 1, 2, and 3 amino acid sequences and the selected VL The amino acid sequences of CDR1, 2, and 3 are one of the following:

[0008] (1) The amino acid sequences of the selected VH CDRs 1, 2, and 3 are represented by SEQ ID NOs: 6, 7, and 8, respectively, and the amino acid sequences of the selected VL CDRs 1, 2, and 3 are represented by SEQ ID NOs: 9, 10, and 11, respectively;

[0009] (2) the amino acid sequences of the selected VH CDRs 1, 2, and 3 are represented by SEQ ID NOs: 12, 13, and 14, respectively, and the amino acid sequences of the selected VL CDRs 1, 2, and 3 are represented by SEQ ID NOs: 15, 16, and 17, respectively;

[0010] (3) the amino acid sequences of the selected VH CDRs 1, 2, and 3 are represented by SEQ ID NOs: 18, 19, and 20, respectively, and the amino acid sequences of the selected VL CDRs 1, 2, and 3 are represented by SEQ ID NOs: 21, 22, and 23, respectively;

[0011] (4) the amino acid sequences of the selected VH CDRs 1, 2, and 3 are represented by SEQ ID NOs: 24, 25, and 26, respectively, and the amino acid sequences of the selected VL CDRs 1, 2, and 3 are represented by SEQ ID NOs: 27, 28, and 29, respectively;

[0012] (5) the amino acid sequences of the selected VH CDRs 1, 2, and 3 are represented by SEQ ID NOs: 30, 31, and 32, respectively, and the amino acid sequences of the selected VL CDRs 1, 2, and 3 are represented by SEQ ID NOs: 33, 34, and 35, respectively;

[0013] (6) the amino acid sequences of these selected VH CDRs 1, 2, and 3 are represented by SEQ ID NOs: 42, 43, and 44, respectively, and the amino acid sequences of these selected VL CDRs 1, 2, and 3 are represented by SEQ ID NOs: 45, 46, and 47, respectively;

[0014] (7) the amino acid sequences of these selected VH CDRs 1, 2, and 3 are represented by SEQ ID NOs: 48, 49, and 50, respectively, and the amino acid sequences of these selected VL CDRs 1, 2, and 3 are represented by SEQ ID NOs: 51, 52, and 53, respectively;

[0015] (8) These selected VH CDR 1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 54, 55, and 56, respectively, and these selected VL CDR 1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 57, 58, and 59, respectively;

[0016] (9) the selected VH CDR 1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 60, 61, and 62, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are represented by SEQ ID NOs: 63, 64, and 65, respectively; or

[0017] (10) The amino acid sequences of these selected VH CDRs 1, 2, and 3 are shown in SEQ ID NOs: 66, 67, and 68, respectively, and the amino acid sequences of these selected VL CDRs 1, 2, and 3 are shown in SEQ ID NOs: 69, 70, and 71, respectively.

[0018] In some embodiments, according to Kabat numbering, VH comprises CDRs 1, 2, 3 having amino acid sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively, and VL comprises CDRs 1, 2, 3 having amino acid sequences set forth in SEQ ID NOs: 9, 10, and 11, respectively.

[0019] In some embodiments, according to Kabat numbering, VH comprises CDRs 1, 2, 3 having amino acid sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively, and VL comprises CDRs 1, 2, 3 having amino acid sequences set forth in SEQ ID NOs: 15, 16, and 17, respectively.

[0020] In some embodiments, according to Kabat numbering, VH comprises CDRs 1, 2, 3 having amino acid sequences set forth in SEQ ID NOs: 18, 19, and 20, respectively, and VL comprises CDRs 1, 2, 3 having amino acid sequences set forth in SEQ ID NOs: 21, 22, and 23, respectively.

[0021] In some embodiments, according to Kabat numbering, VH comprises CDRs 1, 2, 3 having amino acid sequences set forth in SEQ ID NOs: 24, 25, and 26, respectively, and VL comprises CDRs 1, 2, 3 having amino acid sequences set forth in SEQ ID NOs: 27, 28, and 29, respectively.

[0022] In some embodiments, according to Kabat numbering, VH comprises CDRs 1, 2, 3 having amino acid sequences set forth in SEQ ID NOs: 30, 31, and 32, respectively, and VL comprises CDRs 1, 2, 3 having amino acid sequences set forth in SEQ ID NOs: 33, 34, and 35, respectively.

[0023] In some embodiments, the antibody or antigen-binding fragment specifically binds human TNFR2.

[0024] In some embodiments, the antibody or antigen-binding fragment is a human antibody or antigen-binding fragment thereof (eg, a human IgG1 antibody).

[0025] In some embodiments, the antibody or antigen-binding fragment is a single-chain variable fragment (scFV).

[0026] In one aspect, the disclosure relates to a nucleic acid comprising a polynucleotide encoding a polypeptide comprising:

[0027] (1) an immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively, and wherein the VH binds to TNFR2 when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 37;

[0028] (2) an immunoglobulin light chain or fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 10, and 11, respectively, and wherein the VL binds to TNFR2 when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 36;

[0029] (3) an immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively, and wherein the VH binds to TNFR2 when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 39;

[0030] (4) an immunoglobulin light chain or fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 15, 16, and 17, respectively, and wherein the VL binds to TNFR2 when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 38;

[0031] (5) an immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 18, 19, and 20, respectively, and wherein the VH binds to TNFR2 when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 41;

[0032] (6) an immunoglobulin light chain or fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 21, 22, and 23, respectively, and wherein the VL binds to TNFR2 when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 40;

[0033] (7) an immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 24, 25, and 26, respectively, and wherein the VH binds to TNFR2 when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 73;

[0034] (8) an immunoglobulin light chain or fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 27, 28, and 29, respectively, and wherein the VL binds to TNFR2 when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 72;

[0035] (9) an immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 30, 31, and 32, respectively, and wherein the VH binds to TNFR2 when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 75; or

[0036] (10) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 33, 34, and 35, respectively, and wherein the VL binds to TNFR2 when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 74.

[0037] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively.

[0038] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 10, and 11, respectively.

[0039] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively.

[0040] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 15, 16, and 17, respectively.

[0041] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 18, 19, and 20, respectively.

[0042] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 21, 22, and 23, respectively.

[0043] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 24, 25, and 26, respectively.

[0044] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 27, 28, and 29, respectively.

[0045] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 30, 31, and 32, respectively.

[0046] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 33, 34, and 35, respectively.

[0047] In some embodiments, the VH specifically binds human TNFR2 when paired with the VL, or the VL specifically binds human TNFR2 when paired with the VH.

[0048] In some embodiments, the immunoglobulin heavy chain or fragment thereof is a human immunoglobulin heavy chain or fragment thereof, and the immunoglobulin light chain or fragment thereof is a human immunoglobulin light chain or fragment thereof.

[0049] In some embodiments, the nucleic acid encodes a single-chain variable fragment (scFv).

[0050] In some embodiments, the nucleic acid is cDNA.

[0051] In one aspect, the disclosure relates to a vector comprising one or more of the nucleic acids described herein.

[0052] In one aspect, the disclosure relates to a vector comprising two of the nucleic acids described herein, wherein the vector encodes a VH region and a VL region that together bind TNFR2.

[0053] In one aspect, the disclosure relates to a vector pair, wherein each vector comprises one of the nucleic acids described herein, wherein the vector pair collectively encodes a VH region and a VL region that together bind TNFR2.

[0054] In one aspect, the disclosure relates to a cell comprising a vector described herein, or a vector pair described herein. In some embodiments, the cell is a CHO cell.

[0055] In one aspect, the disclosure relates to a cell comprising one or more of the nucleic acids described herein.

[0056] In one aspect, the disclosure relates to a cell comprising two of the nucleic acids described herein. In some embodiments, the two nucleic acids together encode a VH region and a VL region that together bind to TNFR2.

[0057] In one aspect, the disclosure relates to a method of producing an antibody or an antigen-binding fragment thereof, comprising (a) culturing a cell described herein under conditions sufficient for the cell to produce the antibody or antigen-binding fragment; and (b) collecting the antibody or antigen-binding fragment produced by the cell.

[0058] In one aspect, the disclosure relates to an antibody or antigen-binding fragment thereof that binds to TNFR2, comprising: a heavy chain variable region (VH) comprising an amino acid sequence that is at least 80% identical to a selected VH sequence, and a light chain variable region (VL) comprising an amino acid sequence that is at least 80% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

[0059] (1) the selected VH sequence is SEQ ID NO: 36, and the selected VL sequence is SEQ ID NO: 37;

[0060] (2) the selected VH sequence is SEQ ID NO: 38, and the selected VL sequence is SEQ ID NO: 39;

[0061] (3) the selected VH sequence is SEQ ID NO: 40, and the selected VL sequence is SEQ ID NO: 41;

[0062] (4) the selected VH sequence is SEQ ID NO: 72, and the selected VL sequence is SEQ ID NO: 73; or

[0063] (5) The selected VH sequence is SEQ ID NO: 74, and the selected VL sequence is SEQ ID NO: 75.

[0064] In some embodiments, VH comprises the sequence of SEQ ID NO:36, and VL comprises the sequence of SEQ ID NO:37.

[0065] In some embodiments, VH comprises the sequence of SEQ ID NO:72, and VL comprises the sequence of SEQ ID NO:73.

[0066] In some embodiments, the VH comprises the sequence of SEQ ID NO:38, and the VL comprises the sequence of SEQ ID NO:39.

[0067] In some embodiments, the VH comprises the sequence of SEQ ID NO:40, and the VL comprises the sequence of SEQ ID NO:41.

[0068] In some embodiments, the VH comprises the sequence of SEQ ID NO:74, and the VL comprises the sequence of SEQ ID NO:75.

[0069] In some embodiments, the antibody or antigen-binding fragment specifically binds human TNFR2.

[0070] In some embodiments, the antibody or antigen-binding fragment is a human antibody or antigen-binding fragment thereof.

[0071] In some embodiments, the antibody or antigen-binding fragment is a single-chain variable fragment (scFV).

[0072] In one aspect, the disclosure relates to antibodies or antigen-binding fragments thereof that cross-compete with the antibodies or antigen-binding fragments thereof described herein.

[0073] In one aspect, the disclosure relates to an antibody or antigen-binding fragment thereof that binds to TNFR2, comprising: a heavy chain variable region (VH) comprising a VH CDR1, VH CDR2, and VH CDR3 identical to a selected VH sequence; and a light chain variable region (VL) comprising a VL CDR1, VL CDR2, and VL CDR3 identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

[0074] (1) the selected VH sequence is SEQ ID NO: 36, and the selected VL sequence is SEQ ID NO: 37;

[0075] (2) the selected VH sequence is SEQ ID NO: 38, and the selected VL sequence is SEQ ID NO: 39;

[0076] (3) the selected VH sequence is SEQ ID NO: 40, and the selected VL sequence is SEQ ID NO: 41;

[0077] (4) the selected VH sequence is SEQ ID NO: 72, and the selected VL sequence is SEQ ID NO: 73; or

[0078] (5) The selected VH sequence is SEQ ID NO: 74, and the selected VL sequence is SEQ ID NO: 75.

[0079] In one aspect, the disclosure relates to an antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof described herein covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent.

[0080] In one aspect, the disclosure relates to a method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein, or an antibody-drug conjugate described herein.

[0081] In some embodiments, the subject has colorectal cancer, ovarian cancer, acute myeloid leukemia, Lewis lung cancer, breast cancer, hepatocellular carcinoma and colon cancer, glioma. In some embodiments, the subject has renal cell carcinoma, multiple myeloma, colon cancer, ovarian cancer, glioma or cutaneous T-cell lymphoma. In some embodiments, the cancer is colon cancer, glioma or ovarian cancer.

[0082] In one aspect, the disclosure relates to a method of reducing the growth rate of a tumor, comprising contacting a tumor cell with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein, or an antibody-drug conjugate described herein.

[0083] In one aspect, the disclosure relates to a method of killing a tumor cell, comprising contacting the tumor cell with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein, or an antibody-drug conjugate described herein.

[0084] In one aspect, the disclosure relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described herein and a pharmaceutically acceptable carrier.

[0085] In one aspect, the disclosure relates to a pharmaceutical composition comprising an antibody drug conjugate described herein and a pharmaceutically acceptable carrier.

[0086] As used herein, the term "cancer" refers to a cell with the ability to grow autonomously. Examples of such cells include cells with an abnormal state or condition characterized by rapidly proliferating cell growth. The term is intended to include cancerous growth, for example, tumors; carcinogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, regardless of the histopathological type or degree of infiltration. Malignancies of various organ systems are also included, such as malignancies of the head and neck, respiratory system, cardiovascular system, renal system, reproductive system, blood system, nervous system, liver system, gastrointestinal system, and endocrine system; and adenocarcinomas, which include malignancies such as most colon cancers, renal cell carcinomas, prostate cancer, and / or testicular tumors, non-small cell lung cancer, gliomas, and small intestinal cancer. "Naturally occurring" cancers include any cancer other than cancers experimentally induced by implanting cancer cells into a subject, and include, for example, spontaneously occurring cancers, cancers caused by exposure of a patient to one or more carcinogens, cancers caused by insertion of a transgenic oncogene or knockout of a tumor suppressor gene, and cancers caused by infection (e.g., viral infection). The term "cancer" is recognized in the art and refers to a malignancy of epithelial or endocrine tissue. The term also includes carcinosarcoma, which includes malignant tumors consisting of cancerous tissue and sarcomatous tissue. "Adenocarcinoma" refers to a cancer derived from glandular tissue or a cancer in which tumor cells form a recognizable glandular structure. The term "sarcoma" is recognized in the art and refers to a malignant tumor derived from the mesenchyme. The term "hematopoietic neoplastic disorder" includes diseases involving proliferative cells / tumor cells of hematopoietic origin. Hematopoietic neoplastic disorders can be caused by bone marrow, lymphoid or erythroid cells or their precursor cells. Hematological cancer is a cancer that begins in blood-forming tissue (e.g., bone marrow) or in cells of the immune system. Examples of hematological cancers include, for example, leukemia, lymphoma, and multiple myeloma.

[0087] As used herein, the term "antibody" refers to any antigen binding molecule containing at least one (e.g., one, two, three, four, five or six) complementary determining region (CDR) (e.g., any one of the three CDRs from an immunoglobulin light chain or any one of the three CDRs from an immunoglobulin heavy chain) and capable of specific binding to an epitope. Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, chimeric antibodies, human antibodies and humanized antibodies. In certain embodiments, the antibody may contain the Fc region of a human antibody. The term antibody also includes derivatives such as bispecific antibodies, single-chain antibodies, double antibodies, linear antibodies and multispecific antibodies formed by antibody fragments.

[0088] As used herein, the term "antigen-binding fragment" refers to a portion of a full-length antibody that is capable of specifically binding to an antigen. In some embodiments, an antigen-binding fragment contains at least one variable domain (e.g., a heavy chain variable domain or a light chain variable domain). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments.

[0089] As used herein, the term "human antibody" refers to an antibody encoded by endogenous nucleic acids derived from humans (e.g., rearranged human immunoglobulin heavy or light chain loci). In certain embodiments, human antibodies are collected from humans or produced in human cell cultures (e.g., human hybridoma cells). In certain embodiments, human antibodies are produced in non-human cells (e.g., mouse or hamster cell lines). In some embodiments, human antibodies are produced in bacteria or yeast cells. In certain embodiments, human antibodies are produced in transgenic non-human animals (e.g., bovines) containing unrearranged or rearranged human immunoglobulin loci (e.g., heavy or light chain human immunoglobulin loci).

[0090] As used herein, the term "chimeric antibody" refers to an antibody containing sequences present in at least two different species (e.g., antibodies from two different mammalian species, such as human antibodies and mouse antibodies). A non-limiting example of a chimeric antibody is an antibody containing a variable domain sequence (e.g., all or part of a light chain and / or heavy chain variable domain sequence) of a non-human (e.g., mouse) antibody and the constant domains of a human antibody. Additional examples of chimeric antibodies are described herein and are known in the art.

[0091] As used herein, the term "humanized antibody" refers to a non-human antibody containing the minimum sequence derived from a non-human (e.g., mouse) immunoglobulin and containing a sequence derived from a human immunoglobulin. In non-limiting examples, a humanized antibody is a human antibody (receptor antibody), wherein the hypervariable region (e.g., CDR) residues of the receptor antibody are replaced by hypervariable region (e.g., CDR) residues of a non-human antibody (e.g., donor antibody) such as a mouse, rat, or rabbit antibody with desired specificity, affinity, and ability. In certain embodiments, the Fv framework residues of the human immunoglobulin are replaced by corresponding non-human (e.g., mouse) immunoglobulin residues. In certain embodiments, the humanized antibody can contain residues not found in the receptor antibody or the donor antibody. These modifications can further improve antibody performance. In certain embodiments, the humanized antibody contains substantially all of at least one and usually two variable domains, wherein all or substantially all of the hypervariable loops (CDRs) correspond to the hypervariable loops of a non-human (e.g., mouse) immunoglobulin, and all or substantially all of the framework regions are the framework regions of a human immunoglobulin. Humanized antibodies can also contain at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region. Humanized antibodies can be produced using molecular biology methods known in the art. Non-limiting examples of methods for producing humanized antibodies are described herein.

[0092] As used herein, the term "single-chain antibody" refers to a single polypeptide containing at least two immunoglobulin variable domains (e.g., variable domains of mammalian immunoglobulin heavy or light chains) capable of specific binding to an antigen. Non-limiting examples of single-chain antibodies are described herein.

[0093] As used herein, the term "multimeric antibody" refers to an antibody containing four or more (eg, six, eight, or ten) immunoglobulin variable domains.

[0094] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification, and describe a human or non-human animal to which the method according to the present invention provides treatment. This disclosure encompasses both veterinary and non-veterinary applications. Human patients can be adults or minors (e.g., people under 18 years of age). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. Including, for example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, pigs (e.g., piglets, miniature pigs), horses, dogs, felines, bovines, and other domesticated animals, farm animals, and zoo animals.

[0095] As used herein, the phrases "specifically bind to" and "specifically binds to" when referring to an antibody refer to the interaction of the antibody with its target molecule (e.g., TNFR2), preferably with other molecules, because the interaction depends on the presence of a specific structure (i.e., antigenic determinant or epitope) on the target molecule; in other words, the agent generally recognizes and binds to molecules that include a specific structure, rather than all molecules. An antibody that specifically binds to a target molecule may be referred to as a target-specific antibody. For example, an antibody that specifically binds to a TNFR2 molecule may be referred to as a TNFR2-specific antibody or an anti-TNFR2 antibody.

[0096] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to amino acid polymers of any length of at least two amino acids.

[0097] As used herein, the terms "polynucleotide," "nucleic acid molecule," and "nucleic acid sequence" are used interchangeably herein to refer to a nucleotide polymer of any length of at least two nucleotides, and include, but are not limited to, DNA, RNA, DNA / RNA hybrids, and modifications thereof.

[0098] 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 the invention pertains. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. These materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail.

[0099] Other features and advantages of the invention will become apparent from the following detailed description and from the accompanying drawings and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] Figure 1 Graph showing the change in tumor size over time in mice injected with MC38 cancer cells and treated with PBS (phosphate-buffered saline) as a control (G1), anti-hTNFR2 antibodies BC-1F4-IgG1 (G2), BC-3B7-IgG1 (G3), BC-1F10-IgG (G4), 14-1B3-hHvKv-IgG1 (G5), BC-1A8-IgG1 (G6), BC-1C3-IgG1 (G7), 14-4A9-hHvKv-IgG1 (G8), and anti-mPD-1 (G9).

[0101] Figure 2Graph showing the change in tumor size over time in mice injected with MC38 cancer cells and treated with: PBS as control (G1), antibodies BC-1A8-IgG1 (G2), BC-1F10-IgG1 (G3), BC-1F4-IgG1 (G4), anti-mPD-1 (G5), anti-mCTLA4 (G6).

[0102] Figure 3 Graph showing the changes in tumor size over time in mice injected with MC38 cancer cells and treated with: PBS as a control (G1), antibody BC-1C3-IgG1 (G2), anti-mPD-1 (G3), anti-mCTLA4 (G4).

[0103] Figure 4 Graph showing the changes in tumor size over time in mice injected with MC38 cancer cells and treated with: PBS as a control (G1), antibodies BC-1A8-IgG1 (G2), BC-1C3-IgG1 (G3), BC-1F10-IgG1 (G4), BC-1F4-IgG1 (G5), BC-1B6-IgG1 (G6).

[0104] Figure 5 Graph showing the change in tumor size over time in mice injected with MC38 cancer cells and treated with: PBS as control (G1), BC-1A8-IgG1 (G2), BC-1C3-IgG1 (G3), BC-1F10-IgG1 (G4), BC-1F4-IgG1 (G5), BC-1B6-IgG1 (G6).

[0105] Figure 6 Graph showing the change in tumor size over time in hTNFα / hTNFR2 mice injected with MC38 cancer cells and treated with: PBS as a control (G1), BC-1C3-IgG1 (G2), anti-mPD-1 (G3).

[0106] Figures 7A-7B The test results of blood biochemical indicators (AST, ALT) of peripheral blood of mice on the 5th day (D5) after grouping are shown.

[0107] Figure 8 Epitope correlations among anti-hTNFR2 antibodies are shown.

[0108] Figure 9 Cytotoxicity data for BC-1C3-IgG1, BC-1C3-IgG1-SI, BC-1C3-IgG1-LALA, and human IgG1 are shown.

[0109] Figure 10The drug concentration-time curves show the changes in antibody drug concentrations in the serum of humanized TNFR2 mice after injection of different antibody drugs.

[0110] Figure 11A Fluorescent signals indicating activation of reporter cells (Jurkat-GFP-TNFR2 cells) are shown.

[0111] Figure 11B Fluorescent signal indicating activation of reporter cells (Jurkat-GFP-TNFR2 cells) is shown. TNFα is not shown.

[0112] Figure 12 Fluorescent signals indicating activation of reporter cells (Jurkat-GFP-TNFR2 cells) are shown.

[0113] Figures 13A-13B Shows the test results of blood biochemical indicators (AST, ALT) in mouse peripheral blood.

[0114] Figure 14 The CDR sequences of the anti-TNFR2 antibodies BC-1A8 ("1A8"), BC-1B6 ("1B6"), BC-1C3 ("1C3"), BC-1F4 ("1F4"), BC-1F10 ("1F10"), as defined by the Kabat numbering scheme, are listed.

[0115] Figure 15 The CDR sequences of the anti-TNFR2 antibodies BC-1A8 ("1A8"), BC-1B6 ("1B6"), BC-1C3 ("1C3"), BC-1F4 ("1F4"), BC-1F10 ("1F10"), as defined by the Chothia numbering scheme, are listed.

[0116] Figure 16 The amino acid sequences of the heavy and light chain variable regions of the anti-TNFR2 antibodies (1A8, 1B6, 1C3, 1F4, and 1F10) are listed.

[0117] Figure 17 Some related amino acid sequences are listed.

[0118] Figure 18 Figure 2 is a graph showing the change in tumor size over time in mice injected with MC38 cancer cells and treated with PBS (G1), BC-1C3-IgG1 (G2), anti-mPD-1 (G3), a combination of BC-1C3-IgG1 and anti-mPD1 (G4), an atezolizumab analog (G5), or a combination of BC-1C3-IgG1 and an atezolizumab analog (G6).

[0119] Figure 19Graph showing the change in tumor size over time in mice injected with GL261 cancer cells and treated with PBS (G1), 1 mg / kg BC-1C3-IgG1 (G2), 3 mg / kg BC-1C3-IgG1 (G3), 10 mg / kg BC-1C3-IgG1 (G4), or anti-mPD-1 (G5).

[0120] Figure 20 is a graph showing the change in tumor size over time in mice injected with MC38 cancer cells and treated with PBS (G1), BC-1C3-lgG1 (G2), BI-1808 analog (G3), h600-25-108 analog (G4), or HFB3-1hz6-hG1 analog (G5).

[0121] Figure 21 is a graph showing the change in tumor size over time in mice injected with MC38 cancer cells and treated with PBS (G1), BC-1C3-IgG1 (G2), or the h600-25-71 analog (G3).

[0122] Figure 22 is a graph showing the proliferation of hCD8a+ T cells as determined by flow cytometry.

[0123] Figure 23 is a graph showing the proliferation of hCD8a+ T cells as determined by flow cytometry.

[0124] Figure 24A is a graph showing human IL12 release.

[0125] Figure 24B It is a graph showing the release of human IFNγ. DETAILED DESCRIPTION

[0126] Tumor necrosis factor (TNF) is generally considered a major proinflammatory cytokine (Al-Hatamleh et al., “A perspective review on the role of nanomedicine in the modulation of TNF-TNFR2 axis in breast cancer immunotherapy.” Journal of Oncology 2019 (2019)). TNF is one of the first inflammatory mediators produced and secreted during inflammatory processes, including the cancer microenvironment. It promotes the production of cytokine cascades and other inflammatory mediators, such as transcription factors, interleukin (IL)-1, and IL-6. There are two types of TNF receptors (TNFR1 and TNFR2) located on the cell surface. Experiments in inflammation-related cancers have shown that TNFR2 is upregulated preferentially over TNFR1, and anti-TNF monoclonal antibody treatment reduces the number and size of tumors. Therefore, the TNF-TNFR2 axis is involved in the suppression of immune responses and influences tumor progression and metastasis.

[0127] The present disclosure provides examples of antibodies, antigen-binding fragments thereof, that bind to TNFR2 (Tumor Necrosis Factor Receptor 2).

[0128] TNFR2 and cancer

[0129] If T cells are CD8+ cytotoxic T lymphocytes (CTLs), also known as CD8+ effector T cells (Teff), they are an important focus of cancer immunotherapy due to their ability to directly kill tumor cells. However, Treg cells can suppress Teff cells, thereby preventing an appropriate host immune response to eliminate tumors. Therefore, inhibiting suppressive Treg cells and simultaneously activating cytotoxic CD8+ Teff cells could be a potential strategy for treating cancer (Vanamee, S. et al. “TNFR2: a novel target for cancer immunotherapy.” Trends in molecular medicine 23.11 (2017): 1037-1046).

[0130] TNFR2 is a member of the TNFR superfamily (TNFRSF) and is activated by TNF. It is a cell surface receptor that regulates cell survival and proliferation, and targeting this receptor has emerged as a potential next-generation cancer therapy. Certain human tumor cells can aberrantly express TNFR2, and tumor infiltration is dominated by highly suppressive TNFR2+ Treg cells.

[0131] TNFR2 is primarily expressed in cells of the immune system, particularly regulatory T (Treg) cells and endothelial cells, and preferentially binds to transmembrane TNF (tmTNF). TNFR1 and TNFR2 are single-pass transmembrane glycoproteins that share 28% homology primarily in the extracellular domain, which is composed of four cysteine-rich motifs. However, the intracellular domains of the TNF receptors are largely unrelated and lack homologous sequences, suggesting that distinct signaling functions derive from the two distinct receptors. TNFR1 contains an intracellular death domain (DD) that binds to the TNFR1-associated death domain protein (TRADD) of the Fas-associated death domain (FADD) and is primarily involved in cell death signaling. Although TNFR2 does not contain a cytoplasmic DD, it interacts with TNF-associated factor 2 (TRAF2) and primarily contributes to cell survival. The signaling circuitry of TNFR2 differs from that of other TNFRs. TNFR1 contains an intracellular death domain and can activate apoptotic or inflammatory pathways, whereas TNFR2 binds to TNF receptor-associated factor (TRAF) and can activate canonical and noncanonical NF-κB pathways to control cell survival and proliferation in humans and mice.

[0132] TNFR2 has 461 amino acids, with amino acids 1-22 being a signal peptide, amino acids 23-257 being an extracellular domain, amino acids 258-287 being a transmembrane domain, and amino acids 288-461 being a cytoplasmic domain with a TRAF2 binding site. TRAF2 can bind to TRAF1, TRAF3, inhibitor of apoptosis protein 1 (cIAP1), and inhibitor of apoptosis protein 2 (cIAP2).

[0133] Antagonistic anti-TNFR2 antibodies can block ligand binding and lock the membrane receptor in a quiescent (non-signaling), antiparallel dimeric arrangement, whereas agonistic cross-linking antibodies can stabilize the parallel TNF-TNFR2 complex, providing structural stabilization of the active signaling network. Furthermore, it is now well established that TNFR2 contributes to the stabilization of the CD4+Foxp3+Treg phenotype in inflammatory environments.

[0134] A detailed description of TNFR2 and its functions can be found in, for example, Al-Hatamleh et al. "A perspective review on the role of nanomedicine in the modulation of TNF-TNFR2 axis in breast cancer immunotherapy." Journal of oncology 2019 (2019); Vanamee et al. "TNFR2: a novel target for cancer immunotherapy." Trends in molecular medicine 23.11 (2017): 1037-1046; Ortí- et al. “Targeting TNFR2 as a novel therapeutic strategy for Alzheimer's disease.” Frontiers in neuroscience 13(2019):49; Chen et al. “Interaction of TNF with TNF receptor type 2 promotes expansion and function of mouse CD4+CD25+T regulatory cells.” The Journal of Immunology 179.1(2007):154-161; each of which is incorporated by reference in its entirety.

[0135] The present disclosure provides anti-TNFR2 antibodies, antigen-binding fragments thereof, and methods of using these anti-TNFR2 antibodies and antigen-binding fragments to inhibit tumor growth and treat various diseases, including, for example, cancer.

[0136] Anti-TNFR2 antibodies and antigen-binding fragments

[0137] The present disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to TNFR2 (e.g., human TNFR2). The antibodies and antigen-binding fragments described herein are capable of binding to TNFR2. In some embodiments, these antibodies can block the TNFR2 signaling pathway, thereby increasing the immune response. In some embodiments, these antibodies can induce complement-dependent cytotoxicity (CMC) or antibody-dependent cellular cytotoxicity (ADCC).

[0138] The present disclosure provides, for example, anti-TNFR2 antibodies BC-1A8 ("1A8"), BC-1B6 ("1B6"), BC-1C3 ("1C3"), BC-1F4 ("1F4"), BC-1F10 ("1F10"), BC-3B7 ("3B7"), and modified antibodies thereof (including, for example, chimeric antibodies, humanized antibodies, and human antibodies).

[0139] As defined by Kabat numbering, the CDR sequences of 1A8 and 1A8-derived antibodies (e.g., humanized antibodies) include the CDRs of the heavy chain variable domain (SEQ ID NOs: 6, 7, 8) and the CDRs of the light chain variable domain (SEQ ID NOs: 9, 10, 11). CDRs can also be defined by the Chothia system. According to Chothia numbering, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 42, 43, 44, and the CDR sequences of the light chain variable domain are shown in SEQ ID NOs: 45, 46, 47.

[0140] As defined by Kabat numbering, the CDR sequences of 1B6 and 1B6-derived antibodies include the CDRs of the heavy chain variable domain (SEQ ID NOs: 12, 13, 14) and the CDRs of the light chain variable domain (SEQ ID NOs: 15, 16, 17). According to Chothia numbering, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 48, 49, 50, and the CDRs of the light chain variable domain are shown in SEQ ID NOs: 51, 52, 53.

[0141] As defined by Kabat numbering, the CDR sequences of 1C3 and 1C3-derived antibodies include the CDRs of the heavy chain variable domain (SEQ ID NOs: 18, 19, 20) and the CDRs of the light chain variable domain (SEQ ID NOs: 21, 22, 23). According to Chothia numbering, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 54, 55, 56, and the CDRs of the light chain variable domain are shown in SEQ ID NOs: 57, 58, 59.

[0142] As defined by Kabat numbering, the CDR sequences of 1F4 and 1F4-derived antibodies include the CDRs of the heavy chain variable domain (SEQ ID NOs: 24, 25, 26) and the CDRs of the light chain variable domain (SEQ ID NOs: 27, 28, 29). According to Chothia numbering, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 60, 61, 62, and the CDRs of the light chain variable domain are shown in SEQ ID NOs: 63, 64, 65.

[0143] As defined by Kabat numbering, the CDR sequences of 1F10 and 1F10-derived antibodies include the CDRs of the heavy chain variable domain (SEQ ID NOs: 30, 31, 32) and the CDRs of the light chain variable domain (SEQ ID NOs: 33, 34, 35). According to Chothia numbering, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 66, 67, 68, and the CDRs of the light chain variable domain are shown in SEQ ID NOs: 69, 70, 71.

[0144] The amino acid sequence of the heavy chain variable region of the 1A8 antibody is shown in SEQ ID NO: 36. The amino acid sequence of the light chain variable region of the 1A8 antibody is shown in SEQ ID NO: 37.

[0145] The amino acid sequence of the heavy chain variable region of the 1B6 antibody is shown in SEQ ID NO: 38. The amino acid sequence of the light chain variable region of the 1B6 antibody is shown in SEQ ID NO: 39.

[0146] The amino acid sequence of the heavy chain variable region of the 1C3 antibody is shown in SEQ ID NO: 40. The amino acid sequence of the light chain variable region of the 1C3 antibody is shown in SEQ ID NO: 41.

[0147] The amino acid sequence of the heavy chain variable region of the 1F4 antibody is shown in SEQ ID NO: 72. The amino acid sequence of the light chain variable region of the 1F4 antibody is shown in SEQ ID NO: 73.

[0148] The amino acid sequence of the heavy chain variable region of the 1F10 antibody is shown in SEQ ID NO: 74. The amino acid sequence of the light chain variable region of the 1F10 antibody is shown in SEQ ID NO: 75.

[0149] Also provided are amino acid sequences of the heavy chain variable and light chain variable regions of the modified antibodies. In some embodiments, the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 36, 38, 40, 72, or 74. In some embodiments, the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37, 39, 41, 73, or 75. The heavy chain variable region sequence can be paired with the corresponding light chain variable region sequence, and together they bind to TNFR2.

[0150] Percent humanization refers to the percentage identity of the heavy chain or light chain variable region sequence compared to the human antibody sequence in the International Immunogenetics Information System (IMGT) database. The highest hit (tophit) means that relative to other species, the heavy chain or light chain variable region sequence is closer to a specific species. For example, the highest hit with people means that the sequence is closer to people compared to other species. The highest hit with people and cynomolgus monkey (Macaca fascicular) means that the sequence has the same percentage identity as the human sequence and the cynomolgus monkey sequence, and compared with the sequences of other species, these percentage identities are the highest. In certain embodiments, the humanization percentage is greater than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95%. Detailed descriptions of how to determine the percentage of humanization and how to determine the top hits are known in the art and are described, for example, in Jones et al. "The INNs and outs of antibody nonproprietary names," Mabs. Vol. 8, No. 1. Taylor & Francis, 2016, which is incorporated herein by reference in its entirety. A high percentage of humanization generally has various advantages, such as being safer and more effective in humans, being more easily tolerated by human subjects, and / or being less prone to side effects. In some embodiments, the variable region is fully human, e.g., derived from a human heavy chain immunoglobulin locus sequence (e.g., a recombination of human IGHV, IGHD, and IGHJ genes), and / or a human kappa chain immunoglobulin locus sequence (e.g., a recombination of human IGKV and IGKJ genes).

[0151] In addition, in some embodiments, the antibodies or antigen-binding fragments thereof described herein may further contain one, two or three heavy chain variable region CDRs selected from the group consisting of SEQ ID NOs: 6-8, SEQ ID NOs: 12-14, SEQ ID NOs: 18-20, SEQ ID NOs: 24-26 and SEQ ID NOs: 30-32 (Kabat numbering); and / or one, two or three light chain variable region CDRs selected from the group consisting of SEQ ID NOs: 9-11, SEQ ID NOs: 15-17, SEQ ID NOs: 21-23, SEQ ID NOs: 27-29 and SEQ ID NOs: 33-35 (Kabat numbering).

[0152] In some embodiments, the antibody can have a heavy chain variable region (VH) comprising complementarity determining regions (CDR) 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence 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 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 at least 80%, 85%, 90% or 95% identical to a selected VH CDR3 amino acid sequence. In some embodiments, the antibody can have a light chain variable region (VL) comprising CDR 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence 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 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 at least 80%, 85%, 90% or 95% identical to a selected VL CDR3 amino acid sequence. Figure 14 (Kabat CDR) and Figure 15 Selected VH CDR 1, 2, 3 amino acid sequences and selected VL CDR 1, 2, 3 amino acid sequences are shown in (Chothia CDR).

[0153] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain comprising one, two, or three of the following CDRs: SEQ ID NO: 6 with zero, one, or two amino acid insertions, deletions, or substitutions; 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.

[0154] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain comprising one, two, or three of the following CDRs: SEQ ID NO: 12 with zero, one, or two amino acid insertions, deletions, or substitutions; 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.

[0155] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain comprising one, two, or three of the following CDRs: SEQ ID NO: 18 with zero, one, or two amino acid insertions, deletions, or substitutions; 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.

[0156] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain comprising one, two, or three of the following CDRs: SEQ ID NO: 24 with zero, one, or two amino acid insertions, deletions, or substitutions; 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.

[0157] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain comprising one, two, or three of the following CDRs: SEQ ID NO: 30 with zero, one, or two amino acid insertions, deletions, or substitutions; 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.

[0158] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain comprising one, two, or three of the following CDRs: SEQ ID NO: 9 with zero, one, or two amino acid insertions, deletions, or substitutions; 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.

[0159] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain comprising one, two, or three of the following CDRs: SEQ ID NO: 15 with zero, one, or two amino acid insertions, deletions, or substitutions; 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.

[0160] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain comprising one, two, or three of the following CDRs: SEQ ID NO: 21 with zero, one, or two amino acid insertions, deletions, or substitutions; 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.

[0161] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain comprising one, two, or three of the following CDRs: SEQ ID NO: 27 with zero, one, or two amino acid insertions, deletions, or substitutions; 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.

[0162] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain comprising one, two, or three of the following CDRs: SEQ ID NO: 33 with zero, one, or two amino acid insertions, deletions, or substitutions; 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.

[0163] Insertions, deletions, and substitutions can be within the CDR sequence, or at one or both ends of the CDR sequence. In certain embodiments, the CDR is determined based on the Kabat numbering scheme. In certain embodiments, the CDR is determined based on the Chothia numbering scheme. In certain embodiments, the CDR is determined based on a combination of the Kabat and Chothia numbering schemes.

[0164] The present disclosure also provides antibodies or antigen-binding fragments thereof that bind to TNFR2. 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: 36 and the selected VL sequence is SEQ ID NO: 37. In some embodiments, the selected VH sequence is SEQ ID NO: 38 and the selected VL sequence is SEQ ID NO: 39. In some embodiments, the selected VH sequence is SEQ ID NO: 40 and the selected VL sequence is SEQ ID NO: 41. In some embodiments, the selected VH sequence is SEQ ID NO: 72 and the selected VL sequence is SEQ ID NO: 73. In some embodiments, the selected VH sequence is SEQ ID NO:74, and the selected VL sequence is SEQ ID NO:75.

[0165] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps are introduced in one or both of the first and second amino acids or the first and second nucleic acid sequences for optimal comparison, and for comparison purposes, non-homologous sequences can be ignored). The length of the reference sequence for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments, at least 90%, 95%, or 100% of the length of the reference sequence. 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, the molecules are identical at that position. Taking into account the number of gaps and the length of each gap, the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, which need to be introduced for optimal comparison of the two sequences. For example, comparison of two sequences and determination of percent identity between the two sequences can be achieved using a Blosum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0166] The present disclosure also provides nucleic acids comprising polynucleotides encoding polypeptides comprising an immunoglobulin heavy chain or an immunoglobulin light chain. The immunoglobulin heavy chain or immunoglobulin light chain comprises Figure 14 or Figure 15 or having the CDRs shown in Figure 16When the polypeptide is paired with a corresponding polypeptide (eg, a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptides bind to TNFR2.

[0167] Anti-TNFR2 antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) of the antibodies or antibody fragments, as well as multispecific (e.g., bispecific) antibodies or antibody fragments. Additional antibodies provided herein are polyclonal antibodies, monoclonal antibodies, multimeric antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, chimeric antibodies (e.g., human-mouse chimeras), single-chain antibodies, antibodies produced intracellularly (i.e., intrabodies), and antigen-binding fragments thereof. The antibody or antigen-binding fragment 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.

[0168] Antibody fragments are suitable for use in the provided methods as long as they retain the desired affinity and specificity of the full-length antibody. Thus, antibody fragments that bind to TNFR2 will retain the ability to bind to TNFR2. Fv fragments are antibody fragments that contain a complete antigen recognition site and binding site. This region consists of a tightly bound dimer of one heavy-chain variable domain and one light-chain variable domain, which may be covalent in nature, such as in scFv. In this structure, the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. In summary, the six CDRs, or a subset thereof, confer antigen-binding specificity to an antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although generally with lower affinity than the entire binding site. Single-chain Fv or (scFv) antibody fragments comprise the VH and VL domains (or regions) of an antibody, wherein these domains are present in a single polypeptide chain. Typically, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0169] The present disclosure also provides antibodies or antigen-binding fragments thereof that cross-compete with any of the antibodies or antigen-binding fragments described herein. Cross-competition assays are known in the art, for example, as described in Moore et al., "Antibody cross-competition analysis of the human immunodeficiency virus type 1 gp1 20 exterior envelope glycoprotein," Journal of virology 70.3 (1996): 1863-1872, which is incorporated herein by reference in its entirety. In one aspect, the present disclosure also provides antibodies or antigen-binding fragments thereof that bind to the same epitope or region as any of the antibodies or antigen-binding fragments described herein. Epitope binning assays are known in the art, as described, for example, in Estep et al. "High throughput solution-based measurement of antibody-antigen affinity and epitope binning," Mabs. Vol. 5, No. 2. Taylor and Francis, 2013, which is incorporated herein by reference in its entirety.

[0170] Antibodies and antigen-binding fragments

[0171] The present disclosure provides various antibodies and antigen-binding fragments thereof derived from the anti-TNFR2 antibodies described herein. Generally, antibodies (also known as immunoglobulins) are composed of two types of polypeptide chains, light chains and heavy chains. A non-limiting example of an antibody disclosed herein can be a complete four-chain immunoglobulin antibody comprising two heavy chains and two light chains. The heavy chain of the antibody can be any isotype (including IgM, IgG, IgE, IgA or IgD) or subisotype (including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc.). The light chain can be a kappa light chain or a lambda light chain. The antibody can comprise two identical copies of a light chain and two identical copies of a heavy chain. Each contains a variable domain (or variable region, V H ) and multiple constant domains (or constant regions) are bound to each other via disulfide bonds within their constant domains to form the "handle" of the antibody. Each contains a variable domain (or variable region, V L) and a constant domain (or constant region), each bound to a heavy chain via a disulfide bond. The variable region of each light chain pairs with the variable region of the heavy chain to which it is bound. Both the light and heavy chain variable regions contain three hypervariable regions sandwiched between more conserved framework regions (FRs).

[0172] These hypervariable regions, called complementarity determining regions (CDRs), form the loops that comprise the antigen-binding surface of the antibody. The four framework regions primarily adopt a β-sheet conformation, and the CDRs form loops that connect, and in some cases form part of, the β-sheet structure. The CDRs in each chain are brought into close proximity by the framework regions and, together with the CDRs from the other chain, form the antigen-binding region.

[0173] Methods for identifying the CDR regions of an antibody by analyzing its amino acid sequence are well known, and a number of definitions of CDRs are in common use. The Kabat definition is based on sequence variability, while the Chothia definition is based on the location of the structural loop regions. These methods and definitions are described in, for example, Martin, "Protein sequence and structure analysis of antibody variable domains," Antibody Engineering, Springer Berlin Heidelberg, 2001. 422-439; Abhinandan et al. "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains," Molecular immunology 45.14 (2008): 3832-3839; Wu, TT and Kabat, EA (1970) J. Exp. Med. 132: 211-250; Martin et al., Methods Enzymol. 203: 121-53 (1991); Morea et al., Biophys Chem. 68(1-3): 9-16 (October 1997); Morea et al., J Mol Biol. 275(2): 269-94 (January 1998); Chothia et al., Nature 342(6252): 877-83 (December 1989); Ponomarenko and Boume, BMC Structural Biology 7: 64 (2007); each of which is incorporated herein by reference in its entirety.

[0174] CDRs are important for recognizing antigenic epitopes. As used herein, an "epitope" is the smallest portion of a target molecule that can be specifically bound by an antibody's antigen-binding domain. The minimum size of an epitope may be approximately three, four, five, six, or seven amino acids, but these amino acids do not necessarily have to be located in a continuous linear sequence within the primary structure of the antigen, as the epitope may depend on the three-dimensional configuration of the antigen based on its secondary and tertiary structures.

[0175] In some embodiments, the antibody is a complete immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, differing only in their constant regions, particularly their hinge and CH2 upper domains. The sequences and differences of IgG subclasses are known in the art and are described in, for example, Vidarsson et al., "IgG subclasses and allotypes: from structure to effector functions." Frontiers in immunology 5 (2014); Irani et al. "Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases." Molecular immunology 67.2 (2015): 171-182; Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016; each of which is incorporated herein by reference in its entirety.

[0176] Antibodies can also be immunoglobulin molecules derived from any species (e.g., humans, rodents, mice, camelids). Antibodies disclosed herein also include, but are not limited to, polyclonal antibodies, monoclonal antibodies, monospecific antibodies, multispecific antibodies, and chimeric antibodies comprising immunoglobulin binding domains fused to another polypeptide. The term "antigen binding domain" or "antigen binding fragment" is an antibody portion that retains the specific binding activity of a complete antibody, i.e., any portion of an antibody that is capable of specifically binding to an epitope on a target molecule of a complete antibody. It includes, for example, Fab, Fab', F(ab')2 and variants of these fragments. Therefore, in some embodiments, an antibody or its antigen binding fragment can be, for example, scFv, Fv, Fd, dAb, bispecific antibodies, bispecific scFv, diabodies, linear antibodies, single-chain antibody molecules, multispecific antibodies formed by antibody fragments, and any polypeptide comprising an antibody binding domain or a binding domain homologous thereto. Non-limiting examples of antigen binding domains include, e.g., the heavy and / or light chain CDRs of an intact antibody, the heavy and / or light chain variable regions of an intact antibody, the full-length heavy or light chain of an intact antibody, or individual CDRs from the heavy or light chain of an intact antibody.

[0177] Also provided are antibody fragments suitable for use in the methods described herein. Fab fragments contain the variable and constant domains of the light chain and the variable and first constant domains (CH1) of the heavy chain. F(ab')2 antibody fragments comprise a pair of Fab fragments, which are typically covalently linked near their carboxyl termini by hinge cysteines between them. Other chemical couplings of antibody fragments are also known in the art.

[0178] Diabodies are small antibody fragments with two antigen-binding sites that contain a VL linked to a VH (VH and VL) domain on the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

[0179] Linear antibodies contain 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.

[0180] The antibodies and antibody fragments of the disclosure can be modified in the Fc region to provide desired effector function or serum half-life.

[0181] Antibody multimerization can be achieved by natural aggregation of antibodies or by chemical or recombinant linking techniques known in the art. For example, a certain percentage of purified antibody preparations (e.g., purified IgG1 molecules) spontaneously form protein aggregates containing antibody homodimers and other higher-order antibody multimers.

[0182] Alternatively, antibody homodimers can be formed by chemical connection techniques known in the art. For example, heterobifunctional cross-linkers (including but not limited to SMCC (succinimidyl 4- (maleimidomethyl) cyclohexane-1-carboxylate) and SATA (N-succinimidyl S-ethylthio-acetate)) can be used to form antibody multimers. An exemplary scheme for forming antibody homodimers is described in: Ghetie et al. (Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 94: 7509-7514, 1997). Antibody homodimers can be converted into Fab'2 homodimers by digestion with pepsin. Another way to form antibody homodimers is by using the autophilic T15 peptide described in: Zhao et al. (J. Immunol. [Journal of Immunology] 25: 396-404, 2002).

[0183] In certain embodiments, multispecific antibodies are bispecific antibodies. Bispecific antibodies can be prepared by maximizing the percentage of the heterodimer recovered from recombinant cell culture through the interface engineering between a pair of antibody molecules. For example, the interface can contain at least a portion of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the first antibody molecule interface are replaced by larger side chains (for example, tyrosine or tryptophan). By replacing large amino acid side chains with smaller amino acid side chains (for example alanine or threonine), a compensation " cavity " of the same or similar size as one or more large side chains is produced on the interface of the second antibody molecule. This provides a mechanism for increasing the output of heterodimers relative to other unwanted end products such as homodimers. This method is described in, for example, WO 96 / 27011, which is incorporated to its entirety by reference.

[0184] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, while the other can be coupled to biotin. Heteroconjugate antibodies can also be prepared using any conventional cross-linking method. 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.

[0185] Any of the antibodies or antigen-binding fragments described herein can be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in a subject or solution). Non-limiting examples of stabilizing molecules include polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin, such as human serum albumin). Conjugation of a stabilizing molecule can increase the half-life of the antibody or antigen-binding fragment or improve its biological activity in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in humans).

[0186] In some embodiments, the antibodies or antigen-binding fragments described herein can be conjugated to therapeutic agents. Antibody-drug conjugates comprising antibodies or antigen-binding fragments thereof can be covalently or non-covalently bound to therapeutic agents. In some embodiments, the therapeutic agent is a cytotoxic agent or a cell growth inhibitor (e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide (tenoposide), vincristine, vinblastine, colchicine, adriamycin, daunorubicin, dihydroxy anthracin, maytansinoids (e.g., DM-1 and DM-4), diketones, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin and cyclophosphamide and the like).

[0187] In some embodiments, the antigen binding fragment can form a part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor is a fusion of a single-chain variable fragment (scFv) as described herein and a CD3-ζ transmembrane domain and an internal domain. In some embodiments, the chimeric antigen receptor also includes an intracellular signaling domain from a variety of costimulatory protein receptors (e.g., CD28, 41BB, ICOS). In some embodiments, the chimeric antigen receptor includes multiple signaling domains, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to improve effectiveness. Therefore, in one aspect, the disclosure further provides cells (e.g., T cells) expressing chimeric antigen receptors as described herein.

[0188] 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.

[0189] Antibody characteristics

[0190] The antibodies or antigen-binding fragments thereof described herein can block the binding between TNFR2 and a TNFR2 ligand. In some embodiments, by binding to TNFR2, the antibodies can inhibit the TNFR2 signaling pathway. In some embodiments, the antibodies can upregulate an immune response. In some embodiments, the antibodies can reduce tumor volume in animals with tumors that express TNFR2.

[0191] In some embodiments, the antibody (or antigen-binding fragment thereof) specifically binds to TNFR2 (human TNFR2, mouse TNFR2, monkey TNFR2, dog TNFR2, chimeric TNFR2) with an off-rate (koff) of less than 0.1 s. -1 , less than 0.01s -1 , less than 0.001s -1 , less than 0.0001s -1 or less than 0.00001s -1 In some embodiments, the off-rate (koff) is greater than 0.01 s -1 , greater than 0.001s -1 , greater than 0.0001s -1 , greater than 0.00001s -1 or greater than 0.000001s -1 .

[0192] In some embodiments, the kinetic association rate (kon) is greater than 1x10 2 / Ms, greater than 1x10 3 / Ms, greater than 1x10 4 / Ms, greater than 1x10 5 / Ms or greater than 1x10 6 / Ms. In some embodiments, the kinetic association rate (kon) is less than 1x10 5 / Ms, less than 1x10 6 / Ms or less than 1x10 7 / Ms.

[0193] Affinity can be derived from the quotient of the kinetic rate constants (KD = koff / kon). In some embodiments, KD is less than 1x10 -6 M, less than 1x10 -7 M, less than 1x10 -8 M, less than 1x10 -9 M or less than 1x10 -10 In some embodiments, the KD is less than 50 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, the KD is greater than 1 x 10-7 M, larger than 1x10 -8 M, larger than 1x10 -9 M, larger than 1x10 -10 M, larger than 1x10 -11 M, or larger than 1x10 -12 M.

[0194] Common techniques for measuring the affinity of an antibody for an antigen include, for example, ELISA, RIA, and surface plasmon resonance (SPR). In some embodiments, the antibody binds to human TNFR2 (SEQ ID NO: 1), mouse TNFR2 (e.g., SEQ ID NO: 2), monkey TNFR2 (e.g., SEQ ID NO: 3), dog TNFR2 (SEQ ID NO: 4), and / or chimeric TNFR2 (SEQ ID NO: 5). In some embodiments, the antibody does not bind to monkey TNFR2, dog TNFR2, chimeric TNFR2, and / or mouse TNFR2.

[0195] TNFR2 has four cysteine-rich domains (CRDs). CRD1 is 39aa-76aa of SEQ ID NO: 1, CRD2 is 77aa-118aa of SEQ ID NO: 1, CRD3 is 119aa-162aa of SEQ ID NO: 1, and CRD4 is 168aa-196aa of SEQ ID NO: 1. In some embodiments, the antibody (or antigen-binding fragment thereof) specifically binds to CRD1, CRD2, CRD3, and / or CRD4. In some embodiments, the epitope is located at the junction of CRD3 and CRD4.

[0196] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can block the inhibition of CD8+ T cell proliferation by Treg cells.

[0197] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can promote the proliferation of CD8+ T cells. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can promote the proliferation of CD8+ T cells by more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70% or more than 80%.

[0198] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can enhance cytokine release by CD8+T cells. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can enhance the release of IL-2 by CD8+T cells. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can enhance the release of IL-2 by CD8+T cells by more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70% or more than 80%. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can enhance the release of IFN-γ by CD8+T cells. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can enhance the release of IFN-γ by CD8+T cells by more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70% or more than 80%.

[0199] In some embodiments, the tumor growth inhibition percentage (TGI) of the antibodies or antigen-binding fragments thereof described herein is TV In some embodiments, the antibody has an inhibition rate of less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. For example, TGI can be determined 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the start of treatment, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the start of treatment. TV As used herein, the tumor growth inhibition percentage (TGI) was calculated using the following formula: TV %):

[0200] TGI TV (%)=[1-(Ti-T0) / (Vi-V0)]×100%

[0201] Ti is the mean tumor volume of the treatment group on day i. T0 is the mean tumor volume of the treatment group on day 0. Vi is the mean tumor volume of the control group on day i. V0 is the mean tumor volume of the control group on day 0.

[0202] In some embodiments, the antibodies or antigen-binding fragments thereof described herein are cytotoxic, for example, to cells expressing TNFR2. Methods for measuring cytotoxicity are known in the art. In some embodiments, cytotoxicity is calculated by the following formula:

[0203]

[0204] Where experimental refers to the absorbance value of the experimental well (effector cells + target cells + test product); effector refers to the absorbance value of the effector cells (effector cells only). target refers to the absorbance value of the target cells (target cells only). spontaneous refers to the absorbance value of the autofluorescence of the cell culture medium (cell culture medium only, without effector cells or target cells). targetmax refers to the maximum absorbance value of the target cells (target cells + lysate). spontaneous* refers to the absorbance value of the cell culture medium volume control well (culture medium + lysate only). EC50 can also be calculated. In some embodiments, the EC50 is less than 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 μg / mL.

[0205] In some embodiments, the antibodies or antigen-binding fragments thereof as described herein are TNFR2 antagonists. In some embodiments, the antibodies or antigen-binding fragments reduce TNFR2 signaling in target cells expressing TNFR2 (e.g., T cells such as Tregs).

[0206] In some embodiments, the antibodies or antigen-binding fragments thereof as described herein are non-toxic. In some embodiments, no significant body weight difference is observed between the treatment group and the control group, for example at 0.3 mg / kg, 1 mg / kg, 10 mg / kg or 25 mg / kg.

[0207] In some embodiments, the antibody or antigen-binding fragment can bind to tumor cells expressing TNFR2. In some embodiments, the antibody or antigen-binding fragment can induce complement-dependent cytotoxicity (CMC) and / or antibody-dependent cellular cytotoxicity (ADCC) and kill tumor cells.

[0208] In some embodiments, the antibody or antigen-binding fragment has a functional Fc region. In some embodiments, the effector function of the functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector function of the functional Fc region is ADCC and phagocytosis.

[0209] In some embodiments, the antibody or antigen-binding fragment can induce complement-mediated cytotoxicity (CMC).

[0210] In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, the antibody is a human IgG1 antibody.

[0211] In some embodiments, the antibody or antigen-binding fragment does not have a functional Fc region. For example, the antibody or antigen-binding fragment is a Fab, Fab', F(ab')2, and Fv fragment. In some embodiments, the Fc region has a LALA mutation (L234A and L235A mutations in EU numbering) or a LALA-PG mutation (L234A, L235A, P329G mutations in EU numbering).

[0212] In some embodiments, the Fc has S1 mutations (S239D and 1332E mutations by EU numbering).

[0213] Methods for preparing anti-TNFR2 antibodies

[0214] Isolated fragments of human TNFR2 (e.g., the extracellular region) can be used as immunogens to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies can be generated in animals by multiple injections (e.g., subcutaneous or intraperitoneal) of the antigenic peptide or protein. In some embodiments, the antigenic peptide or protein is injected with at least one adjuvant. In some embodiments, the antigenic peptide or protein can be conjugated to an agent that is immunogenic in the species to be immunized. The antigenic peptide or protein can be injected into the animal more than once (e.g., twice, three times, or four times).

[0215] The full-length polypeptide or protein can be used; or alternatively, an antigenic peptide fragment thereof can be used as an immunogen. The antigenic peptide of the protein comprises at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acid sequence of TNFR2 and encompasses an epitope of the protein such that antibodies raised against the peptide form specific immune complexes with the protein. As described above, the full-length sequence of human TNFR2 is known in the art (SEQ ID NO: 1). In some embodiments, an Fc-tagged human TNFR2 protein (an Fc fusion protein containing the extracellular domain of human TNFR2, positions 23-257 of SEQ ID NO: 1) is used as an immunogen.

[0216] Immunogens are typically used to prepare antibodies by immunizing a suitable subject (e.g., a human or transgenic animal expressing at least one human immunoglobulin locus). Suitable immunogenic preparations can contain, for example, recombinantly expressed polypeptides or chemically synthesized polypeptides (e.g., fragments of human TNFR2). The preparations can further comprise an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulant.

[0217] As described above, polyclonal antibodies can be prepared by immunizing a suitable subject with a TNFR2 polypeptide or antigenic peptide thereof (e.g., a portion of TNFR2, such as the extracellular region) as an immunogen. Antibody titers in immunized subjects can be monitored over time by standard techniques, such as enzyme-linked immunosorbent assay (ELISA) using immobilized TNFR2 polypeptide or peptide. If desired, the antibody molecules can be isolated from the mammal (e.g., from blood) and further purified by well-known techniques (e.g., protein A or protein G chromatography) to obtain the IgG fraction. At an appropriate time after immunization, e.g., when specific antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler et al. (Nature 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 the trioma technique. Techniques for producing hybridomas are well known (see generally, Current Protocols in Immunology, 1994, Coligan et al. (eds.), John Wiley & Sons, Inc., New York, NY). Hybridoma cells producing monoclonal antibodies are detected, for example, by screening hybridoma culture supernatants for antibodies that bind to the polypeptide or epitope of interest using standard ELISA assays.

[0218] Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding the human antibodies, humanized antibodies or chimeric antibodies, or antigen-binding fragments thereof described herein or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acid sequence that constitute the antigen-binding site or antigen-binding domain of the antibody. In a population of such variants, some antibodies or antigen-binding fragments will have increased affinity for the target protein (e.g., TNFR2). Deletions, insertions, and / or combinations can be combined in any manner to obtain antibodies or antigen-binding fragments thereof that have increased binding affinity for the target. Amino acid changes introduced into the antibody or antigen-binding fragment can also alter new post-translational modifications 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 so that the enzyme present in the cell attaches to a different sugar), or introducing new glycosylation sites.

[0219] The antibodies disclosed herein can be derived from any animal species, including mammals. Non-limiting examples of natural antibodies include antibodies derived from humans, primates (e.g., monkeys and apes), cows, pigs, horses, sheep, camelids (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits), including transgenic rodents genetically engineered to produce human antibodies.

[0220] Human and humanized antibodies include antibodies having variable and constant regions derived from human germline immunoglobulin sequences (or having an amino acid sequence identical to an amino acid sequence 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.

[0221] Humanized antibodies generally have a human framework (FR) into which non-human CDRs have been transplanted. Therefore, humanized antibodies have one or more amino acid sequences introduced therein from a non-human source. These non-human amino acid residues are generally referred to as "import" residues, which are generally taken from a "import" variable domain. Humanization can essentially be performed by replacing, for example, rodent CDRs or CDR sequences with the corresponding sequences of human antibodies. These methods are described, for example, in Jones et al. "Replacing the complementarity-determining regions in a human antibody with those from a mouse." Nature 321. 6069 (1986): 522; Riechmann et al. "Reshaping human antibodies for therapy." Nature 332. 6162 (1988): 323; Dall'Acqua et al. "Antibody humanization by framework shuffling." Methods 36. 1 (2005): 43-60; each of which is incorporated herein by reference in its entirety. Accordingly, "humanized" antibodies are chimeric antibodies in which substantially less than an intact human V domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically mouse antibodies in which some CDR residues and some FR residues are substituted by residues from analogous sites in human antibodies.

[0222] The selection of human VH and VL domains for preparing humanized antibodies is very important for reducing immunogenicity. According to the so-called "best-fit" method, the sequence of the V domain of the mouse antibody can be screened against the entire library of known human domain sequences. The human sequence that is closest to the mouse sequence is then accepted as the human FR of the humanized antibody (Sims et al. "A humanized CD 18 antibody can block function without cell destruction." The Journal of Immunology 151.4 (1993): 2296-2308; Chothia et al. "Canonical structures for the hypervariable regions of immunoglobulins." Journal of molecular biology 196.4 (1987): 901-917).

[0223] It is further important that antibody is humanized while retaining high specificity and affinity and other favorable biological properties to antigen. In order to achieve this goal, humanized antibodies can be prepared by analyzing the process of parental sequence and multiple conceptual humanized products using the three-dimensional model of the parental sequence and the humanized sequence. The three-dimensional immunoglobulin model is usually available and is familiar to those of ordinary skill in the art. The computer program that illustrates and displays the possible three-dimensional conformational structure of selected candidate immunoglobulin sequences is available. Inspection of these displays allows analysis of the possible effects of residues in the functioning of candidate immunoglobulin sequences, i.e., analyzing the residues that influence the ability of candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from receptor sequences and input sequences, thereby reaching the desired antibody characteristics, as increased affinity for one or more target antigens.

[0224] Generally, amino acid sequence variants of a human, humanized, or chimeric anti-TNFR2 antibody will contain an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence present in the light or heavy chain of the original antibody.

[0225] In certain embodiments, antibodies are produced using mice (e.g., RenMab mice) with humanized heavy chain immunoglobulin loci and humanized kappa chain immunoglobulin loci. The heavy chain immunoglobulin loci are regions on chromosomes containing antibody heavy chain genes. The loci may include, for example, human IGHV (variable) genes, human IGHD (diversity) genes, human IGHJ (connection) genes, and mouse heavy chain constant domain genes. The kappa chain immunoglobulin loci are regions on chromosomes containing genes encoding antibody light chains (kappa chains). The kappa chain immunoglobulin loci may include, for example, human IGKV (variable) genes, human IGKJ (connection) genes, and mouse light chain constant domain genes. A detailed description of RenMab mice is found in PCT / CN2020 / 075698, which is incorporated herein by reference in its entirety. The antibodies produced by mice have complete human VH, complete human VL, and mouse constant regions. In certain embodiments, human VH and human VL are connected to human IgG constant regions (e.g., IgG1, IgG2, IgG3, and IgG4). In some embodiments, the constant region has a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 76, 77, 78 or 90.

[0226] Identity or homology with respect to the original sequence is typically the percentage of amino acid residues present in the candidate sequence that are identical to the sequence present in the human anti-TNFR2 antibody, humanized anti-TNFR2 antibody, or chimeric anti-TNFR2 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.

[0227] Additional modifications can be made to the anti-TNFR2 antibodies or antigen-binding fragments. For example, one or more cysteine ​​residues can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The resulting homodimeric antibodies can have any increased in vitro and / or in vivo half-life. Homodimeric antibodies with increased in vitro and / or in vivo half-life can also be prepared using heterobifunctional cross-linkers as described, for example, by Wolff et al. ("Monoclonal antibody homodimers: enhanced antitumor activity in nude mice." Cancer research 53.11 (1993): 2560-2565). Alternatively, an antibody can be engineered to have two Fc regions.

[0228] In some embodiments, the anti-TNFR2 antibodies or antigen-binding fragments thereof can be covalently modified. These covalent modifications can be performed by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of antibodies or antibody fragments are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with an organic derivatizing agent that is reactive with selected side chains or N-terminal or C-terminal residues.

[0229] In some embodiments, antibody variants having a carbohydrate structure are provided that lack fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such an antibody composition can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. For example, as described in WO 2008 / 077546, the amount of fucose determined by the average amount of fucose within the Asn297 sugar chain relative to the sum of all sugar structures (e.g., complexed, hybrid, and high mannose structures) attached to Asn 297 as measured by MALDI-TOF mass spectrometry is calculated. Asn297 refers to the asparagine residue at approximately position 297 in the Fc region (Eu numbering of Fc region residues, or position 314 by Kabat numbering); however, due to minor sequence variations in antibodies, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function.In some embodiments, to reduce glycan heterogeneity, the Fc region of the antibody may be further engineered to replace asparagine at position 297 with alanine (N297A).

[0230] In some embodiments, in order to improve production efficiency by avoiding Fab-arm exchange, the Fc region of the antibody is further engineered to replace the serine at position 228 (EU numbering) of IgG4 with proline (S228P). A detailed description of the S228 mutation is described in, for example, Silva et al. "The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation." Journal of Biological Chemistry 290.9 (2015): 5462-5469, which is incorporated by reference in its entirety.

[0231] recombinant vector

[0232] The present disclosure also provides recombinant vectors (e.g., expression vectors) comprising the isolated polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein), host cells into which the recombinant vectors have been introduced (i.e., such that the host cells contain the polynucleotides and / or vectors comprising the polynucleotides), and recombinant antibody polypeptides or fragments thereof produced by recombinant techniques.

[0233] As used herein, a "vector" is any construct capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. An "expression vector" is capable of delivering and expressing one or more polynucleotides of interest as encoded polypeptides in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the polynucleotide of interest is located in the vector for expression by being operably linked to regulatory elements such as a promoter, an enhancer, and / or a polyadenylic acid tail, which are located within the vector or in the genome of the host cell at or near or on both sides of the integration site of the polynucleotide of interest, such that the polynucleotide of interest will be translated in the host cell into which the expression vector has been introduced.

[0234] The vector can be introduced into the host cell by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., with a recombinant virus). Thus, non-limiting examples of vectors include viral vectors (which can be used to produce recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0235] In some embodiments, a polynucleotide disclosed herein (e.g., a polynucleotide encoding a polypeptide disclosed herein) is introduced using a viral expression system (e.g., vaccinia or other poxviruses, retroviruses, or adenoviruses), which may involve the use of non-pathogenic (defective), replication-competent viruses, or replication-defective viruses. In the latter case, viral propagation typically occurs only in complementing virus packaging cells. Suitable systems are disclosed, for example, in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86:317-321; Flexner et al., 1989, Ann. NY 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 can 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 can be increased by coating the DNA on biodegradable beads that can be efficiently transported into cells.

[0236] For expression, the DNA insert sequence comprising the polynucleotide encoding the antibody or encoding polypeptide disclosed herein can be operably linked to a suitable promoter (e.g., a heterologous promoter), such as bacteriophage lambda PL promoter, E. coli lac promoter, E. coli trp promoter, and E. coli tac promoter, SV40 early and late promoters, and promoters of retroviral LTRs, to name a few. Other suitable promoters are known to those skilled in the art. In certain embodiments, the promoter is a cytomegalovirus (CMV) promoter. The expression construct can further contain sites for transcription initiation and termination, and (in the transcribed region) a ribosome binding site for translation. The mature transcribed coding portion expressed by the construct can include a translation initiation codon at the beginning and a termination codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated.

[0237] As noted, the expression vector may 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 culture in Escherichia coli and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells, such as Escherichia coli (E. coli) cells, Streptomyces (Streptomyces) cells, and Salmonella typhimurium (Salmonella typhimurium) cells; fungal cells, such as yeast cells; insect cells, such as Drosophila (Drosophila) S2 cells and Spodoptera (Spodoptera) Sf9 cells; animal cells, such as CHO cells, COS cells, Bowes (Bowes) melanoma cells, and HK293 cells; and plant cells. Suitable culture media and conditions for the host cells described herein are known in the art.

[0238] 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, and 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 apparent to the skilled artisan.

[0239] Suitable non-limiting bacterial promoters include the E. coli lacI and lacZ promoters, T3 and T7 promoters, gpt promoter, lambda PR and PL promoters, and trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the SV40 early and late promoters, promoters from retroviral LTRs, such as the Rous sarcoma virus (RSV) promoter, and metallothionein promoters, such as the mouse metallothionein-I promoter.

[0240] In the yeast Saccharomyces cerevisiae, a variety of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH are available.

[0241] The construct can be introduced into the host cell 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, for example, Davis et al., Basic Methods In Molecular Biology (1986), which is incorporated herein by reference in its entirety.

[0242] The transcription of the DNA encoding the disclosed antibodies by higher eukaryotic organisms can be increased by inserting an enhancer sequence into the vector. An enhancer is a cis-acting element of DNA, typically about 10 to 300 bp, that acts to increase the transcriptional activity of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer (which is located on the rear side of the replication origin at base pairs 100 to 270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the rear side of the replication origin, and adenovirus enhancers.

[0243] In order to secrete the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space, or the extracellular environment, an appropriate secretion signal can be incorporated into the expressed polypeptide. The signal can be endogenous to the polypeptide or a heterologous signal.

[0244] Polypeptides (e.g., antibodies) can be expressed in modified forms, such as fusion proteins (e.g., GST fusions) or with histidine tags, and can include not only secretion signals but also additional heterologous functional regions. For example, additional amino acids, particularly charged amino acid regions, can be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell during purification or subsequent processing and storage. Similarly, peptide moieties can be added to the polypeptide to promote purification. Such regions can be removed before the final preparation of the polypeptide. Adding peptide moieties to polypeptides to cause secretion or excretion, thereby improving stability and promoting purification, is especially a familiar and conventional technique in the art.

[0245] Treatment

[0246] The antibodies or antigen-binding fragments thereof disclosed herein can be used for a variety of therapeutic purposes.

[0247] In one aspect, the present disclosure provides methods for treating cancer in a subject, methods for reducing the rate at which a tumor volume in a subject increases over time, methods for reducing the risk of developing metastasis, or methods for reducing the risk of a subject developing additional metastasis. In some embodiments, treatment can stop, slow, delay, or inhibit the progression of cancer. In some embodiments, treatment can result in a decrease in the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0248] In one aspect, the present disclosure is characterized in that following methods, these methods include administering a therapeutically effective amount of antibody disclosed herein or its antigen-binding fragment to a subject in need (e.g., suffering from or being identified as suffering from or being diagnosed as suffering from cancer), the cancer is such as breast cancer (e.g., triple negative breast cancer), carcinoid, cervical cancer, endometrial cancer, neural cancer, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer or hematological malignancies. In certain embodiments, cancer is unresectable melanoma or metastatic melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer or metastatic hormone-refractory prostate cancer. In certain embodiments, the subject suffers from solid tumors. In certain embodiments, cancer is head and neck squamous cell carcinoma (SCCHN), renal cell carcinoma (RCC), triple negative breast cancer (TNBC) or colorectal cancer. In certain embodiments, the subject suffers from Hodgkin lymphoma. In some embodiments, the subject has triple negative breast cancer (TNBC), gastric cancer, urothelial carcinoma, Merkel cell carcinoma, or head and neck cancer. In some embodiments, the cancer is melanoma, pancreatic cancer, mesothelioma, glioma, hematological malignancy, particularly non-Hodgkin's lymphoma, lymphoma, chronic lymphocytic leukemia or advanced solid tumors.

[0249] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk for cancer.A variety of methods known in the art can be used to identify patients with cancer.

[0250] In some aspects, the disclosure relates to a method of treating an autoimmune disease or inflammation, comprising administering to a subject an effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described herein, or an antibody-drug conjugate as described herein.

[0251] In one aspect, present disclosure provides methods for treating, preventing, or reducing the risk of developing an abnormal or undesirable immune response disorder (e.g., autoimmune disorder) by affecting the function of Treg. 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 nodularis, cardiomyopathy, polychondritis, celiac disease-dermatitis, polyglandular syndrome, chronic fatigue syndrome (CFIDS), polymyalgia rheumatica, chronic inflammatory demyelinating disease, polymyositis and dermatomyositis, chronic inflammatory polyneuropathy, primary agammaglobulinemia, Churg-Strauss syndrome (Churg-Strauss syndrome), inflammatory bowel disease, myelopathy ... syndrome), primary biliary cirrhosis, cicatricial pemphigoid, psoriasis, CREST syndrome, Raynaud's phenomenon, cold agglutinin disease, Reiter's syndrome, Crohn's disease, rheumatic fever, discoid lupus, rheumatoid arthritis, cryoglobulinemia, sarcoidosis, fibromyalgia, scleroderma, Graves' disease, Sjögren's syndrome, Guillain-Barré disease, stiff-person syndrome, Hashimoto's thyroiditis, Takayasu arteritis, idiopathic pulmonary fibrosis, temporal arteritis / giant cell arteritis, idiopathic thrombocytopenic purpura (ITP), ulcerative colitis, IgA nephropathy, uveitis, diabetes mellitus (e.g., type 1), vasculitis, lichen planus, and vitiligo. Anti-TNFR2 antibodies or antigen-binding fragments thereof can also be administered to a subject to treat, prevent, or reduce the risk of developing a disorder associated with an abnormal or undesirable immune response associated with cell, tissue, or organ transplantation (e.g., kidney transplantation, liver transplantation, and heart transplantation), such as 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 antibodies or antigen-binding fragments can be used to treat inflammation.

[0252] In some embodiments, the antibody or antigen-binding fragment thereof is a TNFR2 agonist.

[0253] In some aspects, the present disclosure relates to a method of suppressing an immune response in a subject, the method comprising administering to the subject an effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described herein, or an antibody-drug conjugate as described herein. In some embodiments, the subject suffers from an autoimmune disease.

[0254] As used herein, an "effective amount" refers to an amount or dosage sufficient to achieve a beneficial or desired result, including stopping, slowing, delaying, or inhibiting the progression of a disease (e.g., cancer). The effective amount will depend, for example, on the age and weight of the subject to whom the antibody, antigen-binding fragment, polynucleotide encoding the antibody, vector comprising the polynucleotide, and / or composition thereof is to be administered, the severity of the symptoms, and the route of administration, and thus can be determined on an individual basis.

[0255] Effective amount can be administered once or repeatedly.For example, the effective amount of an antibody or antigen binding fragment is enough to improve, stop, stabilize, reverse, suppress, slow down and / or delay the amount of patient's autoimmune disease or cancer progression, or is enough to improve, stop, stabilize, reverse, slow down and / or delay the amount of in vitro cell (for example, biopsy cell, any cancer cell as described herein or cell line (for example, cancer cell line)) propagation.As understood in the art, the effective amount of an antibody or antigen binding fragment may be different, specifically can depend on patient's medical history and other factors, such as the type (and / or dosage) of antibody used.

[0256] The effective amount and schedule for administering the antibodies, polynucleotides encoding the antibodies, and / or compositions disclosed herein can be determined empirically, and making such determinations is within the skill of the art. One skilled in the art will appreciate that the dosage that must be administered will depend, for example, on the mammal to which the antibodies, polynucleotides encoding the antibodies, and / or compositions disclosed herein will be administered, the route of administration, the specific type of antibodies, polynucleotides encoding the antibodies, antigen-binding fragments, and / or compositions disclosed herein used, and other drugs administered to the mammal.

[0257] A typical daily dosage of an effective amount of an antibody is 0.01 mg / kg to 100 mg / kg (mg / kg patient body weight). In some embodiments, the dosage can be less than 100 mg / kg, 50 mg / kg, 40 mg / kg, 30 mg / kg, 25 mg / kg, 20 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, 0.3 mg / kg, or 0.1 mg / kg. In some embodiments, the dose may be greater than 50 mg / kg, 40 mg / kg, 30 mg / kg, 20 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, 0.3 mg / kg, 0.1 mg / kg, 0.05 mg / kg, or 0.01 mg / kg. In some embodiments, the dose is about 50 mg / kg, 40 mg / kg, 30 mg / kg, 25 mg / kg, 20 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, 0.9 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.

[0258] In any of the methods described herein, at least one antibody, its antigen-binding fragment or pharmaceutical composition (e.g., any antibody, antigen-binding fragment or pharmaceutical composition 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 with the same composition (e.g., a liquid composition). In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered with the same composition (e.g., a liquid composition containing at least one antibody or antigen-binding fragment and a solid oral composition containing at least one additional therapeutic agent). In some embodiments, at least one additional therapeutic agent is administered in the form of a pill, tablet, or capsule. In some embodiments, at least one additional therapeutic agent is administered as a sustained-release oral formulation.

[0259] In some embodiments, one or more additional therapeutic agents can be administered to a subject before or after administration of at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any described herein). In some embodiments, one or more additional therapeutic agents and at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any described herein) are administered to a subject such that the biological activity periods of the one or more additional therapeutic agents and the at least one antibody or antigen-binding fragment (e.g., any described herein) in the subject overlap.

[0260] In some embodiments, at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) can be administered to a subject over an extended period of time (e.g., over a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years). A skilled medical professional can determine the length of the treatment period using any of the methods described herein for diagnosing or following up on the effectiveness of treatment (e.g., observing at least one symptom of cancer). As described herein, a skilled medical professional can also change the type and amount (e.g., increase or decrease) of the antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) administered to the subject, and can also adjust (e.g., increase or decrease) the dose or frequency of at least one antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) administered to the subject based on an assessment of the effectiveness of treatment (e.g., using any of the methods described herein and known in the art).

[0261] In certain embodiments, one or more other therapeutic agents can be administered to a subject.Other therapeutic agents can include one or more inhibitors selected from the group consisting of: B-Raf inhibitors, EGFR inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c-Met inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, Akt inhibitors, mTOR inhibitors, PI3K / mTOR dual inhibitors, Bruton (Bruton) tyrosine kinase (BTK) inhibitors and isocitrate dehydrogenase 1 (IDH1) inhibitors and / or isocitrate dehydrogenase 2 (IDH2) inhibitors.In certain embodiments, other therapeutic agents are indoleamine 2,3-dioxygenase -1 (IDO1) inhibitors (e.g., epacadostat).

[0262] In some embodiments, the additional therapeutic agent may comprise one or more inhibitors selected from the group consisting of: a HER3 inhibitor, a LSD1 inhibitor, an MDM2 inhibitor, a BCL2 inhibitor, a CHK1 inhibitor, an activated hedgehog signaling pathway inhibitor, and an agent that selectively degrades the estrogen receptor.

[0263] In some embodiments, the additional therapeutic agent can include one or more therapeutic agents selected from the group consisting of: Trabectedin, Nab-paclitaxel, trebananib, pazopanib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Axinda, ceritinib, sunitinib, temsirolimus, axitinib, everolimus, sorafenib, Votrient, pazopanib, IMA-901, AGS-003, cabozantinib, vinflunine, Hsp90 inhibitors, Ad-GM-CSF, temozolomide, IL-2, interferon nab, vinblastine, thalidomide, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomid, amrubicin, carfilzomib, pralatrexate, and enzastaurin.

[0264] In some embodiments, the additional therapeutic agent may comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, IL-1, HMGB 1, an IL-10 antagonist, an IL-4 antagonist, an IL-6 antagonist (e.g., IL-6 receptor), an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a CX3CL1-targeted therapy, a CXCL9-targeted therapy, a CXCL10-targeted therapy, a CCL5-targeted therapy, an LFA-1 agonist, an ICAM1 agonist, and a select agonist.

[0265] In some embodiments, the subject is administered carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI.

[0266] In some embodiments, the additional therapeutic agent is an anti-OX40 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, an anti-GITR antibody, an anti-TIM-3 antibody, or an anti-CD40 antibody.

[0267] Pharmaceutical compositions and routes of administration

[0268] Also provided herein are pharmaceutical compositions containing 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 the pharmaceutical composition in any combination. The pharmaceutical composition can be formulated in any manner known in the art.

[0269] Pharmaceutical composition can be formulated into and is compatible with its expected route of administration (for example intravenous, intra-arterial, intramuscular, intradermal, subcutaneous or intraperitoneal). Composition can include sterile diluent (for example sterile water or sterile saline), fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvents, antibacterial or antifungal (for example benzyl alcohol or methyl parahydroxybenzoate, chlorobutanol, phenol, ascorbic acid, thimerosal etc.), antioxidant (for example ascorbic acid or sodium bisulfite), chelating agent (for example ethylenediaminetetraacetic acid), buffer (for example acetate, citrate or phosphate) and isotonic agent (for example sugar, such as glucose), polyol (for example mannitol or sorbitol) or salt (for example sodium chloride) or its any combination. Liposomal suspension can also be used as pharmaceutically acceptable carrier. The preparation of composition can be prepared and it is packaged in ampoule, disposable syringe or multiple dose bottle. If desired (e.g., in the form of an injectable formulation), suitable fluidity can be maintained by, for example, using a coating such as lecithin or a surfactant. The absorption of the antibody or its Fab can be prolonged by integrating agents that delay absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled release can be achieved by implants and microencapsulated delivery systems that can include biodegradable biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid).

[0270] 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).

[0271] The pharmaceutical composition for parenteral administration is preferably sterile and substantially isotonic and manufactured under good manufacturing practice (GMP) conditions. The pharmaceutical composition can be provided in unit dosage form (i.e., a single dose). The pharmaceutical composition can be formulated using one or more physiologically acceptable carriers, diluents, excipients or adjuvants. The formulation depends on the selected route of administration. For injection, the antibody can be formulated in an aqueous solution, preferably in a physiologically compatible buffer, to reduce discomfort at the injection site. The solution can contain a preparaton, such as a suspending agent, a stabilizer and / or a dispersant. Alternatively, the antibody can be in a lyophilized form before use for composition with a suitable vehicle (e.g., sterile pyrogen-free water).

[0272] The toxicity and therapeutic efficacy of the composition can be determined by standard pharmaceutical procedures in cell culture or experimental animals (e.g., monkeys). For example, the LD50 (lethal dose for 50% of the population) and the ED50 (therapeutically effective dose for 50% of the population) can be determined: the therapeutic index is the ratio of LD50:ED50. Agents that exhibit a high therapeutic index are preferred. When an agent exhibits adverse side effects, care should be taken to minimize potential harm (i.e., reduce undesirable side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

[0273] The data obtained from cell culture assays and animal studies can be used to prepare any given medicament for use in a subject (e.g., people) of appropriate dose. The therapeutically effective amount of one or more (e.g., one, two, three or four) antibodies or their antigen-binding fragments (e.g., any antibody or antibody fragment as described herein) will be in a subject (e.g., a human subject identified as having cancer or identified as having a subject in a risk of developing a disease, such as a subject previously suffering from cancer but now cured) to treat the disease of the subject (e.g., killing cancer cells), reducing the severity, frequency and / or duration of one or more symptoms of the subject (e.g., people). Methods known in the art can be used, and by observing one or more symptoms of the subject (e.g., people), the effectiveness and administration of any antibody or antigen-binding fragment as described herein can be determined by a healthcare professional or veterinary professional. Certain factors (e.g., the severity of the disease or disorder, previous treatment, the general health status and / or age of the subject, and other diseases present) can affect the dosage and time course required for the effective treatment of the subject.

[0274] Exemplary dosages include milligram or microgram amounts per kilogram of subject body weight of any antibody or antigen-binding fragment described herein (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; about 1 μg / kg to about 50 μg / kg; about 0.3 mg / kg to about 25 mg / kg, about 1 mg / kg to about 10 mg / kg; or about 1 mg / kg to about 5 mg / kg). Although these dosages cover a wide range, it will be understood by those of ordinary skill in the art that therapeutic agents, including antibodies and antigen-binding fragments thereof, vary in their efficacy and that an effective amount can be determined by methods known in the art. Typically, a relatively low dose is administered first, and the attending healthcare professional or veterinary professional (in the case of therapeutic applications) or researcher (when still in the development stage) can subsequently and gradually increase the dose until an appropriate response is obtained. Furthermore, it will be understood that the specific dosage level for any particular subject will depend on a variety of factors, including the activity of the specific compound used, the subject's age, weight, general health, sex, and diet, time of administration, route of administration, rate of excretion, and in vivo half-life of the antibody or antibody fragment.

[0275] The pharmaceutical composition can be included in a container, pack, or dispenser together with instructions for administration.The present disclosure also provides methods of making antibodies or antigen-binding fragments thereof for the various uses as described herein.

[0276] Examples

[0277] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0278] Example 1. Generation of anti-hTNFR2 antibodies

[0279] To generate antibodies against human TNFR2 (TNFR2; SEQ ID NO: 1), RenMab mice were immunized with human TNFR2. Anti-TNFR2 antibodies were prepared as described below.

[0280] RenMab mice have both humanized heavy chain immunoglobulin loci and humanized kappa chain immunoglobulin loci. The heavy chain immunoglobulin loci are regions on chromosomes containing antibody heavy chain genes. The loci include IGHV (variable), IGHD (diversity), IGHJ (connection) and heavy chain constant domain genes. The kappa chain immunoglobulin loci are regions on chromosomes containing genes encoding antibody light chains (kappa chains). The kappa chain immunoglobulin loci include IGKV (variable), IGKJ (connection) and light chain constant domain genes. A detailed description of RenMab mice can be found in PCT / CN2020 / 075698, which is incorporated herein by reference in its entirety.

[0281] Mouse immunization

[0282] RenMab mice were immunized with an Fc-tagged human TNFR2 protein (Fc fusion protein containing the extracellular domain of human TNFR2, positions 23aa-257aa of SEQ ID NO: 1). The Fc-tagged human TNFR2 protein was emulsified with adjuvant and injected into four locations on the back of the mice. For the first subcutaneous (sc) injection, the diluted antigen was emulsified with an equal volume of complete Freund's adjuvant (CFA). For subsequent sc injections, the protein was emulsified with an equal volume of incomplete Freund's adjuvant (IFA). At least four injections were performed, with at least 14 days between each injection. Seven days after the third injection or booster immunization, blood (serum) was collected and analyzed for antibody titer using fluorescence-activated cell sorting (FACS).

[0283] In another experiment, several mice were immunized by injecting an expression plasmid encoding human TNFR2 into the mice. The plasmid encoding the antigen was injected into the tibialis anterior muscle (intramuscular injection; im injection). At least four injections were performed, with at least 14 days between each injection. Blood (serum) was collected seven days after the last immunization and the serum was tested for antibody titer by ELISA.

[0284] In addition, a booster immunization procedure is performed at least fourteen days after immunization (by plasmid injection or protein injection). CHO cells expressing the TNFR2 antigen on their surface are injected intravenously into the mice via the tail vein. Immune system organs (e.g., bone marrow, lymph nodes, spleen, etc.) are then collected four days after injection.

[0285] Spleen cells were collected and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines. Hybridoma cells were screened and selected to identify cell lines that produced TNFR2-specific antibodies. Using this technology and the above-mentioned immunogen, several anti-TNFR2 chimeric antibodies (i.e., antibodies having human variable domains and mouse constant domains) were obtained. Specifically, after pulse immunization, mouse immune organs were harvested and plasma cells were separated by magnetic beads. Monoclonal hybridoma cells that secreted antigen-specific monoclonal antibodies were screened using hybridoma fusion technology. Antibody light chain and heavy chain V region sequences were obtained from selected monoclonal hybridoma cells by reverse transcription and PCR sequencing. The antibody light chain and heavy chain V region sequences were constructed into antibody expression vectors and verified using the ExpiCHO-STM cell expression system. Cells were transfected in a 24-well system, and antibodies were collected in the supernatant on day 3. FACS was used to verify the specificity of binding between the antibody and TNFR2. Using this technology, several anti-TNFR2 chimeric antibodies (i.e., antibodies having human variable domains and mouse constant domains) were obtained. The constant domains in these antibodies can be easily replaced to obtain fully human anti-TNFR2 antibodies (anti-hTNFR2 antibodies). Exemplary fully human antibodies obtained by this method are named as follows: 14-1B3-hHvKv ("14-1B3" or "1B3") and 14-4A9-hHvKv ("14-4A9" or "4A9"), etc. Taking 14-4A9-hHvKv as an example, when the antibody VH\VL is linked to a different subtype, such as IgG1, the antibody is named 14-4A9-hHvKv-IgG1.

[0286] Antigen-positive B cells were also isolated directly from immunized mice without fusion with myeloma cells. Anti-TNFR2 antibodies were further isolated from antigen-positive B cells. The light chain and heavy chain V region sequences of the antibodies were obtained directly from antigen-positive B cells. For example, single cell techniques (e.g., The Optofluidic system, Digital Cell Biology, Inc. (Berkeley Lights Inc.) is used to screen and discover plasma cells that secrete antigen-specific monoclonal antibodies. The antibody V region sequence is obtained using reverse transcription and PCR sequencing. The antibody is expressed. FACS is used to verify the binding between the antibody and TNFR2. Exemplary antibodies obtained by this method include: BC-1A8 ("1A8"), BC-1B6 ("1B6"), BC-1C3 ("1C3"), BC-1F4 ("1F4"), BC-1F10 ("1F10"), and BC-3B7 ("3B7"). Taking BC-1F4 as an example, when the antibody VH\VL is linked to different subtypes such as the IgG1 subtype, the antibody is named BC-1F4-IgG1. Examples of other subtypes are as follows: BC-1F4-IgG1-SI, BC-1F4-IgG1-LALA.

[0287] The heavy and light chain CDR1, CDR2, and CDR3 amino acid sequences of 1A8 are shown in SEQ ID NOs: 6-11 (Kabat numbering) or SEQ ID NOs: 42-47 (Chothia numbering). The human heavy chain variable region and human light chain variable region of the antibody are shown in SEQ ID NO: 36 or SEQ ID NO: 37.

[0288] The heavy and light chain CDR1, CDR2, and CDR3 amino acid sequences of 1B6 are shown in SEQ ID NOs: 12-17 (Kabat numbering) or SEQ ID NOs: 48-53 (Chothia numbering). The human heavy chain variable region and human light chain variable region of the antibody are shown in SEQ ID NO: 38 or SEQ ID NO: 39.

[0289] The heavy and light chain CDR1, CDR2, and CDR3 amino acid sequences of 1C3 are shown in SEQ ID NOs: 18-23 (Kabat numbering) or SEQ ID NOs: 54-59 (Chothia numbering). The human heavy chain variable region and human light chain variable region of the antibody are shown in SEQ ID NO: 40 or SEQ ID NO: 41.

[0290] The heavy and light chain CDR1, CDR2, and CDR3 amino acid sequences of 1F4 are shown in SEQ ID NOs: 24-29 (Kabat numbering) or SEQ ID NOs: 60-65 (Chothia numbering). The human heavy chain variable region and human light chain variable region of the antibody are shown in SEQ ID NO: 72 or SEQ ID NO: 73.

[0291] The heavy and light chain CDR1, CDR2, and CDR3 amino acid sequences of 1F10 are shown in SEQ ID NOs: 30-35 (Kabat numbering) or SEQ ID NOs: 66-71 (Chothia numbering). The human heavy chain variable region and human light chain variable region of the antibody are shown in SEQ ID NO: 74 or SEQ ID NO: 75.

[0292] Antibody preparation

[0293] Positive antibody sequences from the sequence verification phase were plasmid-extracted and transfected into a 25 mL system. After 10-12 days of cell culture, the expression supernatant was collected and subjected to affinity chromatography. The resulting antibody samples were used for the following in vitro testing and screening.

[0294] Example 2. In vitro testing of anti-TNFR2 antibodies

[0295] Blocks the binding of human TNFR2 to TNFα

[0296] Blocking assays were performed to determine whether anti-TNFR2 antibodies could block the binding between TNFR2 and its ligand hTNFα.

[0297] Specifically, 30 μl of CHO cells (1×10 5 Cells were added to each well of the plate. Purified antibodies were titrated to final concentrations of 10, 2.5, 0.625, 0.1565, and 0.039 μg / mL. 30 μl / well of titrated antibodies were added to each well and incubated at 4°C for 30 minutes.

[0298] 30 μl of biotin-hTNFα (AcroBiosystems, catalog number: TNA-H82E1) was added to each well (final concentration in each well was 0.5 μg / mL) and the cells with biotin-hTNFα and the antibody were incubated at 4° C. for 30 minutes.

[0299] After washing twice with phosphate buffered saline (PBS), 50 μl of PE-labeled anti-human IgG Fc antibody (PE anti-human IgG Fc, Jackson ImmunoResearch, catalog number: 109-115-098) diluted 1:100 and Alexa Fluor 500 diluted 1:500 were added. 647-labeled streptavidin (AF647 Streptavidin, Jackson ImmunoResearch, catalog number: 016-600-084) was added to each well and incubated at 4°C for 15 minutes, followed by washing with PBS. The signals of AF647 and PE were determined by flow cytometry (ThermoAttuneNX).

[0300] Table 1 below shows the percentage of cells with the test of streptavidin signal in flow cytometry analysis.If the percentage of test cells with streptavidin signal (AF647) increases and antibody concentration decreases, then the antibody has blocking affinity (showing that binding affinity is strong).According to the data, BC-1A8-IgG1, BC-1F4-IgG1, BC-3B7-IgG1 and BC-1F10-IgG1 have a strong blocking effect.But BC-1B6-IgG1, BC-1C3-IgG1, 14-1B3-hHvKv-IgG1 and 14-4A9-hHvKv-IgG1 can not effectively block the combination between TNFR2 and TNFα.

[0301] Table 1

[0302]

[0303]

[0304] Binding affinity of anti-TNFR2 antibodies to human TNFR2 and TNFR1

[0305] The binding affinity of anti-TNFR2 antibodies to human TNFR2 and TNFR1 (tumor necrosis factor receptor 1) was measured by surface plasmon resonance (SPR) using a Biacore (Biacore, INC, Piscataway, NJ) 8K biosensor equipped with a pre-immobilized Protein A sensor chip.

[0306] Purified anti-TNFR2 antibody was diluted to 1 μg / mL and then injected into the Biacore 8K biosensor at 10 μL / min for approximately 50 seconds to achieve the desired protein density (e.g., approximately 50 response units (RU)). His-tagged human TNFR1 (human TNFR1 / CD120a / TNFRSF1A protein, His-tag, Beijing Acrobiosystems Co., Ltd., catalog number: TN1-5222) or TNFR2 (human TNFR2 / CD120b / TNFRSF1B protein, His-tag, Beijing Acrobiosystems Co., Ltd., catalog number: TN2-5227) was then injected at 30 μL / min for 120 seconds at a concentration of 200, 100, 50, 25, 6.25, or 1.56 nM. Dissociation was monitored for 600 seconds. The chip was regenerated after the last injection of each glycine titration (pH 2.0, 30 μL / min, for 30 seconds).

[0307] The data were fitted to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. Methods Enzymology 6.99-110) using Biacore 8K Evaluation Software 3.0, thereby simultaneously obtaining kinetic association rates (k on ) and dissociation rates (k off ). Affinity was derived from the quotient of the kinetic rate constants (K D = k off / k on ).

[0308] As will be understood by one of ordinary skill in the art, the same method was followed for each antibody tested, with parameters (eg, antibody concentration) adjusted appropriately. Table 2 below summarizes the results for the antibodies tested.

[0309] Table 2

[0310]

[0311]

[0312] The results showed that these human antibodies had very high binding affinity to human TNFR2. None of the eight antibodies (BC-1A8-IgG1, BC-1B6-IgG1, BC-1C3-IgG1, BC-1F4-IgG1, BC-1F10-IgG1, 14-1B3-hHvKv-IgG1, and 14-4A9-hHvKv-IgG1) could bind to TNFR1.

[0313] Binding affinity of anti-TNFR2 antibodies to monkey TNFR2

[0314] Similar to the binding affinity experiments described above, the binding affinity of the anti-TNFR2 antibodies BC-1B6-IgG1, BC-1C3-IgG1, and BC-1F4-IgG1 to His-tagged monkey (Crab-eating monkey) TNFR2 (fINFR2-His, Sino Biological, Catalog No. 90102-C08H) was measured. The results are summarized in Table 3, which show that all three anti-TNFR2 antibodies bind to monkey TNFR2 with good binding affinity.

[0315] Table 3

[0316]

[0317] Cross-reactivity of anti-TNFR2 antibodies to mouse, canine, and monkey TNFR2

[0318] In each experiment, CHO cells were transfected with EGFP and human TNFR2 (TNFR2, SEQ ID NO: 1), mouse TNFR2 (mTNFR2, SEQ ID NO: 2), fINFR2, or dog (canine) TNFR2 (dTNFR2, SEQ ID NO: 4).

[0319] 30 μl CHO cells (1×10 5 30 μl of purified anti-TNFR2 antibody (10 μg / mL) (as listed in Table 4) was added to each well and incubated at 4°C for 30 minutes.

[0320] After washing twice with PBS (1600 rpm, 6 min), 50 μl of Alexa Fluor-labeled anti-human IgG Fc antibody (BC-1A8-IgG1, BC-1B6-IgG1, BC-1C3-IgG1, BC-1F4-IgG1, BC-1F10-IgG, BC-3B7-IgG1, 14-1B3-hHvKv-IgG1, 14-4A9-hHvKv-IgG1) was added to each well at a dilution of 1:500, incubated at 4°C for 15 minutes, and subsequently washed with PBS (1200 rpm, 5 min). The signal of AF647 was detected by flow cytometry.

[0321] The table below summarizes the cross-reactivity of the antibodies tested with human (TNFR2), mouse (mTNFR2), monkey (fTNFR2), and dog (dTNFR2) TNFR2.

[0322] Table 4

[0323]

[0324] Epitope correlation analysis of purified anti-hTNFR2 antibodies

[0325] Surface plasmon resonance (SPR) competition experiments were performed to analyze the relative positions of target protein epitopes between a pair of purified anti-TNFR2 monoclonal antibodies. Five monoclonal antibodies were used to investigate the binding inhibition (blocking) of each antibody against the other: BC-1A8-IgG1, BC-1F4-IgG1, BC-3B7-IgG1, BC-1C3-IgG1, and BC-1F10-IgG1. HBS-EP+ buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 150 mM NaCl, 3 mM ethylenediaminetetraacetic acid (EDTA), and 0.05% P20, pH 7.4) was used as the running buffer throughout the experiment. Anti-His antibodies were immobilized on the surface of an S-series sensor chip CM5 via amino conjugation to generate an anti-His chip (i.e., CM5-anti-His-channel 1, 8-chip). Then, 1M ethanolamine (pH 8.5) was injected to block the remaining active carboxyl groups on the chip surface, followed by balancing with HBS-EP+ buffer for 2 hours. Recombinant human TNFR2 protein (1 μg / mL) with a His tag was injected into the Biacore 8K biosensor at 10 μL / min for 50 seconds and captured on the anti-His chip to achieve the desired protein density (i.e., 200 RU). A pair of antibodies (200 nM each) was continuously injected onto the chip at 30 μL / min. The binding time of the first injected antibody (analyte 1) was 250 seconds, and the binding time of the second antibody (analyte 2) was then 250 seconds. After the antibody was injected in each analysis cycle, the chip was regenerated twice with glycine buffer (pH 1.7; 30 μL / min for 30 seconds). When each monoclonal antibody was paired with another antibody, the same experimental steps were performed for each pair of monoclonal antibodies to obtain binding inhibition data.

[0326] The binding value of each antibody was obtained using Biacore Insight evaluation software. To quantify the interference of one antibody with the binding of another antibody, the binding ratio was calculated to compare each pair of antibodies. The binding ratio was defined as the binding value of the second antibody (analyte 2) divided by the binding value of the first antibody (analyte 1). Statistical software was also used for cluster analysis. Epitope correlation was analyzed and the five anti-hTNFR2 antibodies were classified into four epitope clusters ( Figure 8 In summary, 1A8 and 1F4 share the same or overlapping epitopes. 3B7, 1C3, and F10 do not show epitope correlation with other antibodies.

[0327] TNFR2 has four cysteine-rich domains (CRDs). In another experiment, FACS was used to detect the binding of anti-hTNFR2 antibodies to TNFR2 proteins with different domains. 647AffiniPure F(ab′)2 fragment goat anti-human IgG, Fcγ fragment specific, Jackson ImmunoResearch Laboratories, Inc. (Cat. No. 151524). A fluorescently labeled secondary antibody can be attached to the Fc region of the anti-hTNFR2 antibody, allowing detection of anti-hTNFR2 antibody binding to ΔCRD cells by FACS. Specifically, ΔCRD1-TNFR2 protein (CRD1 deletion), ΔCRD2-TNFR2 protein (CRD2 deletion), ΔCRD3-TNFR2 protein (CRD3 deletion), or ΔCRD4-TNFR2 protein (CRD4 deletion) are expressed in CHO-S cells, and the binding between the cells and the different anti-hTNFR2 antibodies is detected. The TNFR2 protein is shown in SEQ ID NO: 1, CRD1 is sequence 39aa-76aa, CRD2 is sequence 77aa-118aa, CRD3 is sequence 119aa-162aa, and CRD4 is sequence 168aa-196aa. The results of the flow cytometry experiments are shown in Table 5 below.

[0328] The results showed that 1A8 and 1F4 did not bind to TNFR2 lacking CRD3, and the positive rate of binding to TNFR2 lacking CRD4 was also significantly reduced, indicating that 1A8 and 1F4 have similar binding epitopes for human TNFR2, and these epitopes are likely to be located at CRD3 (class I epitope). 1B6 and 1C3 did not bind to TNFR2 lacking CRD3 or CRD4, indicating that 1B6 and 1C3 have similar binding epitopes for human TNFR2, and these epitopes are likely to be located at the junction of CRD3 and CRD4 (class III epitope). The positive rate of binding between 1F10 and TNFR2 lacking CRD3 or CRD4 was significantly reduced, indicating that the binding epitope of 1F10 is different from the class I epitope and the class III epitope (belonging to the class IV epitope). The results are consistent with the SPR epitope characterization.

[0329] Table 5

[0330]

[0331] In vitro ADCC assay

[0332] Experiments were performed to evaluate the ADCC effect of anti-TNFR2 antibodies. BC-1A8-IgG1, BC-1C3-IgG1, BC-1F4-IgG1, BC-1F10-IgG1, BC-1B6-IgG1, and isotype control human IgG1 (Crown Bioscience Inc., C0001-3) were used in the experiments.

[0333] Lactate dehydrogenase (LDH) is a cytoplasmic enzyme found in many different cell types and is released into the cell culture medium upon plasma membrane damage. The CyQUANT LDH Cytotoxicity Assay Kit (Invitrogen, catalog number: C20301) is used to accurately quantify extracellular LDH to assess the ADCC effect of antibodies.

[0334] The anti-TNFR2 antibody was serially diluted (10-fold) to a maximum concentration of 100 μg / mL. Target cells (MC38 cells overexpressing human TNFR2) were seeded in 96-well plates (cell density of 2×10 4 Cells / well, 100 μL) were added and incubated at 37°C for 3-4 hours. Effector cells (peripheral blood mononuclear cells (PBMC)) were revived (cell density was 2×10 4 Cells / well). The same volume (100 μL) of effector cells was added to each well of a 96-well plate along with 10 μL of antibody. The 96-well plate was incubated overnight at 37°C. The absorbance at 490 nm and 680 nm was measured using a microplate reader and used to calculate the target cell killing (% cytotoxicity) of each antibody group. EC50 values ​​were calculated using nonlinear fitting, using antibody concentration as the horizontal axis and cytotoxicity as the vertical axis.

[0335]

[0336] In the above formula, Experimental refers to the absorbance value of the experimental well; Effector refers to the absorbance value of the effector cells. Target refers to the absorbance value of the target cells. Spontaneous refers to the autofluorescence absorbance value of the cell culture medium. TargetMax refers to the maximum absorbance value of the target cells. Spontaneous* refers to the absorbance value of the cell culture medium volume control well.

[0337] The EC50 results are shown in Table 6. Compared to the isotype control hIgG1, the killing of target cells (% cytotoxicity) by the anti-TNFR2 antibodies (BC-1A8-IgG1, BC-1C3-IgG1, BC-1F4-IgG1, BC-1F10-IgG1, or BC-1B6-IgG1) increased with increasing doses of these antibodies, indicating that BC-1A8-IgG1, BC-1C3-IgG1, BC-1F4-IgG1, BC-1F10-IgG1, and BC-1B6-IgG1 all have ADCC activity.

[0338] Table 6

[0339] protein ADCC EC50 value (ng / mL) BC-1A8-IgG1 have 86.41 BC-1B6-IgG1 have 16.00 BC-1C3-IgG1 have 96.94 BC-1F4-IgG1 have 40.25 BC-1F10-IgG1 have 161.50 hIgG1 none -

[0340] In another similar experiment, the ADCC effect of anti-TNFR2 antibodies (BC-1C3-IgG1, BC-1C3-IgG1-SI and BC-1C3-IgG1-LALA) was evaluated using target cells (MC38 cells overexpressing human TNFR2) and effector cells (FcR-TANK (CD16a-158V) cell line, ImmuneOncoBiopharmaceuticals (Shanghai) Co., Ltd.).

[0341] Cytotoxicity data were Figure 9 The EC50 results are shown in Table 7. Compared with the isotype control hIgG1, the killing (% cytotoxicity) of target cells by anti-hTNFR2 antibodies (BC-1C3-IgG1 and BC-1C3-IgG1-SI) increased with increasing doses of these antibodies, indicating that BC-1C3-IgG1-SI has strong ADCC activity.

[0342] Table 7

[0343]

[0344] Example 3. In vivo testing of anti-hTNFR2 antibodies

[0345] To test antibodies in vivo and predict their effects in humans, a humanized TNFR2 mouse model was generated. The humanized TNFR2 mouse model was engineered to express a chimeric TNFR2 protein (SEQ ID NO: 5) in which the extracellular domain of the mouse TNFR2 protein was replaced with the corresponding extracellular domain of human TNFR2. Amino acid residues 33-260 of mouse TNFR2 (SEQ ID NO: 2) were replaced with amino acid residues 33-259 of human TNFR2 (SEQ ID NO: 1).

[0346] Humanized TNFR2 mouse models (e.g., B-TNFR2 mice) provide a new tool for testing new therapeutic treatments in a clinical setting by significantly reducing the difference between the clinical outcomes of humans and ordinary mice expressing mouse TNFR2. A detailed description of the humanized TNFR2 mouse model can be found in PCT / CN2020 / 113618; which is incorporated herein by reference in its entirety.

[0347] Anti-hTNFR2 antibodies were tested in TNFR2 humanized mice (B-TNFR2) to demonstrate their effects on tumor growth in vivo.

[0348] In vivo efficacy in B-TNFR2 mice with MC38, 10 mg / kg

[0349] MC-38 tumor cells (colon adenocarcinoma cells) were injected subcutaneously into B-TNFR2 mice. When the tumor volume of the mice reached 100 mm 3 -150mm 3 At the same time, the mice were randomly divided into different groups (six mice in each group) according to the volume of the tumor.

[0350] Mice were then injected with PBS as a control (G1), anti-hTNFR2 antibodies BC-1F4-IgG1 (G2), BC-3B7-IgG1 (G3), BC-1F10-IgG (G4), 14-1B3-hHvKv-IgG1 (G5), BC-1A8-IgG1 (G6), BC-1C3-IgG1 (G7), 14-4A9-hHvKv-IgG1 (G8), and anti-mPD-1 (G9) (BIO X CELL, catalog number: BE0146). Anti-mPD-1, which has been shown to be effective in mice, was used as a positive control. The antibodies were administered intraperitoneally at 10 mg / kg on the first and fourth days of each week for 3 weeks (a total of 6 injections).

[0351] The body weight of the mice was monitored throughout the treatment period. The weight of the mice increased across the different groups, with no statistically significant differences (P>0.05). On the day of grouping (day 0; "D0"), the average body weight of each group ranged from 20.3 g to 21.4 g. At the end of the experiment (28 days after grouping, D28), the average body weight of each group ranged from 22.7 g to 24.7 g, with a body weight change of 107.4% to 116.5%. The results demonstrate that the anti-hTNFR2 antibody was well tolerated and non-toxic to mice.

[0352] In the group treated with anti-hTNFR2 antibody, tumor size ( Figure 1 ) showed significant differences. TGI of each treatment group on day 28 (28 days after grouping) TV The percentages are shown in Table 8 below. The positive control (anti-mPD-1, G9) showed good therapeutic effects at the above doses and frequencies (TGITV% = 78.5%). Various antibodies including BC-1F4-IgG1 (G2), BC-1F10-IgG1 (G4), BC-1A8-IgG1 (G6), and BC-1C3-IgG1 (G7) also showed good therapeutic effects (TGITV% = 78.5%). TV %>60). Except for G3 and G5, the TGI of TNFR2 antibodies TV % is greater than 80%. The TGI TV % was better than the positive control group (G19 / anti-mPD-1 TGI TV %=78.5%).

[0353] Table 8

[0354]

[0355] In vivo efficacy in B-TNFR2 mice with MC38, 1 mg / kg

[0356] Similar to the aforementioned in vivo efficacy experiments, after establishing a tumor model, mice were injected with PBS as a control (G1), anti-hTNFR2 antibodies BC-1A8-IgG1 (G2), BC-1F10-IgG1 (G3), BC-1F4-IgG1 (G4), anti-mPD-1 (G5), and anti-mCTLA4 (G6) (BIO X CELL, catalog number: BE0164). Antibodies were administered intraperitoneally at 1 mg / kg on the first and fourth days of each week for 3 weeks (a total of 6 injections).

[0357] The body weight of the mice was monitored throughout the experiment. The weight of the mice increased across all groups, with no significant differences between the groups (P>0.05). At the time of grouping (D0), the mean body weight of each group ranged from 20.4 g to 20.8 g. At the end of the experiment (24 days after grouping, D24), the mean body weight of each group ranged from 21.6 g to 23.7 g, with a body weight change of 102.5% to 114.7%. Similar to previous experiments, the results showed that the anti-hTNFR2 antibody was well tolerated and non-toxic to mice.

[0358] Tumor size showed significant differences in the groups treated with anti-TNFR2 antibody ( Figure 2 In particular, the tumor sizes of G3, G4, and G5 were significantly smaller than those of G1 (G3: P = 0.005, G7: P = 0.001, G8: P = 0.011). As shown in Table 9 below, the TGI of each treatment group on day 24 (24 days after grouping) was also calculated. TV Compared with the positive control (G5, G6), G2, G3 and G4 showed better tumor inhibition (TGI TV %).

[0359] Table 9

[0360]

[0361] In vivo efficacy in B-TNFR2 mice with MC38, 0.3 mg / kg

[0362] In another similar experiment, the dose of the anti-hTNFR2 antibody to be tested was reduced to 0.3 mg / kg. When the tumor volume of the mice reached 100 ± 50 mm 3 Mice (bearing MC38 tumors) were randomly divided into different groups (six mice per group).

[0363] Mice were then injected with PBS as a control (G1), anti-hTNFR2 antibody BC-1C3-IgG1 (G2), anti-mPD-1 (G3), and anti-mCTLA4 (G4). Antibodies were administered intraperitoneally at 0.3 mg / kg on the first and fourth days of each week for 3 weeks (6 injections in total).

[0364] The body weight of the mice was monitored during the experiment. Weight gain was observed across all groups, with no significant differences between groups (P>0.05). At the time of grouping (D0), the mean body weight of each group ranged from 19.5 g to 19.8 g. At the end of the experiment (25 days after grouping, D25), the mean body weight ranged from 21.6 g to 23.3 g, with a body weight change of 108.9% to 118.2%.

[0365] Similar to previous results, the results showed that the anti-TNFR2 antibody was well tolerated and non-toxic to mice.

[0366] In the group treated with anti-hTNFR2 antibody, tumor size ( Figure 3 ) showed significant differences. As shown in Table 10 below, the TGI of each treatment group on day 25 (25 days after grouping) was calculated. TV %. BC-1C3-lgG1 (G2) showed the best efficacy at this dose and was superior to the positive controls (G3, G4).

[0367] Table 10

[0368]

[0369]

[0370] In vivo efficacy in B-TNFR2 mice with B16F10, 10 mg / kg

[0371] B16F10 tumor cells (melanoma cells) were subcutaneously injected into B-TNFR2 mice. When the tumor volume of the mice reached 100 ± 50 mm 3 At the same time, the mice were randomly divided into different groups (six mice in each group) according to the volume of the tumor.

[0372] Mice were then injected with PBS as a control (G1), anti-hTNFR2 antibodies BC-1A8-IgG1 (G2), BC-1C3-IgG1 (G3), BC-1F10-IgG1 (G4), BC-1F4-IgG1 (G5), and BC-1B6-IgG1 (G6). Antibodies were administered intraperitoneally at 10 mg / kg on the first and fourth days of each week for two weeks (a total of three injections).

[0373] The body weight of mice was monitored during the experiment. The body weight of mice increased across all groups, with no significant differences between groups (P>0.05). At grouping (D0), the average body weight of each group ranged from 19.2 g to 19.7 g. On day 10 (D10) after grouping, the average body weight of each group ranged from 22.1 g to 24.5 g, with a body weight change of 114.3% to 126.8%. These results demonstrate that the anti-hTNFR2 antibody was well tolerated and nontoxic to mice.

[0374] Tumor size showed significant differences in the groups treated with anti-TNFR2 antibody ( Figure 4 ). BC-1A8-IgG1 (G2), BC-1C3-IgG1 (G3), BC-1F10-IgG1 (G4) and BC-1B6-IgG1 (G6) all showed anti-tumor efficacy in the B16F10 melanoma model. The TGI of each treatment group at day 10 (10 days after grouping) was TV The % are shown in Table 11 below.

[0375] Table 11

[0376]

[0377] In vivo efficacy and toxicity in B-TNFR2 mice with MC38, 25 mg / kg

[0378] In another similar experiment, the dose of the anti-hTNFR2 antibody to be tested was adjusted to 25 mg / kg to test the efficacy and toxicity in vivo. MC38 cancer cells (colon adenocarcinoma cells) were injected subcutaneously into B-TNFR2 mice. When the tumor volume of the mice reached 100 ± 50 mm 3 At the same time, the mice were randomly divided into different groups (four mice in each group) according to the volume of the tumor.

[0379] Mice were then injected with PBS as a control (G1), anti-hTNFR2 antibodies BC-1A8-IgG1 (G2), BC-1C3-IgG1 (G3), BC-1F10-IgG1 (G4), BC-1F4-IgG1 (G5), and BC-1B6-IgG1 (G6). Antibodies were administered intraperitoneally at 25 mg / kg on the day of grouping (D0) and on day 3 (D3) after grouping (two injections in total).

[0380] The weight of mice was monitored during the experiment. The weight of mice in different groups increased, and there was no significant difference between the groups. The average weight of each group was in the range of 18.5g-19.2g. At the end of the experiment (21 days after grouping), the average weight of each group was in the range of 21.1g-23.3g, and the weight change was between 110.0%-122.7%. On the 5th day after grouping (D5), peripheral blood of mice was collected to test blood biochemical indicators (AST, ALT). The biochemical index test results on D5 ( Figures 7A-7B ) showed that ALT and AST did not change significantly compared with the control. Similar to the previous results, the results showed that 25 mg / kg of anti-TNFR2 antibody was well tolerated and non-toxic to mice.

[0381] In the group treated with anti-TNFR2 antibody, tumor size ( Figure 5 ) showed significant differences. Compared with the control group G1, the tumor volumes of the treatment groups treated with anti-hTNFR2 antibodies BC-1A8-IgG1 (G2), BC-1C3-IgG1 (G3), BC-1F10-IgG1 (G4), BC-1F4-IgG1 (G5) and BC-1B6-IgG1 (G6) were significantly reduced. TV The TGI of each treatment group on day 21 (21 days after grouping) was also calculated as shown in Table 12 below. TV %.

[0382] Table 12

[0383]

[0384]

[0385] In vivo efficacy and toxicity of hTNFa / hTNFR2 mice with MC38, 3 mg / kg

[0386] The humanized TNFα mouse model was engineered to express human TNFα protein (SEQ ID NO: 79), in which the coding sequence of the mouse TNFR2 protein was replaced by the corresponding human coding sequence. A double humanized TNFα / TNFR2 mouse model (B-hTNFα / hTNFR2 mouse) was also generated by crossing the TNFα humanized mouse with the TNFR2 humanized mouse. A detailed description of the humanized TNFα mouse model can be found in PCT / CN2020 / 072714; which is incorporated herein by reference in its entirety.

[0387] Similar to previous in vivo drug efficacy experiments, the effects of these anti-hTNFR2 antibodies on in vivo tumor growth were tested in a dual humanized TNFα / TNFR2 mouse model. In each group, dual humanized TNFα / TNFR2 mice were injected with phosphate-buffered saline (PBS, G1), BC-1C3-IgG1 (G2), and anti-mPD-1 (G3) by intraperitoneal (ip) administration. The antibodies were administered at 3 mg / kg by intraperitoneal injection twice a week for 3 weeks (a total of 6 injections).

[0388] Weight and tumor size were monitored throughout the experiment. Mice in all groups gained weight, with no significant differences between groups (P > 0.05). At the time of group assignment (D0), the mean body weight in each group ranged from 20.0 g to 20.6 g. At the end of the experiment (D28), the mean body weight ranged from 23.5 g to 25.1 g, with a body weight change of 115.7% to 127.2%. Similar to previous results, these results demonstrate that the anti-hTNFR2 antibody was well tolerated and nontoxic to mice.

[0389] In the group treated with anti-hTNFR2 antibody, tumor size ( Figure 6 ) showed significant differences. As shown in Table 13 below, the TGI of each treatment group at day 28 was calculated. TV %. BC-1C3-lgG1 (G2) showed the best efficacy at this dose and was superior to the positive control (G3).

[0390] Table 13

[0391]

[0392] In vivo efficacy in B-TNFR2 mice with MC38, 3 mg / kg

[0393] Similar to the above in vivo efficacy experiments, MC38 cancer cells were subcutaneously injected into B-TNFR2 mice. When the tumor volume of the mice reached 100 mm 3 At 4 hr, mice were randomly divided into different groups (six mice per group) according to the volume of the tumor. Mice were injected with PBS as a control (G1), 3 mg / kg BC-1C3-IgG1 (G2), 3 mg / kg anti-mPD-1 (G3), a combination of 3 mg / kg BC-1C3-IgG1 and 3 mg / kg anti-mPD-1 (G4), 3 mg / kg atezolizumab analog (G5), or 3 mg / kg BC-1C3-IgG1 and 3 mg / kg atezolizumab analog (G6). The administration frequency was twice a week (a total of 6 administrations).

[0394] Atezolizumab is a humanized anti-PD-L1 monoclonal antibody developed by Genentech (VH SEQ ID NO: 80; VL SEQ ID NO: 81).

[0395] Weight and tumor size were monitored throughout the experiment. Mice in all groups gained weight, with no significant differences between groups (P>0.05). At the time of group assignment (D0), the mean body weight in each group ranged from 18.9 g to 19.3 g. At the end of the experiment (D24), the mean body weight ranged from 22.4 to 24.2 g, with a body weight change of 118.6% to 125.3%. Similar to previous results, these results demonstrate that the anti-hTNFR2 antibody was well tolerated and nontoxic to mice.

[0396] The tumor sizes in the groups treated with the antibody are shown in Figure 18 As shown in Table 14 below, the TGI of each treatment group on day 24 was calculated. TV %. Compared with the control group, the tumor growth of the treatment groups was inhibited to varying degrees. In the treatment group, the combination of anti-PD-1 antibody and anti-hTNFR2 antibody (G4) inhibited tumor growth with excellent efficacy. Similarly, compared with anti-hTNFR2 antibody (G2) and anti-PD-L1 antibody atezolizumab analog (G5), the combination of anti-PD-L1 antibody atezolizumab analog and anti-hTNFR2 antibody (G6) also showed better tumor growth inhibition.

[0397] Table 14

[0398]

[0399] In vivo efficacy in B-TNFR2 mice with GL261, 1-10 mg / kg

[0400] GL261 tumor cells (glioblastoma cells) were injected subcutaneously into B-TNFR2 mice. When the tumor volume of the mice reached about 80 mm 3 At the same time, the mice were randomly divided into different groups (six mice in each group) according to the volume of the tumor.

[0401] Mice were then injected with PBS as a control (G1), 1 mg / kg BC-1C3-IgG1 (G2), 3 mg / kg BC-1C3-IgG1 (G3), 10 mg / kg BC-1C3-IgG1 (G4), or 1 mg / kg anti-mPD-1 (G5). Antibodies were administered by intraperitoneal injection on the first and fourth days of each week for 3 weeks (6 injections in total).

[0402] The body weight of the mice was monitored throughout the treatment period. The body weight of the mice increased across the different groups, with no statistically significant differences (P>0.05). On the day of grouping (day 0; "D0"), the average body weight of each group ranged from 19.9 g to 20.6 g. At the end of the experiment (24 days after grouping, D24), the average body weight of each group ranged from 22.5 g to 24.2 g, with a body weight change of 109.4% to 118.3%. The results demonstrate that the anti-hTNFR2 antibody was well tolerated and non-toxic to mice.

[0403] The tumor size data in the antibody-treated groups are shown in Figure 19 As shown in Table 15 below, the TGI of each treatment group on day 24 was calculated. TV %. BC-1C3-IgG showed better anti-tumor effects than anti-mPD-1 antibodies at a dose of 1 mg / kg. In addition, BC-1C3-IgG1 showed a dose-dependent anti-tumor effect (the higher the dose level, the better the anti-tumor effect).

[0404] Table 15

[0405]

[0406] In vivo efficacy in B-TNFR2 mice with MC38, 3 mg / kg

[0407] BI-1808 (VH SEQ ID NO: 82; VL SEQ ID NO: 83) is a monoclonal antibody developed by BioInvent that targets tumor necrosis factor receptor superfamily member 1B (TNFR2). The product is in early clinical development as a single agent and in combination with pembrolizumab for the treatment of solid tumors and cutaneous T-cell lymphoma (CTCL).

[0408] h600-25-108 (VH SEQ ID NO: 84; VL SEQ ID NO: 85) is a humanized anti-TNFR2 monoclonal antibody developed by Apexigen.

[0409] HFB3-1hz6-hGl (VH SEQ ID NO: 86; VL SEQ ID NO: 87) is a monoclonal anti-TNFR2 agonist antibody in a Phase I clinical trial at HiFiBiO Therapeutics for the treatment of advanced solid tumors.

[0410] Similar to the above in vivo efficacy experiments, MC38 tumor cells were subcutaneously injected into B-TNFR2 mice. 3At 4 hr, mice were randomly divided into different groups (six mice per group) according to tumor volume. Mice were injected with PBS (G1), 3 mg / kg BC-1C3-IgG1 (G2), 3 mg / kg BI-1808 analog (G3), 3 mg / kg h600-25-108 analog (G4), or 3 mg / kg HFB3-1hz6-hG1 analog (G5). The administration frequency was twice a week (a total of 6 administrations).

[0411] Weight and tumor size were monitored throughout the experiment. Mice in all groups gained weight, with no significant differences between groups (P>0.05). At the time of group assignment (D0), the mean body weight in each group ranged from 20.1 g to 20.4 g. At the end of the experiment (D28), the mean body weight ranged from 22.9 to 24.9 g, with a body weight change of 113.2% to 122.4%. Similar to previous results, these results demonstrate that the anti-hTNFR2 antibody was well tolerated and nontoxic to mice.

[0412] Tumor size Figure 20 As shown in Table 16 below, the TGI of each treatment group on day 28 was calculated. TV The results showed that the BC-1C3-IgG1 antibody (G2) exhibited significantly better in vivo efficacy at the same dose level compared to the BI-1808 analog (G3), h600-25-108 analog (G4), or HFB3-1hz6-hG1 analog (G5).

[0413] Table 16

[0414]

[0415] In vivo efficacy in Bh TNFa / h TNFR2 mice with MC38, 3 mg / kg

[0416] h600-25-71 (VH SEQ ID NO: 88; VL SEQ ID NO: 89) is a humanized anti-TNFR2 monoclonal antibody developed by Apexigen.

[0417] Similar to the above in vivo efficacy experiments, MC38 tumor cells were subcutaneously injected into B-hTNFa / hTNFR2 mice. 3 At 4 hr, mice were randomly divided into different groups (six mice per group) according to the volume of the tumor and injected with PBS (G1), 3 mg / kg BC-1C3-IgG1 (G2), or 3 mg / kg h600-25-71 analog (G3). The administration frequency was twice a week (a total of 6 administrations).

[0418] Weight and tumor size were monitored throughout the experiment. Mice in all groups gained weight, with no significant differences between groups (P>0.05). At the time of group assignment (D0), the mean body weight for each group ranged from 21.0 g to 21.1 g. At the end of the experiment (D24), the mean body weight ranged from 23.8 g to 24.8 g, with a body weight change of 113.3% to 117.5%. Similar to previous results, these results demonstrate that the anti-hTNFR2 antibody was well tolerated and nontoxic to mice.

[0419] Tumor size Figure 21 The TGI of each treatment group on day 24 is shown in TV The % values ​​are shown below in Table 17. The results show that BC-1C3-IgG1 exhibited significantly better in vivo efficacy than the h600-25-71 analog.

[0420] Table 17

[0421]

[0422] Example 4. PK and TILS Analysis

[0423] PK analysis of B-TNFR2 in mice with MC38 model at 10 mg / kg

[0424] The pharmacokinetic clearance of anti-TNFR2 antibodies was determined in humanized TNFR2 mice. MC38 cells (5×105) were injected subcutaneously into humanized TNFR2 mice and the cells were inoculated after the tumors grew to 300 mm. 3 Mice were divided into 8 groups (n=4) at 4 hr. 10 mg / kg of BC-1C3-IgG1 (G2), BC-1F4-IgG1 (G3), BC-1B6-IgG1 (G4), BC-1C3-IgG1-SI (G5), BC-1F4-IgG1-SI (G6), BC-1B6-IgG1-SI (G7), or isotype control IgG1 (G1) were administered by intravenous injection. Blood samples were collected 15 min, 6 hours, 24 hours, 3 days, 5 days, and 7 days after administration.

[0425] Serum levels of human antibodies were determined by sandwich ELISA enzyme-linked immunosorbent assay. Briefly, goat polyclonal anti-human IgG (Fc specific) capture antibody (Jackson ImmunoResearch, catalog number: 109-036-098) was diluted to a final concentration of 2.0 μg / mL with 0.1 M carbonate buffer (pH 9.6), added to a 96-well plate (ELISA plate) at 100 μL / well, and incubated overnight at 4°C. Then, 200 μL of blocking buffer (2% BSA) was added to each well. The wells were sealed and incubated at room temperature for 1 hour. After washing the plate with a plate washer, horseradish peroxidase (HRP)-conjugated goat polyclonal anti-human IgG (Fc specific) antibody (Jackson ImmunoResearch, catalog number: 109-005-088) was added to each well of the ELISA plate at 100 μL / well and incubated at 37°C for 1 hour. After washing the plate, tetramethylbenzidine (TMB) solution was added to a 96-well plate at 100 μL / well to react with HRP as a substrate. After incubation at room temperature in the dark, 100 μL of stop solution (Beyotime, catalog number: P0215) was added to each well. The absorbance of each well at 450 nm and 630 nm wavelengths was read using a microplate reader. Data analysis software Gen5 was used to analyze the data. A standard curve of four parameters was created using the absorbance and corresponding concentration of the calibration samples prepared from each test product. The antibody concentration of each serum sample was calculated using the standard curve. A drug concentration-time curve was created using the sample concentration calculated at each time point. Pharmacokinetic parameters were calculated using Phoenix winnolin 8.3. The results are shown in Table 18 below.

[0426] Table 18

[0427]

[0428] T 1 / 2 : Terminal half-life

[0429] C max : Maximum concentration

[0430] AUC 0-7天 : Area under the blood concentration-time curve from 0 to 7 days

[0431] CL: Clearance

[0432] The above results showed that after injection of different antibodies, the antibody concentration in the serum of TNFR2 humanized mice decreased over time (see Figure 10), which is consistent with the pharmacokinetic characteristics. The longest half-life (T1 / 2) of the BC-1C3-IgG1(G2) antibody in mice was 2.99 days. The shortest half-life of the BC-1B6-IgG1-SI(G7) antibody in mice was 0.86 days. The half-lives of the other antibodies in mice ranged from 1.69 days to 2.38 days, which was relatively close. By the end of the seventh day of sampling, the area under the curve (AUC) of the drug concentration-time curve for the BC-1C3-IgG1(G2) antibody was 250.93 hr*ug / mL, which was greater than the AUCs of the other antibodies (AUC0-7 days: 77.90-168.89 hr*ug / mL). The clearance (CL) of the BC-1C3-IgG1(G2) antibody was 28.85 ml / hr / kg, while the CLs of the other antibodies ranged from 47.07-61.19 ml / hr / kg. The results showed that BC-1C3-IgG1(G2) was cleared less efficiently than other antibodies and that the BC-1C3-IgG1(G2) antibody was metabolized more slowly in mice.

[0433] Example 5. Reporter Cell Activation and Binding Assays

[0434] Activation of reporter cells

[0435] This experiment was performed to test whether anti-TNFR2 antibodies could activate the TNFR2 pathway.

[0436] Human TNFα protein (Sino Biological Inc., catalog number: 10602-HNAE) was serially diluted (3-fold) to a maximum concentration of 10 ng / mL as a positive control. Anti-TNFR2 antibodies BC-1F4-IgG1, BC-1C3-IgG1, BC-1B6-IgG1, and BC-1F10-IgG1 were serially diluted (3-fold) to a maximum concentration of 60 μg / mL. Jurkat-GFP-TNFR2 cells were seeded in 96-well plates (cell density of 1×10 5 Cells were plated in a 96-well plate (100 μL per well, 100 μL of either human TNFα protein or anti-TNFR2 antibody) and incubated overnight at 37°C. After incubation, the plate was removed and transferred to a 96-well plate. Each well was washed with 150 μL of PBS. The supernatant was discarded. 100 μL of PBS was added to each well to resuspend the cells. The plate was then placed in a luminescence detector to detect the fluorescent signal. If the antibody can activate TNFR2, the reporter cells will report a GFP signal.

[0437] like Figure 11AAs shown, no fluorescence signal was detected in the presence of anti-TNFR2 antibodies BC-1F4-IgG1, BC-1C3-IgG1, BC-1B6-IgG1, and BC-1F10-IgG1. However, BC-1C3-IgG1 showed weak reporter cell activation ( Figure 11B ).

[0438] Blocking effect on reporter cells

[0439] Experiments were performed to test whether anti-TNFR2 antibodies could block the binding between TNFR2 and its ligand TNFα.

[0440] Jurkat-GFP-TNFR2 reporter cells were seeded in 96-well plates (cell density was 1×10 5 cells / well). TNFα protein was diluted to 1 ng / mL. Anti-TNFR2 antibodies BC-1F4-IgG1, BC-1C3-IgG1, BC-1B6-IgG1, and BC-1F10-IgG1 were serially diluted (3-fold) to a maximum concentration of 10 μg / mL. 50 μl of human TNFα protein and 50 μl of antibody were added to each well and incubated at 37°C for 24 hours. After incubation, the plate was removed and transferred to a 96-well plate. Each well was washed with 150 μl of PBS. The supernatant was discarded. 100 μL of PBS was added to each well to resuspend the cells. The GFP signal was determined by flow cytometry.

[0441] like Figure 12 As shown, when the concentrations of the anti-TNFR2 antibodies BC-1F10-IgG1 and BC-1F4-IgG1 were increased, the GFP signal (indicating that cells bound to TNFα) decreased (y-axis), indicating that the binding between human TNFα and TNFR2 was blocked by the anti-TNFR2 antibodies BC-1F10-IgG1 and BC-1F4-IgG1.

[0442] Example 6. In vivo toxicity experiment (non-tumor bearing model)

[0443] TNFR2 humanized mice (6-8 weeks) were randomly divided into a control group and a treatment group (4 mice per group) according to their body weight. The control group was injected with an equal volume of PBS, and the treatment group was injected with anti-hTNFR2 antibody (BC-1C3-IgG1, BC-1F4-IgG1 or BC-1B6-IgG1) or CTLA4 antibody (anti-mCTLA4). The injection dose of anti-hTNFR2 antibody and CTLA4 antibody was 30 mg / kg or 100 mg / kg. The frequency of administration was once a week for a total of 4 times. The specific administration dose, mode and frequency are shown in Table 19 below. During the experiment, weight changes and any abnormalities were monitored. Blood biochemical indicators were monitored on the 1st, 8th, 15th, 22nd and 28th day after grouping. Blood biochemical indicators included aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatine kinase (CK), albumin (ALB), total protein (TP), amylase (AMY), urea (UREA), creatinine (CREA), glucose (GLU), triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), calcium (Ca), and inorganic phosphorus (P). Routine blood tests were performed on day 28 after grouping. Routine blood tests included the following tests: white blood cell count (WBC), red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin content (MCH), mean corpuscular hemoglobin concentration (MCHC), PLT (platelet count), lymphocytes (LYMPH#), lymphocyte percentage (LYMPH%), monocytes (MONO#), monocyte percentage (MONO%), neutrophil percentage (NEUT%). At the end of the experiment, the heart, liver, spleen, lung, kidney, and intestine of the mice were placed in formalin for HE staining, and the weights of the liver, spleen, and kidney were measured.

[0444] Table 19

[0445]

[0446]

[0447] The results showed that the body weight of all mice in the control group and the treatment group showed an upward trend throughout the experiment, and there was no significant difference in body weight changes among the groups. Figures 13A-13B The results of exemplary blood biochemical parameters and blood routine test on day 28 showed no significant difference compared with the control. Similar to the previous results, the in vivo toxicity results showed that the anti-hTNFR2 antibody was well tolerated and non-toxic to mice.

[0448] Example 7. TNFR2 Antibody Blocks Treg Cells from Inhibiting CD8+ T Cell Proliferation

[0449] Human IgG1 (control) and anti-TNFR2 antibodies BC-1C3-IgG1 and HFB3-1hz6-hG1 analogs were diluted to a final concentration of 20 ng / mL. Anti-CD3 antibody (CD3, Biopsies Biotech Co., Ltd., catalog number: CDE-M120a) was used to coat a 96-well plate (10 μg / mL, 100 μl / well) overnight at 4°C. 100 μl of CFSE-labeled (CellTrace TM PBMC cells (A11Cells, catalog number: PB003F-C) and 50 μl Treg cells (OriCell, catalog number: FPB009-4F-2) containing CFSE cell proliferation kit, Thermo Fisher, catalog number: CDE-M120a) were added to each well. 40 μl BC-1C3-IgG1, 40 μl human IgG1 or 40 μl HFB3-1hz6-hGl analogs were added to each well and incubated at 37°C, 5% CO2 for 120 hours. After incubation for 48 hours, 10 μl IL-2 (Bipsys Biotech Co., Ltd., catalog number: IL-2-H4113) was added to each well. After incubation for 120 hours, cells were collected and hCD8+ cells were determined by flow cytometry.

[0450] like Figure 22 As shown, BC-1C3-IgG1 blocked the inhibition of CD8+ T cell proliferation by Treg cells.

[0451] Example 8. CD8+ T cell activation assay

[0452] Human IgG1 (control), anti-TNFR2 antibody BC-1C3-IgG1 and HFB3-1hz6-hG1 analog were serially diluted to 0.1 μg / mL, 1 μg / mL and 10 μg / mL. Anti-CD3 antibody and anti-TNFR2 antibody were used to coat 96-well plates overnight at 4°C. TMCD8+ T cells (Animal Biotech) containing a CFSE cell proliferation kit (Thermo Fisher Scientific, catalog number: CDE-M120a) were added to each well (1×105 cells / well, 100 μl / well), and hCD28 (BioXcell, catalog number: BE0248) was added to each well (1 μg / mL, 100 μl / well) and incubated at 37°C, 5% CO2 for 72 hours. The supernatant was collected to detect the secretion levels of human IL2 and human IFNγ, and the cells in the pellet were detected by flow cytometry.

[0453] like Figure 23 As shown, CD8+ T cells increased with increasing concentrations of the anti-TNFR2 antibody BC-1C3-IgG1. Figures 24A-24B The results showed that the secretion of human IL-2 and IFNγ increased after adding anti-TNFR2 antibody. These results indicate that BC-1C3-IgG1 can promote the proliferation and activation of CD8+ T cells.

[0454] Other embodiments

[0455] It should be understood that although the invention has been described in conjunction with specific embodiments, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. An antibody or antigen-binding fragment thereof that binds to TNFR2, comprising: A heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, and a light chain variable region (VL) comprising CDRs 1, 2, and 3; according to Kabat numbering, the VH comprises CDRs 1, 2, and 3 as represented by amino acid sequences of SEQ ID NOs: 18, 19, and 20, respectively, and the VL comprises CDRs 1, 2, and 3 as represented by amino acid sequences of SEQ ID NOs: 21, 22, and 23, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment specifically binds to human TNFR2.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof according to claim 3 , wherein the antibody or antigen-binding fragment is a human IgG1 antibody.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment is a single-chain variable fragment (scFV).

6. A nucleic acid comprising a polynucleotide encoding an antibody or antigen-binding fragment thereof that binds TNFR2, the polypeptide comprising: (1) an immunoglobulin heavy chain comprising a heavy chain variable region (VH) comprising CDRs 1, 2, and 3 of the amino acid sequences set forth in SEQ ID NOs: 18, 19, and 20, respectively, and wherein the VH binds to TNFR2 when paired with a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID NO: 41; and (2) An immunoglobulin light chain comprising a VL comprising CDRs 1, 2, and 3 of the amino acid sequences set forth in SEQ ID NOs: 21, 22, and 23, respectively, and wherein the VL binds to TNFR2 when paired with a VH of the amino acid sequence set forth in SEQ ID NO:

40.

7. The nucleic acid of claim 6, wherein the VH specifically binds to human TNFR2 when paired with the VL, or the VL specifically binds to human TNFR2 when paired with the VH.

8. The nucleic acid of claim 6, wherein the immunoglobulin heavy chain is a human immunoglobulin heavy chain, and the immunoglobulin light chain is a human immunoglobulin light chain.

9. The nucleic acid of claim 6, wherein the nucleic acid encodes a single-chain variable fragment (scFv).

10. The nucleic acid according to any one of claims 6 to 9, wherein the nucleic acid is cDNA.

11. A vector comprising the nucleic acid according to any one of claims 6 to 10.

12. A vector comprising two of the nucleic acids of any one of claims 6-10, wherein the vector encodes a VH region and a VL region that together bind to TNFR2.

13. A vector pair, wherein each vector comprises one of the nucleic acids of any one of claims 6-10, wherein the vector pair collectively encodes a VH region and a VL region that together bind to TNFR2.

14. A cell comprising the vector according to claim 11 or 12, or the vector pair according to claim 13; the cell is not a plant cell.

15. The cell according to claim 14, wherein the cell is a CHO cell.

16. A cell comprising the nucleic acid according to any one of claims 6 to 10; the cell is not a plant cell.

17. A cell comprising two of the nucleic acids according to any one of claims 6 to 10; said cell is not a plant cell.

18. The cell of claim 17, wherein the two nucleic acids together encode a VH region and a VL region, and the VH region and VL region together bind TNFR2.

19. A method for producing an antibody or an antigen-binding fragment thereof, the method comprising (a) culturing the cell according to any one of claims 14 to 18 under conditions sufficient for the cell to produce the antibody or antigen-binding fragment; and (b) collecting the antibody or antigen-binding fragment produced by the cell.

20. An antibody or antigen-binding fragment thereof that binds to TNFR2, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH is the sequence of SEQ ID NO: 40 and the VL is the sequence of SEQ ID NO:

41.

21. The antibody or antigen-binding fragment thereof of claim 20, wherein the antibody or antigen-binding fragment specifically binds to human TNFR2.

22. The antibody or antigen-binding fragment thereof according to claim 20, wherein the antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof.

23. The antibody or antigen-binding fragment thereof according to any one of claims 20 to 22, wherein the antibody or antigen-binding fragment is a single-chain variable fragment (scFV).

24. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 and 20 to 23 in the preparation of a medicament for treating cancer; The cancer is selected from the group consisting of colorectal cancer, ovarian cancer, acute myeloid leukemia, Lewis lung cancer, breast cancer, hepatocellular carcinoma, glioma, renal cell carcinoma, multiple myeloma and cutaneous T-cell lymphoma.

25. The use according to claim 24, wherein the cancer is colon cancer, glioma or ovarian cancer.

26. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-5 and 20-23, and a pharmaceutically acceptable carrier.

Images